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Synthyra 5

The beta-glucosidase encoding gene bglA was cloned from Bacillus polymyxa 1.794. The bglA gene was inserted in expression vector pET28a(+) and transformed into Escherichia coli BL21 (DE3), finally the recombinant strain BL1979 was obtained. Induced by IPTG, the expression P-glucosidase activity reached to 24.7 IU/mL. The optimum temperature and optimum pH of the recombinant expression P-glucosidase in BL1979 were 37 degrees C and 7.0 respectively,the purity can reach to 92.7%. Analysis of the fusion protein by nondenaturing gradient gel electrophoresis, we found the fusion protein exists in dimmer, tetramer,hexamer and octamer, they all have hydrolase activity.

Without serum to provide adherent factors, CHO-dhfr- cells grow in suspension when cultured in serum-free medium. Although this offers advantages in some applications, in most production systems adherent cell growth is preferable. Gene transfection, clonal selection and amplification can be easier for adherent cells; the density of immobilized cells is often higher than those in suspension culture, which results in a higher protein productivity; washout of cells by perfused medium during continuous fermentation can be avoided; for high-throughput microplate assays, adherent cells are preferred to facilitate medium changes and cell washing. It has been proved that purified vitronectin alone was able to mediate attachment and spreading of CHO cells in serum-free medium. So we constructed a tricistronic expression vector expressing Igf-1, Vitronectin and Bel-2 at the same time. The vector was transfected into CHO-dhfr- cells and one clone, namely CHO-IVB2, expressing high level of the three proteins was screened out by Western blot. The cell line showed similar apoptosis-resistant and serum-independent properties to CHO-IB, an engineered cell line constructed before. When cultured in IMEM protein-free medium without any components supplemented, CHO-IVB can grow adherently. The viable cell numbers and growth rate of CHO-IVB were much higher than CHO-IB, making CHO-IVB an apoptosis-resistant host for production of recombinant proteins which can grow adherently in protein-free medium.

After the cell enters into its programmed cell death, xylanases from grass plants gradually matured through its N-terminal and C-terminal sequence been cut by acid proteases several times. They could not be expressed by conventional protein expression system. Search the GenBank database, xynIII from a mutant of T. reesei QM9414(ATCC26921)was found. It is similar to grass plants' xylanase in their families and structures. It couldn't express in T. reesei QM9414, but its gene exist in genomic DNA as one copy. Through overlap-PCR method, 4 exons of xynIII were cloned, sequenced, spliced, and the whole cDNA of mature xynIII was acquired. The cDNA was inserted into pETBlue-2 vector and transformed into E. coli DE3 pLacI cell. Xyn III could be expressed in the transformed cell under the conditions of 37 degrees C, 1 mmol/L IPTG induced for 3h. Low temperature (15 degrees C), long time(64h) induction(0.2 mmol/L IPTG) could enhance xynIII activity.

It is well known that the immune reactivity is modulated by gender. In fact, women show a more effective immune response as well as a more frequent development of autoimmune diseases. In particular, 17 beta-estradiol (E2) in patients with systemic inflammatory diseases leads to an higher production of IgG and IgM in peripheral blood mononucleated cells (PBMC) and the secretion of metalloproteinases and IL-6 by synovial fibroblasts. The effect of E2 seems to be partially related to its concentration. In fact, at the physiological concentration, E2 seems to exert a pro-inflammatory effect, while at pharmacological concentrations shows anti-inflammatory effects. Steroid hormones can be converted in downstream hormones along defined pathways. The conversion of dehydroepiandrosterone (DHEA) in peripheral macrophages leads to the androgen production. Subsequently the enzyme aromatase converts androgens in estrogens, and its activity is increased by some inflammatory cytokines such as IL-1beta, IL-6 and TNF-alpha. In the synovial fluids of rheumatoid arthritis (RA) patients the levels of estrogens result significantly increased compared with controls, showing the consequence of this unbalanced steroid metabolism. Furthermore, the metabolism of estrogens leads to some downstream hydroxylated metabolites, that are not waste products, but still active molecules in the inflammatory response. In fact, it has been found that synovial fluids of RA patients present a different ratio of 16-hydroxylated estrogen metabolites/ 2-hydroxylated metabolites, confirming that also the unbalanced metabolism of estrogens and not only the estrogen concentration seems to be related to the development and worsening of rheumatoid arthritis.

It was found that the level of Calponin h1 (CaP h1) mRNA was significantly up-regulated by Estrogen in the myometrium of sheep towards the end pregnancy. Although the CaP h1 has been widely used as a reference gene to observe the changes of expression level of other genes, the full-length gene in sheep has not been obtained. With the oligo nucleotide primers according to human, mouse and pig CaP h1 mRNA, the full-length cDNA of CaP h1 was cloned by 5'- and 3'-RACE (Genbank accession number = AY327118). The cDNA was 1499bp in length and contained a complete open reading frame of 891 bp, encoding a protein of 297 amino acid residues. 5'-and 3'-UTR was 79 bp and 529bp, respectively. With PCR-SSP approaches,the genomic DNA of sheep CaP h1 was obtained .It showed that the gene has 7 exons and 6 introns, spanning over 8kb(Genbank accession number of introns : AY771807,AY771808, AY771809, AY771810.) Homologous comparison indicated that the cDNA sequences are highly conserved across the species. The highest homology was found in wild pig (92%), followed by human (88%), rat (81%), mouse (81%) and chicken (79%). The intron sequence and length showed a large variation among species (>50%).

Generation of fungal protoplast is an essential tool for genetic transformation system. To establish protoplast-mediated genetic transformation system of Monascus purpureus, conditions for the protoplast isolation and regeneration of the mycelia of various enzymes and osmotic stabilizers were examined. To investigate suitable cell age for the protoplast preparation of mycelia of M. purpureus, the mycelia were cultured in different ways at 30oC. Mycelia obtained through cellophane - mediated culture for 30~40h were adequate to protoplast preparation. When lysing enzyme, cellulase and snailase were added to the mycelia in combination or alone, combination of lysing enzyme, cellulase and snailase accordingly at the concentration of 0.3%, 0.1% and 1% was most benefit for protoplast yield. When we applied various osmotic stabilizers at different concentrations to protoplast preparation, 1 mol/L MgSO4 was most effective for the protoplast release. The suitable incubation time with enzyme for the maximum release of protoplasts was 2.5-hr. When we investigate various osmotic stabilizers for the regeneration of the protoplasts of mycelia of strain M34 and N18, the complete medium containing 0.6 mol/L sucrose induced highest hyphal growth with regeneration frequency of 8.5% and 36.4%, respectively. PEG and CaCl2- mediated protoplast co-transformation of strain M34 with pBC-Hygro and pNL1, hygromycin B as selective marker, was fulfilled and 100 stable transformants per microgram DNA were obtained.

Eleven subcloned DNA fragments from the 5'- upstream region of lipA, lipC and lipF of Phanerochaete chrysosporium were assayed by using the gel mobility shift assay(GMSA). The total proteins extracted from P.chrysosporium mycelia grown in Kirk medium and natural fir wood chip were used to identify the segments in these 11 DNA fragments which are controlled by some regulatory proteins. The results showed that two DNA segments LG2P3(396bp) and LG6S1-2 (738bp) in the 5'-noncoding regions of lipC and lipF were able to specifically bind total mycelial proteins of P. chrysosporium incubated in Kirk medium, separately. One DNA segment LG6S2 (226bp) from the 5'-noncoding region of lipF was found to specifically bind total mycelial proteins of this fungus on natural fir wood chip. Analysis of the sequences showed that there were many cis-regulatory elements in these DNA segments, implying that these sequences may be bound by some transcriptional regulation protein factors.

A fusion protein, Interferon-BLA (IFN-BLA), was constructed with IFN-beta-1b and IFN-alpha-2b separating by a linker -GGGS-. The laboratory-scale expression conditions in E.coli BL21 CodonPlus (DE3)-RIL had been optimized and IFN-BLA was expressed higher than 35% of total protein in the cells mainly as inclusion body. The inclusion body of IFN-BLA was denatured and refolded by dialysis and purified by ion-exchange chromatography. The overall yield of IFN-BLA was about 45 mg/L with purity higher than 90%. Antiviral activity assay suggested that this newly fused protein may have synergetic or additive antiviral activities.

The National Toxicology Program (NTP) Center for the Evaluation of Risks to Human Reproduction (CERHR) conducted an evaluation of the potential for di-isononyl phthalate (DINP) to cause adverse effects on reproduction and development in humans. DINP is one of 7 phthalate chemicals evaluated by the NTP CERHR Phthalates Expert Panel. These phthalates were selected for evaluation because of high production volume, extent of human exposures, use in children's products, and/or published evidence of reproductive or developmental toxicity. DINP is a mixture of branched, C-9 phthalate isomers used to add flexibility to a wide variety of plastic products such as toys, garden hoses, flooring tiles, tarps, and pool liners. The results of this evaluation on DINP are published in a NTP-CERHR monograph which includes: 1) the NTP Brief, 2) the Expert Panel Report on the Reproductive and Developmental Toxicity of Di-isononyl Phthalate, and 3) public comments received on the Expert Panel Report. As stated in the NTP Brief, the NTP reached the following conclusions regarding the possible effects of exposure to DINP on human development and reproduction. First, although DINP could possibly affect human development if exposures are sufficiently high, there is minimal concern for DINP causing adverse effects to human reproduction or fetal development. There is no direct evidence that exposure of people to DINP adversely affects reproduction or development, but studies show that oral exposure of pregnant rats to high doses (500 and 1000 mg/kg bodyweight/day) of DINP can adversely affect fetal development. Effects on pup growth were noted in a 2-generation reproductive toxicity study in rats at doses of 143-285 mg/kg body weight/day. Human exposure information for DINP was not available but it was assumed that the general US population would be exposed to 3-30 mug/kg body weight/day, based upon the range of estimated exposures for DEHP, a more widely used phthalate. Second, based on estimates of exposure of children to DINP from mouthing toys and other objects, the NTP has minimal concern for developmental effects in children. After the expert panel meeting, a US Consumer Products Safety Commission panel estimated that the majority of children exposed to DINP had a "minimum to non-existent risk of injury" from mouthing toys. Children's exposure was estimated at 70-280 mug/kg body weight/day, a level 1000-fold lower than exposures resulting in developmental effects in rats. NTP-CERHR monographs are transmitted to federal and state agencies, interested parties, and the public and are available electronically in PDF format on the CERHR web site (http://cerhr.niehs.nih.gov) and in printed text or CD-ROM from the CERHR (National Institute of Environmental Health Sciences, P.O. Box 12233, MD EC-32, Research Triangle Park, NC; fax: 919-316-4511).

The National Toxicology Program (NTP) Center for the Evaluation of Risks to Human Reproduction (CERHR) conducted an evaluation of the potential for propylene glycol (PG) to cause adverse effects on reproduction and development in humans. PG was selected for evaluation because of the potential for widespread human exposure through its use in food, tobacco, pharmaceutical products, cosmetics, various paints and coatings and as an antifreeze and de-icing solution. PG is a small, hydroxy-substituted hydrocarbon used as a chemical intermediate in the production of unsaturated polyester resins and in the production of plasticizers. The results of this evaluation on PG are published in an NTP-CERHR monograph which includes: 1) the NTP Brief, 2) the Expert Panel Report on the Reproductive and Developmental Toxicity of Propylene Glycol, and 3) public comments received on the Expert Panel Report. As stated in the NTP Brief, the NTP reached the following conclusions regarding the possible effects of exposure to PG on human development and reproduction. There is negligible concern for adverse developmental and reproductive effects in humans at current, proposed, or estimated exposure levels. There is no direct evidence that exposure of people to PG adversely affects reproduction or development. Studies in pregnant laboratory animals at oral doses of PG greater than 1,200 mg/kg body weight/ day and up to 10,400 mg/kg body weight/day in mice, did not produce developmental toxicity in offspring. In a continuous breeding study, no effects on fertility were observed in male or female mice at doses up to 10,100 mg/kg body weight/day in drinking water. The pharmacokinetics of PG indicates that the lack of adverse effects observed in laboratory animals is relevant to humans. The rate-limiting step in PG metabolism is conversion to the more toxic lactaldehyde product by alcohol dehydrogenase. Studies indicate that this enzyme saturates in humans at doses 8-10- fold lower than in rats and rabbits, thus affording less toxicity in humans. It is estimated that the average daily intake of PG from food products in the US is 34 mg/kg body weight/day for a 70 kg person, which is over 300 -fold lower than the highest dose tested in laboratory animals. NTP-CERHR monographs are transmitted to federal and state agencies, interested parties, and the public and are available in electronic PDF format on the CERHR web site (http://cerhr.niehs.nih.gov) and in printed text or CD-ROM from the CERHR (National Institute of Environmental Health Sciences, P.O. Box 12233, MD EC-32, Research Triangle Park, NC; fax: 919-316-4511).

To investigate the cell type which express IL-10 mRNA and the protein in gingival tissues of patients with chronic periodontitis.

To observe the effect of immunization with the fusion protein of GBD of Streptococcus mutans glucan binding protein-A against dental caries.

Molecular biology is an exciting, rapidly expanding field, which has enabled enormously greater understanding of the biology of diseases and malfunctions in many fields. It chiefly concerns itself with understanding the interactions between the various systems of a cell, including the interrelationship of DNA, RNA and protein synthesis and how these interactions are regulated. Since the introduction of molecular biology into modern science, numerous other fields have been enabled to go "molecular". Advanced molecular biological techniques showed us new avenue towards finding answers to the questions asked for decades. The first part of this article described the history of molecular biology. It started as a joined discipline of other areas of biology, i.e. genetics and biochemistry in the 1930s and 1940s, and enjoyed its classical period and became institutionalized in the 1950s and 1960s. Major molecular techniques manipulating proteins, DNA and RNA were introduced and their mechanisms were concisely illustrated. The current knowledge of molecular biology and their applications in orthodontic and oral and maxillofacial surgery, i.e. osteoclast differentiation and function, regulation of tooth movement, mechanotransduction/cell-signalling, bone fracture healing, oral cancer as well as craniofacial/dental anomalies and distraction osteogenesis were discussed. Although the problems of introducing molecular technologies are still substantial, it is anticipated that the future of medicine/dentistry will be "molecular": molecular prevention, molecular diagnosis and molecular therapy.

To investigate the changes in non-protein respiratory quotient (NPRQ) and oxidation rate of protein after burn injury, and the effect of growth hormone (GH) administration on metabolism.

To investigate the effect of intensive insulin therapy on serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and C reaction protein (CRP), all of which reflected the inflammatory status in patients with severe trauma.

To investigate the expression of human telomerase reverse transcriptase (hTERT) mRNA and protein in cervix cancer, cervical intraepithelial neoplasia (CIN) and normal cervix.

To examine the therapeutic effect of inducible costimulator fusion protein (ICOS-Ig) on airway inflammation in a murine model of allergic asthma.

By transformation mediated by Agrobacterium tumefacien, we successfully transferred the chimeric gene of GFP-mTn ( mTn is the binding domain of microfilament binding protein talin from mouse, which can show the microfilament in living cell ) into Torenia fournieri. Using confocal laser scanning microscopy (CLSM), the distribution of fusion protein in different kinds of tissues and cell in transgenic Torenia fournieri was observed. GFP fluorescence was found in leaf epidermal cell, stomatal guard cell and root epidermal cell. Actin filaments can be visualized clearly only in guard cells. In the guard cells of open stomata under light, actin filaments arrange reticularly and randomly in cortical cytoplasm. In the guard cells of closed stomata under darkness, actin filaments arrange curly along the longitude of guard cell, and some helix and ring structures were found. GFP fluorescence was not found in other cell types, including stem epidermal cell, root hair cell and reproductive organs. The transgenic Torenia fournieri we got provides a suitable material to study dynamics of actin filament in stomatal guard cell.

The nm23 gene family was involved in cellular multiphysiopathological processes including differentiation, development, apoptosis and cancer promotion, progression or metastasis. Some data indicate that nm23 plays an important role in regulating reproductive processes. In the present study, we analyzed the proteome of the implantation sites and the peri-implantation sites in NIH. mice on Day 5 of gestation by using two-Dimensional gel electrophoresis (2-D PAGE), while the virgin mice as the control. A protein spot with pI 7.1, Mr 18 kDa showed up-regulated expression in endometrium during the blastocysts adhesiveness. Using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS), this protein was identified as nm23-M2/NDPK B. The nm23-M2 expression in mice endometrium was shown progressive increase on Day 5 of gestation by RT-PCR, which was consistent with the result obtained by immunohistochemistry. These findings suggest that nm23-M2/NDPK B was involved in the process of blastocyst implantation.

The dimorphic transition of yeast and hyphal forms is one of most determinants in Candida albicans for its pathogenicity. This transition is regulated by several signal transduction pathways. Transcriptional factor Flo8 plays an important role in morphogenesis of Saccharomyces cerevisiae. In this work, a C. albicans genomic DNA library was introduced into a S. cerevisiae flo8/flo8 mutant and genes which could suppress invasive growth defect were isolated. A novel gene was isolated and designated CaPPE1 (Candida albicans PPE1 gene). CaPPE1 encoded for a 361 amino acid protein CaPpe1, shared highest similarity in amino acids (35% identity) with the protein phosphatase methylesterase Ppel of S. cerevisiae. In haploid of S. cerevisiae, ectopic expressed CaPPE1 could partially suppress the invasive growth defect of the flo8 mutant but failed to suppressed the invasive growth defects of the mutants in MAPK pathway(ste12/ste12 and tec1/ tecl). Ectopic expression of the CaPPE1 in diploid of S. cerevisiae suppress the filamentous growth defect of some mutants in MAPK pathway, but not in flo8/flo8 mutant under nitrogen starvation condition. It is suggested that CaPpe1 may be involved in different regulating pathways in diploid filamentous growth and in haploid invasive growth.

To investigate microsatellite instability (MSI) and loss of heterozygosity (LOH) of locus D17S396, D17S579 and D17S855, and their effect on the expression of nm23H1 and BRCA1 of gastric cancer, which would provide experimental basis for clinical treatment and prognosis analysis of gastric cancer. DNA was extracted from paraffin-embedded materials. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used to analyze MSI and LOH. Expression of nm23H, and BRCA1 was detected by Envision immuno-histochemistry and Leica-Qwin computer imaging techniques. In the forty cases of gastric cancer, the frequency of MSI, LOH and nm23H1 protein were 20.00%, 17.50% and 55.00% respectively at locus D17S396, while at locus D17S579, the frequency of MSI, LOH and BRCA1 protein were 22.50%, 15.00% and 37.50% respectively; at locus D17S855, the frequency of MSI, LOH and BRCA1 of thirty-seven cases were 18.92%, 18.92%, 37.84% respectively. In tumor node metastasis (TNM) staging, at locus D17S396, D17S579 and D17S855, MSI in stages I + II appeared more frequently than that in stages III + IV, while LOH appeared the contrary tendency. In the group of metastasis of gastric cancer, MSI had a less frequency (5.00%) than that with no metastasis (35.00%, P < 0.05) at locus D17S396, but LOH appeared more frequently (30.00%) than that with no metastasis (5.00%, P < 0.05). At locus D17S579, MSI had an increasing tendency with the degree of tumor differentiation (50.00% in high differentiation cases, 20.00% in middle differentiation cases, and 0% in low differentiation cases, P < 0.05). The frequency of nm23H1 and BRCA1 protein in stages TNM I + II was higher than that in stages TNM III + IV; and that in higher differentiation cases was higher than in poor differentiation cases. The frequency of nm23H1 protein in the group of metastasis (30.00%) was less than that with no metastasis significantly (80.00%, P<0.01). The frequency of nm23H1 protein in the group positive to MSI (87.50%) was higher than that in the group negative to MSI (46.88%, P < 0.05). However, nm23H1 protein in group positive to LOH (14.29%) was lower than that in the group negative to LOH (63.64%, P < 0.05). The frequency of BRCA1 protein in the group positive to MSI (66.67%) was more than that in the group negative to MSI (29.03%, P < 0.05). The results of experiments indicate that MSI and LOH may separately control the development of sporadic colon cancer with different pathways. MSI may be an early period molecule marker for sporadic colon cancer, enhanced expression of nm23H1 protein can effectively inhibit colon cancer metastasis and improve prognosis of sporadic colon cancer patients. By comparison, LOH mostly arises in the late period of sporadic colon cancer and endows a high aggressive and poor prognostic phenotype. nm23H1 protein could effectively restrain gastric cancer metastasis and development; and BRCA1 protein could restain tumor from becoming lower differentiation.

To investigate the expression of CTP encoding gene in the methylotropic yeast, pichia pastoris and the possibility of CTP acting as an antifertility vaccine or anti-cancer vaccine, we strung two, three or four CTP cDNA to construct CTP polymeric cDNA in order to enhance the immunogenicity of the CTP. Then, the recombinant genes were subcloned into a pichia pastoris expression vector pPIC9K to construct pPIC9K-(hCGbeta-CTP37)n(n = 2,3,4). After identified by restriction endonuclease digestion and DNA sequencing, the recombinant vectors were linearized and transferred into GS115 by electroporation. The induced culture supernatant was precipitated by PEG6000 and the precipitate was washed by 75% alcohol. SDS-PAGE and RIA analysis suggested GS115 expressed the recombinant genes successfully and the recombinant protein had anti-hCG antibody binding activity. In addition, ANTHEPROT 4.3 software was used to analyze the protein structure of CTP quadrigeminum. We found that CTP quadrigeminum had similar secondary structure with hCGbeta, but the speciality of antigen better than that of the latter. Therefore, we conclude that this study prepared basic necessary data for developing antifertility vaccines or anticancer vaccines basing on hCGbeta--CTP37.

This study was aimed at investigating the effect of glutamate on motor neurons in organotypic cultured spinal cord slices treated by threohydroxyaspartate (THA), an inhibitor of glutamate transporter. The spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentration of THA(50 micromol/L,100 micromol/L,500 micromol/L) was added into the culture medium respectively. Ventral alpha-motor neurons survival was evaluated by immunohistochemistry staining monoclonal antibody SMI-32, a nonphosphorylated neurofilament marker, and interneurons in dorsal horn were identified by monoclonal anti-calretinin antibody staining. Lactate dehydrogenase (LDH) level in the culture medium was also measured. The spinal cord slices in the control group could maintain excellent organotypic cellular organization and a stable population of ventral alpha-motor neurons. THA caused a slow dose-dependent loss of alpha-motor neurons and an increase in LDH enzyme activity in the culture medium while dorsal interneurons were less damaged. 100 micromol/L THA resulted in a significant decrease in (alpha-motor neurons after cultured for 4 weeks. On the contrary, the interneurons in the dorsal horn were less affected. It was also observed in patients with amyotrophic lateral sclerosis (ALS). Excellular Glu mainly caused selective alpha-motor neuron death, and motoneurons were more sensitive to glutamate excitotoxicity than sensory neurons in the spinal cord.

The purpose of present study is to determine the location of the novel gene hbrp which is related to bovine seminal plasma (BSP) proteins in the human chromosome by the FISH (fluorescent in situ hybridization) technique, and to investigate the relation of HBRP to TPK( tyrosine protein kinase)by genetic engineering. The result is that the novel gene hbrp was successfully localized on human chromosome 19q1.3; HBRP protein obviously inhibited the activity of PKC. It is concluded that the structure of BSP contained two tandemly arranged fibronectin type II(Fn2)--domains which were related to the function of binding protein BSP. Amino acid sequence of HBRP protein with four tandemly arranged Fn2-domains showed that the protein might be binding protein functionly relating to BSP protein. We have concluded that HBRP protein obviously inhibited the activity of TPK. The location of the novel gene on human chromosome 19q1.3 is very important to study the other biological functions of the protein encoded by gene hbrp.

To explore primarily the reconstruction of biological tissue engineered blood vessel in vitro, the decellularised scaffolds were obtained from swine common carotid arteries by enzyme digestion, and vascular smooth muscle cells (VSMCs) were isolated from canine thoracic aorta and subcultured and purified. The VSMCs were seeded into the inner surface of scaffolds and they were cocultured in vitro for 4 weeks. Histological staining and transmission electron microscopy were used to observe the growth of canine VSMCs on swine decellularized arterial scaffolds. The results showed the seeded VSMCs grew well on the decellularized scaffold throughout the duration of 4 weeks, which suggests that the method in this article is practicable for reconstructing complete biological tissue engineering blood vessel.

In order to evaluate diamond like carbon film (DLC), DLC containing Si, graphite, diamond film (DF), low temperature isotropic carbon film (LTIC) and SiC, we investigated the correlations between surface energy parameters and hemocompatibility indices such as kinetic clotting time, hemolysis and platelet consumption. An analysis of T-type correlation degree in the Grey system theory was performed. The results showed: (1) all of correlation degrees between kinetic clotting time and polar surface energy parameters were positive, but for critical surface tension, the correlation degree was negative; among five of surface energy parameters, interface tension had the highest relation degree (0.63) with kinetic clotting time, and critical surface tension (-0.43) took the second place; (2) on the contrary, all of correlation degrees between hemolysis and polar surface energy parameters were negative, but for critical surface tension, the correlation degree was positive; and that which had closer correlations with hemolysis were still interface tension (-0.43) and critical surface tension (0.29); (3) critical surface tension had the highest relation degree (0.68) with platelet consumption, and surface tension (0.32) took the second place; (4) kinetic clotting time possessed higher negative correlation degrees with hemolysis (-0.57) and platelet consumption (-0.36). These data indicate that kinetic clotting time depended on a balance between the polarity of surface and the limited humidifying of water on the surface, and that platelet consumption was based on good humidification and power polarity of surface, while hemolysis was promoted by the aid of chromatic dispersion action stemming from the surface and fully humidifying of water on the surface. There was "seesaw effect" between kinetic clotting time and hemolysis or platelet consumption, hence the hemocompatibility of carbonaceous biomaterials could be equivalently evaluated by using kinetic clotting time as an index. It has been confirmed: (1) successive occurrences, including adhesion, deformation and collection of platelets on the material surfaces as results of protein adsorption, are the major mechanism of blood coagulation of carbonaceous materials; (2) the hemocompatibility of carbonaceous biomaterials can be evaluated by using critical surface tension as an index. These findings may underpin the hemocompatibility evaluation of carbonaceous biomaterials based on surface properties.

In order to study the cytocompatibility of nanophase hydroxyapatite ceramic in vitro, we prepared hydroxyapatite by use of the wet chemistry techniques. The grain size of hydroxyapatite of interest to the present study was determined by scanning electron microscopy and atomic force microscopy with image analysis software. Primary culture of osteoblast from rat calvaria was established. Protein content, synthesis of alkaline phosphatase and deposition of calcium-containing mineral by osteoblasts cultured on nanophase hydroxyapatite ceramics and on conventional hydroxyapatite ceramics for 7, 14, 21 and 28 days were examined. The results showed that the average surface grain size of the nanophase and that of the conventional HA compact formulations was 55 (nanophase) and 780 (conventional) nm, respectively. More importantly, compared to the synthesis of alkaline phosphatase and deposition of calcium-containing mineral by osteoblasts cultured on nanophase was significantly greater than that on conventional ceramics after 21 and 28 days. The cytocompatibility was significantly greater on nanophase HA than on conventional formulations of the same ceramic.

nm23-H1 is a proven tumor metastasis suppressive gene, tumor metastasis phenotype could be reversed by transfected nm23-H1 cDNA. This study was conducted to transfect nm23-H1 cDNA into L9981 cells and to explore the function of nm23-H1 in reversing the malignant phenotype of L9981 cells. The plasmid of pLXSN-nm23-H1-EGFP was constructed by gene clone technique, and the transfected nm23-H1 cDNA cell lines of L9981-nm23-H1 was established. The protein expression of nm23-H1 was detected by Western blot. The biologic features of L9981-nm23-H1 cells were studied in vitro and in vivo. The results showed that the fusion protein of nm23-H1-EGFP was stable, continuous and expressed with high efficiency in L9981-nm23-H1 cells. The cell proliferation, colon formation and invasive ability are significantly lowered in L9981 cells transfected nm23-H1 cDNA (P < 0.01); the tumorgenesis and the lung metastasis incidence was lower in tranfected nm23-H1 cells than in L9981 and L9981-Plxsn in nude mice (P < 0.01); the rate for inhibiting tumorgenesis of nm23 -H1 was 82.56%. These data suggest that the malignant phenotype could be reversed by wild nm23-H1 gene in L9981 cells.

Interferon gamma-inducible protein 10, a member of the family of CXC chemokines, is secreted by interferon gamma-stimulated, monocytes, endothelial cells and keratinocytes. Interferon gamma-inducible protein 10 plays an important role in recruiting activated T cells into sites of tissue inflammation. In this experiment, PCR products of Interferon gamma-inducible protein 10 were cloned into prokaryote expression vector pET 32(a) to generate recombinant pET-IP10 with S-Tag at the N-terminus, and expressed successfully in E. coli BL21 (DE3). The total expressed products amounted to 25.3% in all bacterion proteins. pET-IP10 mainly formed inclusion body in E. coli. Soluble recombinant protein accounted for 20% among IP-10 fusion protein. The soluble recombinant proteins were purified by using S-Tag affinity chromatography effectively with purity of over 90%. The chemotaxis biological activity of purified Interferon gamma-inducible protein 10 could specifically exhibit the directional migration of stimulated T cells at concentration of 100 ng/ml. The results indicated that the strategy we used in this experiment was effective for recombinant Interferon gamma-inducible protein 10 production with biological activity.

For the purpose of detecting the HBD-2 expression at protein level, the recombinant prokaryotic expression vector pGEX-1lambdaT-HBD-2 was constructed and the E. coli-based product of GST-HBD-2 fusion protein was prepared. When rabbit was immunized with the fusion protein, the anti-serum against HBD-2 was produced. After caprylic acid and ammonium sulfate precipitation, high titer of specific polyclonal antibody against HBD-2, which was detected by ELISA and Western blot, was obtained. This result suggests that recombinant peptide fusion protein could be used instead of the conjugate of peptide-albumin or peptide-thyroid globulin to produce antibody. The obtained antibodies could be used for revealing the tissue distribution of HBD-2 and the regulation of its gene expression.

The purpose of the study is to explore the in vitro refolding protocol for humanized anti-CTLA4-scFv expressed in E. coli. The inclusion bodies are denatured and then diluted or dialyzed into a refolding buffer. We analyzed several factors affecting the refolding yield, including refolding time, temperature, and redox environment. The refolded target proteins are analyzed by non-reducing SDS-PAGE, and the concentration of refolded proteins are examined by Bio-Rad Dc Protein Assay kit. The result shows that a high yield of the protein with natural conformation can be acquired in the condition of 0.15 mol x L(-1) sodium chloride, 50 mmol x L(-1) Tirs-HCl, pH 8.0 buffer containing 1 micromol x L(-1) reduced glutathione and 3 micromol x L(-1) oxidized glutathione. The refolding time is 48 to 54 h at 4 degrees C. 28 mg refolded proteins are produced from 3.9g E. coli.

Endothelialization of artificial vascular graft is considered as one of the most promising methods to improve its antithrombotic ability and long-term patency. Endothelialization of artificial vascular graft includes harvesting endothelial cells, choosing some materials with better compliance and seeding endothelial cells. The methods such as immobilization of extracellular matrix protein and growth factor to substrates, stimulation of chronic in vitro shear stress for endothelial cell retention on artificial vascular graft in bioactor, genetic modificatioin of ECs, and changes of electric charge of ECs are used to increase the adherence ability of endothelial cells. This paper reviews the process of endothelialization of artificial vascular graft and makes brief comments on the methods of endothelialization of artificial vascular graft.

The localization of the activity of immobilized urease on chitosan membrane was studied by X-ray microanalysis. BaCl2 and urea were selected as the capture and substrate respectively. The substrate was hydrolyzed by immobilized urease to produce NH3 and CO2 in Tris-HCl buffer (pH 7.0), and the latter was captured by BaCl2 to form precipitate. The precipite was deposited on the active site of immobilized urease. It is shown that the method is practicable and reliable. The optimum condition for the localization of activity of immobilized urease was studied.

In order to study the renaturation mechanism of denatured protein in denaturant solution, the renaturation and separation process for three kinds of lysozyme molecules, which were separately denatured by urea and guanidine hydrochloride in the presence of reducing agents, was studied by size exclusion chromatography. When initial lysozyme concentration in denaturant solution was more than 10 g/L, the denatured lysozyme molecules were renatured and isolated in a size exclusion chromatographic column. A refolded lysozyme intermediate, a bi-molecular aggregate, was found. This result was confirmed by non-reducing sodium dodecyl sulfate-polyacrylamide gel electrohoresis (SDS-PAGE) analysis of renatured lysozyme molecules with the dilution method. Compared with the dilution method, the amount of the bi-molecular aggregate found by size exclusion chromatography was far less than that found in the dilution method. This result shows that the process for the renaturing of denatured lysozyme molecules in solution can be well described with three-state model in the presence of reducing agents.

The purified anti-carbofuran antibody was conjugated to carbonyl diimidazole (CDI)-activated Sepharose CL-4B to synthesize the immunosorbent for the immunoaffinity chromatographic (IAC) column specific to carbofuran. The conditions of IAC were optimized as follows: pH 7.2 phosphate buffer (PB) was used as equilibrium and adsorbent medium, and methanol-water (60:40, v/v) as eluent. The results showed that the dynamic column capacity was up to 1.58 mg/L bed volume. The efficiency of enrichment of IAC was more than 167 times when the initial concentration of carbofuran in a standard sample solution was lower than 2 microg/L. The spiked river water was cleaned up and enriched by IAC, and carbofuran in eluate was determined by enzyme linked immunosorbent assay (ELISA). The average recovery of carbofuran from river water was 89.8% with the relative standard deviation of 4.8% at the spiked level of 0.1 mg/L. Meanwhile the eluate was determined by high performance liquid chromatography (HPLC), the results from HPLC correlated well with those from ELISA. The IAC method of carbofuran was successfully established.

Optically active axially dissymmetric binaphthyl derivatives have been extensively used as excellent chiral ligands in asymmetric catalysis and chiral recognition. The review describes the applications of binaphthyl derivatives as chiral stationary phases in high performance liquid chromatography. Fifty four references are cited.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. A combination therapy comprising pegylated interferon and ribavirin currently represents the most effective therapy for chronic HCV infection. The limitations of this current therapy mainly its efficacy and significant side effects have prompted the development of new drugs. Few categories of therapeutic agents appear promising for future therapy, e.g. novel interferons, ribavirin analogs, antisense oligonucleotides, short interfering RNAs, ribozymes, enzyme inhibitors, immunomodulatory agents, antifibrotic agents, therapeutic vaccines and antibodies. Few drugs belong to afore-mentioned categories have already reached the different clinical phases of development. The present article highlights the status of current available therapies and emerging drugs for the treatment of hepatitis C.

The vaccine made of recombinant envelope protein (rVp28) of white spot syndrome virus (WSSV) expressed in silkworm (Bombyx mori) pupae using a baculovirus vector was used to investigate the efficacy of oral administration on WSSV disease resistance of Procambarus clarkii. Vaccine was mixed with diet at a ratio of 2% (w/w), and Procambarus clarkii were orally administered throughout 75 days. Vaccination with rVP28 showed the significantly higher cumulative survival compared with positive and negative control (P < 0.05) following an oral challenge on the 35th day post-vaccination (dpv), with PRP values 54.16% and 59.26%, respectively. rVP28 induced higher resistance via IM (intramuscular) injection challenge with WSSV stock, with PRP value of 46.12% and 49.99%, respectively. The survivors were subsequently re-challenged on the 55th dpv. rVP28 induced the significantly higher resistance to oral re-challenge (P < 0.05), with both PRP values 55.80% and 63.16%, respectively. rVP28 induced higher resistance to IM injection re-challenge, with both PRP values 31.25%. A DIG labeled WSSV DNA probe was used to detect WSSV by in situ hybridization. The positive cells were observed in epithelial cells of stomach, hepatopancreas and gut of the infected control crayfish, while negative reaction were observed in the tissues of survivors-vaccinated. These results indicated that vaccination of crayfish with recombinant protein had significant effect on oral infection, and had higher resistance against intramuscular injection challenge. This suggested the protection against WSSV could be induced in crayfish by recombinant protein rVp28 expressed in silkworm pupae.

The aim of this study was to examine microsatellite instability (MSI) and loss of heterozygosity (LOH) of locus D17S396 on chromosome 17 and their influence on the expression of nm23H1 in the epithelial ovarian tumors, which may provide experimental basis for the mechanism of nm23H1 gene and tumor metastasis. Techniques such as DNA extraction from formalin-fixed paraffin-embedded tissues, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), ordinary silver stain were used to study MSI and LOH of locus D17S396. Envision immunohistochemistry and Leica-Qwin computer imaging techniques were used to assess the expression of nm23H1 gene. In our experiments, the frequency of heredity instability of malignant ovarian tumors was 40%, which is higher than that of borderline ovarian tumors, while there were no heredity instability occurred in benign ovarian tumors and normal ovarian tissue. Among 25 epithelial ovarian carcinomas, the frequency of LOH in lymph node metastasis cases (66.67%) was significantly higher than those without metastasis (10.53%). Moreover, the frequency of LOH was higher in FIGO stage III and IV than in stage I and II. However, the frequency of MSI showed no correlation with any clinicopathologic characteristics. The positive frequency of nm23H1 protein in the ovarian epithelial carcinoma and borderline tumors were 56.00% and 57.14%, respectively. They were both higher than those of the benign tumors and normal ovarian tissue. In the epithelial ovarian carcinomas, the positive frequency of nm23H1 protein in lymph node metastasis case was significantly lower than those without metastasis. FIGO stage III and IV also exhibited lower positive frequency of nm23H1 protein compared with stage I and II. Furthermore, there was no difference in nm23H1 protein expression intensity analyzed by computer imaging. In the epithelial ovarian carcinomas, the positive frequency of nm23H1 protein in LOH positive group was 0.00%, which is lower than that of LOH negative group (P < 0.01). The results indicated that the heredity instability of nm23H1 gene might be implicated in pathogenesis and progression of epithelial ovarian tumor. The occurrence of LOH might be the molecule marker of the deteriorism of ovarian tissue. Both MSI and LOH of nm23H1 gene controlled development of the epithelial ovarian tumor independently in different paths. LOH could inhibit the expression of nm23H1 in local tissue of the epithelial ovarian carcinoma, which endowed it with high aggressive and poor prognosis. Increasing the amount of nm23H1 protein expression could effectively restrain metastasis of the ovarian epithelial carcinoma and improve prognosis of patients.

The repressor of bacteriophage lambda is a protein containing two domains of approximately equal size. Fragments containing the amino-terminal domain of repressor bind specifically to lambda operator DNA and mediate positive and negative control of lambda transcription both in vitro and in vivo.

IT has been suggested that the bacterial flora of the rumen should be considered as three distinct, interacting populations-the bacteria of rumen fluid (the population which has been studied most extensively), the bacteria associated with food particles, and the bacteria adhering to the epithelial wall of the organ(1). Until now, studies of the 'epithelial' population have been restricted to examination of postmortem samples of wall tissue and its attached bacterial flora(2-5). A recently developed technique(6) for feeding young sheep for long periods solely by infusion of protein and other essential nutrients into the abomasum, and of volatile fatty acids and bicarbonate buffer into the rumen, has provided us with an opportunity to study in isolation the role of the bacterial population of the wall in the ecomicrobiology of the rumen in the living animal. Our studies show that this population can exist independently of the other two populations, that it is primarily responsible for urea digestion in the rumen and that it initiates breakdown of dead epithelial tissue. Furthermore, our results point to an inverse relationship between ammonia concentration and ureolytic activity in rumen fluid, which may account for the control which ammonia exerts over flux of urea across the rumen wall(7-9).

THE regulatory systems controlling cell division have not been identified, but it has been shown that growth factors such as epidermal growth factor, fibroblast growth factor (FGF), and serum initiate rapid changes in cellular metabolism, probably involving post-transcriptional control mechanisms(1-5). As phosphorylation has been shown to be an important regulatory mechanism in several metabolic pathways, we initiated experiments to determine whether factors which stimulate DNA synthesis also stimulate endogenous phosphorylation. We find mat, within 5 min of addition of FGF or serum to (32)P-labelled Swiss 3T3 cells, there is a specific increase in the phosphorylation of a membrane protein with an apparent molecular weight of 33,000. Experiments with isolated cell fractions demonstrate that the phosphorylation of this protein is stimulated by cyclic AMP. This rapid and specific response to mitogens raises the possibility that this phosphorylation might be part of the initial, cellular signal for DNA synthesis.

AFTER exposure to secretagogues the small intestine changes from a tissue that absorbs fluid and electrolyte from lumen to blood into a tissue that secretes electrolyte and fluid into the lumen(1-4). It has been shown that this secretion results from an increase in the passive Cl(-) permeability of the mucosal border, which permits Nad to leak passively from the lateral intercellular spaces, where it is present at hypertonic concentrations(5), into the mucosal bathing solution. Na(+) and water, electroosmotically coupled to Na(+) movement, leak through the tight junctions(1,2), and Cl(-) leaks through relatively anhydrous anion-selective channels, induced withira the mucosal border by secretagogues. The increased reflux of NaCl from the lateral intercellular space accounts for both the apparent decrease in electroneutral NaCl uptake across the mucosal border induced by secretagogues and the apparent increase in active CP secretion and short-circuit current(3,6,7). We have investigated the mechanism by which intestinal secretagogues increase passive Cl(-) permeability and thereby cause secretion. Cl(-) permeability is increased by several secretagogues, some of which, such as theophylline and choleragen, increase intracellular cyclic AMP concentration, and others, such as A23187, the Ca(2+) ionophore, or carbachol, do not(8). Thus there has been no known common mode of secretory induction. To investigate this problem we used two drugs that prevent intestinal secretion in vitro, RMI 12330A (Richardson Merrell), and the antipsychotic pheno-thiazine trifluoperazine (Stelazine, Smith, Kline and French). RMI 12330A prevents secretion by inhibiting choleragen-induced adenylyl cyclase activity(9). Stelazine inhibits phosphodiesterase in tissues(11,12) by preventing the activation of the enzyme by Ca(2+)-dependent regulator protein, CDR. We report here that it also inhibits Cl(-) secretion and binds to CDR.

CHANGES in transcriptional activity at defined loci are often correlated with significant local structural changes in the genome(1), and in polytene chromosomes, such changes are thought to be associated with compositional or conformational changes in the protein complement at these particular bands(2,3). Thus, various studies on Balfoiani rings and specific 'puffs' in such chromosomes are useful for elucidating the role of defined chromosomal components in both chromosome structure and gene activity. Such studies require specific probes which will allow in situ localisation of a chromosomal component during the various stages of puffing. Antibodies specific to purified histone fractions(4-7), HMG proteins(8), RNA polymerase(9) and non-histone protein subfractions(10) have been used in studies on chromatin and chromosome structure. We reported previously that concanavalin A (Con A) specifically binds to three types of non-histone proteins present in chromatin purified from rat liver nuclei and suggested that derivatives of Con A might serve as specific probes to study the in situ organisation of these non-histone proteins(11). We have now reacted fluorescein-labelled Con A with polytene chromosomes isolated from different developmental stages of Chironomus thummi and visualised the location of the bound Con A by fluorescence microscopy. We observed that the fluorescent lectin, which has an affinity for glucose- and mannose-containing molecules, specifically bound to the transcriptionally active regions of chromosome IV. The extent of binding of Con A to the Balbiani rings present in regions b and c of chromosome IV is proportional to the size of the respective ring. Our results indicate that glucose- or mannose-containing molecules are present in these Balbiani rings and that the availability of these sugars to interact with Con A can be correlated with the developmental stage of a puff. We suggest that lectins can be useful cytological tools with which to study the in situ organisation of defined chromosomal components during various functional states of the genome.

To investigate the effect of the treatment of periapical diseases with recombinant human netic protein (rhBMP-2) composite in dog models so as to provide basis for its clinical application.

Second mitochondria-derived activator of caspase (Smac) is a recently identified, novel pro-apoptotic molecule, which is released from mitochondria into the cytosol during apoptosis. Smac promotes activation of caspases by neutralizing members of the inhibitor of apoptosis proteins (IAPs) family, such as X-linked inhibitor of apoptosis protein (XIAP). The objective of the study was to examine the pro-apoptotic effect of human Smac gene on Burkitt's lymphoma Raji cells.

An approximately 43kD protein has been isolated and purified from the herb Cajanus indicus L and believed to be the most active principle for its hepatoprotective action. In this study, experiments have been performed to evaluate the effectiveness of that protein for the preventive and curative action against thioacetamide-induced toxicity in vivo using a murine model. Mice were treated with the protein intraperitoneally at a dose of 2mg/kg body weight for 2 and 6 days before and separately 1-5 days after thioacetamide administration to evaluate its preventive and curative role, respectively. Thioacetamide was administered once at a dose of 150mg/kg body weight and after 48h of its application, the animals were sacrificed. Levels of various markers related to physiological and pathological conditions of the liver, e.g., glutamate pyruvate transaminase (GPT), alkaline phosphatase (ALP), etc. were determined in the murine sera under different experimental conditions. In addition, antioxidant enzymes glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) as well as thiobarbituric acid reactive substances (TBARS), were measured from the liver homogenates. The antioxidant property of the protein was compared with the potent antioxidant, vitamin E (used as a positive control). The active principle effectively reduced the elevated GPT and ALP levels in serum and lipid peroxidation in the liver tissue. The reduced levels of SOD, CAT and GST by thioacetamide were again brought back to almost normal levels upon pre- and post-treatment with the protein. Histopathological changes in the liver of TAA control and protein-treated groups also prove that the protein possesses hepatoprotective activity. The protein acts dose-dependently and maximum hepatoprotectivity was obtained when administered at a dose of 2mg/kg body weight. Data suggest that the active principle plays an important preventive and curative role against thioacetamide-induced hepatotoxicity.

A family of at least eleven genes called Mar related to long terminal repeat retrotransposons from the Ty3/gypsy group, including two genes previously identified as such, is present in human and mouse genomes. Single orthologous copies were identified for most Mar genes in different mammals. All of them have lost essential structural features necessary for autonomous retrotransposition before divergence between mouse and human. Three Mar genes also have introns at identical positions in human and mouse. Hence, Mar genes do not correspond to functional retrotransposons. Mar genes evolved under purifying selection, strongly suggesting that they are not pseudogenic relics but rather neofunctionalized retrotransposon genes. All putative Mar proteins display sequence similarity to the capsid-like domain of the Gag protein of Tf1/Sushi retrotransposons. In addition, three Mar proteins have conserved the Gag CCHC zinc finger motif, suggesting a role in nucleic acid binding. Some Mar genes have also retained from their retrotransposon origin a -1 ribosomal frameshifting between the gag-related open reading frame and a region encoding a putative aspartyl protease domain. EST analysis revealed that the majority of Mar genes are expressed in brain as well as in other tissues and organs. Some Mar proteins might function as transcription factors or be involved in the control of cell proliferation and apoptosis. Strikingly, as many as eight Mar genes are located on the X chromosome in human, mouse and other mammals, and at least two of the autosomal genes are subject to imprinting. We suggest that retrotransposons might be a source for epigenetically regulated genes. Epigenetic regulation of these neogenes might be derived from the cellular defense mechanisms having controlled their retrotransposon ancestor.

The aim of the present study was to determine whether angiotensin-converting enzyme inhibitors (ACEI) could contribute to the protective effects of preconditioning, and to explore its underlying mechanism. The Langendorff model of isolated rat heart was used. Cardiac contractility and lactate dehydrogenase (LDH) in the coronary effluent were measured, and infarct area of hearts after 30 min of ischemia followed by 120 min of reperfusion was analyzed. We found that: (1) The subthreshold preconditioning (2 min of ischemia and 10 min of reperfusion), captopril (an ACEI with sulfhydryl groups) or perindoprilate (an ACEI without sulfhydryl groups) alone did not protect the hearts from being injured by 30 min of ischemia and 120 min of reperfusion. (2) However, the combination of captopril or perindoprilate with subthreshold preconditioning could decrease left ventricular end-diastolic pressure (LVEDP), increase left ventricular developed pressure (LVDP) and coronary flow compared with the subthreshold preconditioned group. The combination treatments also inhibited the release of LDH from ischemia/reperfusion hearts, and reduced the infarct area in ischemic heart after 2 h of reperfusion (P<0.05). (3) By using NOS inhibitor L-NAME (100 mumol/L) before combined administration of ACEI with subthreshold preconditioning, the protection effect triggered by the combination treatment was significantly reduced. Pretreatment of the hearts with mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel inhibitor 5-HD (100 mumol/L) also abolished the protection effect (P<0.05). (4) Subthreshold preconditioning, captopril or perindoprilate alone could enhance the NO content in coronary effluent (P<0.05), but the combination of captopril or perindoprilate with subthreshold preconditioning could further augment the NO content compared with the subthreshold preconditioned group (P<0.05). The results indicate that ACEIs with or without sulfhydryl groups may potentiate the subthreshold preconditioning to trigger cardiac protection effect against the ischemia/reperfusion injury. This protection effect in the heart is possibly mediated by the generation of NO and the activation of mitoK(ATP) channel.

To investigate if glycogen synthase kinase 3 (GSK3) is involved in squamous differentiation of airway (tracheobronchial) epithelial cells, primary pig airway epithelial cells were treated with lithium chloride, a highly selective inhibitor of GSK3. Change in morphology of cells was monitored under microscopy, and expression of beta-catenin, phosphorylated GSK3 and involucrin, a squamous differentiation marker, were dectected by Western blotting, while expression of mRNA of another squamous differentiation marker, small proline-rich protein, was detected by RT-PCR. Further, luciferase reporter assay was used to assess the activation of beta-catenin/Tcf signaling. The results demonstrated that lithium was able to induce a squamous morphology of the cells, and to enhance the expression of involucrin and small proline-rich protein mRNA. Moreover, lithium increased inhibitory phosphorylation of GSK3, augmented nuclear translocation of beta-catenin in a dose- and time-dependent manner. Activation of beta-catenin/Tcf signaling was observed after the elevation of squamous differentiation markers. Taken together, these data suggest that GSK3 is possibly involved in squamous differentiation of pig airway epithelial cells.

To investigate the role of found in inflammatory zone 1 (FIZZ1) protein in the pathogenesis of experimental pulmonary fibrosis, 48 male Sprague-Dawley rats were randomly divided into two groups, the pulmonary fibrosis group and the control group. Rat pulmonary fibrosis was reproduced by an intratracheal injection of bleomycin (5 mg/kg body weight). Normal saline (1 ml/kg body weight) was given intratracheally injection in the control group. There were 24 rats in each group, and 6 animals were separately killed on the 7th, 14th, 21th and 28th day after treated with bleomycin or normal saline. Then the following tests were undertaken: (1) HE and Masson staining of lung section;(2) Determination of lung tissue hydroxyproline (HYP);(3) Immunohistochemical staining of protein of FIZZ1 in the lung;(4) In situ hybridization of FIZZ1 mRNA in the lung. The results showed: (1) There were full of inflammatory cells in the lung, the interval of alveoli enlarged and many alveolar spaces disappeared on the 7th day after treated with bleomycin in the fibrosis group. Collagen began to proliferate after 14 d. The pulmonary fibrosis was stably established on the 28th day, full of green fibers in the Masson staining of lung section. (2) The expression of FIZZ1 protein in the lung increased after 7 d in bleomycin-treated animals (3.013+/-0.326 vs 0.473+/-0.056, P<0.01 vs control), but was slightly decreased on the 14th day (2.124+/-0.197) and expensively decreased on the 21st day (1.760+/-0.105) and the 28th day (0.691+/-0.081). (3) The expression of FIZZ1 mRNA in the lung also increased after 7 d by treated with bleomycin (3.795+/-0.338 vs 0.678+/-0.087, P<0.01 vs control), but decreased on the 14th day (1.276+/-0.104) and further decreased on the 28th day (0.896+/-0.084). The expression of FIZZ1 protein and mRNA in fibrosis group was higher than that in the control group (P<0.05 or P<0.01). The results suggest that FIZZ1 protein and FIZZ1 mRNA are dynamically changed in the lung with experimental pulmonary fibrosis, which may contribute to the pathogenesis of pulmonary fibrosis.

Soy isoflavones have been reported to be natural chemopreventive in several types of human cancer. Daidzein and genistein are two main components of soy isoflavones. In our previous study, they were shown to be anti-proliferative and induce cell cycle arrest at S phase of SHZ-88 rat breast cancer cells. We hypothesized that soy isoflavones might exert its anticancer effect by activating cAMP/PKA pathway. The present study was designed to analyze the effect of soy isoflavones on the cAMP/PKA pathway in SHZ-88 cells. Daidzein and genistein were dissolved in DMSO. Cells were treated with 50 mug/ml daidzein and 15 mug/ml genistein, respectively, and with only equal DMSO in the culture medium as control. The cellular cAMP content was tested by radioimmunoassay (RIA). The activity of adenylate cyclase (AC), phosphodiesterase (PDE) and PKA were measured by RIA and (gamma-(32)P) ATP incorporation. Reverse transcript-polymerase chain reaction (RT-PCR) was used to analyze the expression of cAMP response element binding protein (CREB) mRNA of the cells. The results showed that the concentration of cAMP in the cells treated with 50 mug/ml daidzein and 15 mug/ml genistein was significantly increased by 9.5%and 11.0%, respectively, 5 min later (P<0.05), then increased by 31.0%and 40.3%, respectively, 10 min later (P<0.01), compared with that of the control group cells. The activity of AC was not affected during the course of experiment, but that of PDE was decreased to 71.8%and 71.6%, respectively, in the control group 5 min later (P<0.05). The PKA activity was increased to 125.8%and 122.3%, respectively, in the control group 20 min after the cells were treated with daidzein and genistein (P<0.05), and kept at high level till 40 min after treatment. CREB mRNA of the cells treated with daidzein and genistein was increased by 31.6%and 51.1%, respectively, 3 h later (P<0.05), then began to decrease 6 h after treatment. The current study suggests that soy isoflavones activate the cAMP/PKA pathway in SHZ-88 rat breast cancer cells by inhibiting the activity of phosphodiesterase.

The effects of neuroglobin (NGB) gene transfer in vivo mediated by GeneJamer on the hearing response properties of the inferior colliculus (IC) neurons in mice after administration of sodium salicylate were studied. Forty-eight Kunming mice were divided into 4 groups (n=12 in each group): Group A1 (negative control);Group A2 (positive control);Group B, sodium salicylate (450 mg/kg every day) + pEGFP-C1;Group C, sodium salicylate (450 mg/kg every day) + pEGFP-NGB. The GeneJamer and pEGFP-NGB were mixed and injected into IC neurons in mice. The expression of NGB mRNA and protein of IC neurons in mice was detected by using RT-PCR and Western blot methods. The intensity-rate functions, intensity-latency functions and frequency-turning curves in IC neurons were recorded by extracellular electrophysiological recording techniques and the effects of pEGFP-NGB transfer following injection of sodium salicylate on them were studied. It was found that: (1) The GeneJamer-mediated pEGFP-NGB could be effectively transferred into the IC brain tissues in mice and NGB could be expressed intensively. (2) The intensity-rate functions of IC neurons were raised after administration of sodium salicylate. The non-monotonic styles of intensity-rate functions in groups A1, A2 and C were accounted for 74.6%, 72.2 %, 59.3 %, respectively, and the function in group B for 47%. There were significant differences between group B and groups A1, A2 or C (P<0.01, P<0.01, P<0.05). (3) The intensity-latency functions in IC neurons were reduced after administration of sodium salicylate. The non-monotonic styles of intensity-latency functions in groups A1, A2 and C were accounted for 3.2 %, 5.1 %and 21 %, respectively, and that in group B for 45.5 %. There were significant differences between group B and groups A1, A2 or C (P<0.01, P<0.01, P<0.05, respectively). (4) The frequency-turning curves in groups A1 and A2 were sharpened. In 72 acoustic neurons recorded in the group B, the frequency-turning curves from 53 neurons were broadened while those of the rest were sharpened. In group C the frequency-turning curves recorded from 12 of 67 acoustic neurons were broadened while those of the remaining were sharpened. These results suggest that in vivo transfer of NGB gene is highly expressed in IC neurons in mice. In vivo transfer of NGB gene reverses the change of intensity-rate functions, intensity-latency functions and the code styles after administration of sodium salicylate in IC neurons in mice.

Despite scientific advances, the therapeutic options for hepatitis C virus (HCV) are limited by poor response rates. HCV1b is particularly resistant to standard interferon therapy. The inhibition of the progression of chronic hepatitis and liver cirrhosis and the prevention of the occurrence and recurrence of hepatocellular carcinoma (HCC) are important and thus, there is a need for new therapeutic modalities for HCV1b infection. We, therefore, investigated highly immunogenic peptides and report in this study three novel candidate peptides (at positions 711-720 in envelope 2 protein, 885-893 in non-structural protein 2 and 1716-1724 in non-structural protein 4B) among 35 peptides of conserved regions of HCV1b proteins containing HLA-A24 binding motifs tested. Namely, HCV(711-720), HCV(885-893) and HCV(1716-1724) induced HLA-A24-restricted and peptide-specific cytotoxic T lymphocytes (CTLs) activity in peripheral blood mononuclear cells (PBMCs) from 7, 6 and 5 of 12 patients and also were recognized by plasma of 8, 5 and 7 of 12 HCV1b(+) patients, respectively. These results may provide new insight into the development of a peptide-based specific immunotherapy for HCV1b(+) HLA-A24(+) patients.

A review on the recent research progress in spectroscopic probes of protein was presented and discussed. The review included categories of spectroscopic probes of protein, interaction models of protein and spectroscopic probes, interaction force of protein and spectroscopic probes, and protein conformation research. Future research direction of spectroscopic probes of protein was forecast.

A new method to determine Chinese traditional medicine activity eliminating superoxide anion radical by kinetic spectrophotometry was developed. It is shown that the optimum determination condition may be obtained when wavelength is 520 nm, the concentration of enzyme is 4 x 10(-3) microg x mL(-1), the reaction time is between 2-7 min. Vitamin C activity eliminating superoxide anion radical was determined under the condition, and the results were identical with literature. The method is used to determine IC50 of Cortex Magnoliae Officinalis and Cortex Eucommia, and they are 7.580 and 323.800 mg x L(-1) respectively.

In genetic modification of plants, once the transformants are obtained, selection markers are no longer required in mature plants. At present, the Cre/lox site-specific recombination system is most widely used to eliminate the selectable marker genes from the transgenic plants. In this study, attempt was made to favour the selection of marker-free plants in the re-transformation method. Green fluorescent protein (GFP) can be directly visualized in living cells, tissues or organisms under UV illumination. This advantage of GFP is exploited in the development of a practical approach in which GFP is used as a visual marker to monitor the removal of the selectable marker gene from transgenic plants. For that purpose, the pGNG binary vector was constructed, in which the GFP gene (gfp) was linked to the expression cassette Nos P-nptII-NosT and the two units were cloned between two directly-orientated lox sites. The CaMV 35S promoter was placed before the first lox site and used to drive GFP expression. The beta-glucuronidase gene (gus) of Escherichia coli was cloned behind the second lox site without a promoter, thus would not be expressed in this position. Tobacco plants were first transformed with pGNG and selected on kanamycin (Kan)-containing media. Regenerated transgenic shoots were readily singled out by GFP fluorescence. The GFP-expressing plants were then re-transformed with pCambia1300-Cre containing hygromycin phosphotransferase gene (hpt) as a selectable marker gene. The Cre-mediated recombination resulted in the elimination of lox-flanked genes, herein gfp and nptII, from the plant genome and brought the GUS gene next to the 35S promoter. Our data demonstrated that transgenic plants free of nptII were easily selected by monitoring the loss of green fluorescence, and at the same time, GUS (here as a target protein) was expressed in the nptII-free plants. Finally, hpt and cre were removed from the progenies of the nptII-free plants by gene segregation.

Metabolic engineering provide powerful tools for the systematic manipulation of cellular metabolic activities. The ptsG gene for glucose-specific transporter Enzyme II CBGlc of the phosphotransferase system was knock-out so as to reduce the accumulation of acetic acid in the high cell-density culture of Escherichia coli on excess glucose. The chloramphenicol-resistant cassette with short shared sequences on both ends generated by PCR was electroporated into Escherichia coli DH5alpha and JM109. Recombination between linear DNA cassettes and Escherichia coli chromosomes took place by Red recombinase functions. Therefore, the ptsG gene was disrupted to construct the mutants called DH5alphaP and JM109P. There was no difference between the mutants and parent strains in LB media.However, in LB media supplemented with glucose, the mutants of Escherichia coli deficient in ptsG showed greater biomass, together with exploiting more glucose. The maximal cell density obtained with DH5alphaP was approximately 3 times more than that of DH5alpha, then the result of JM109P increased fourfold. The products of recombinant protein TNF respectively accounted for 24.3% of total cellular protein in DH5alphaP with A600 8.28 and 20.8% of total cellular protein in JM109P with A600 7.62. The specific volume expression amount of TNF was greater in the ptsG mutant than in its parent strain. These results demonstrate that the ptsG-mutant strains will be available for high cell-density culture.

The study of mammary gland bioreactor is in the ascendant. In order to generate transgenic goats of well-controlled expression of exogenic genes, we constructed a human lactoferrin (hLF) gene targeting vector containing promoter, exon 1, intron1 and some of exon 2 (about 6.1 kb fragment) and exon 6 approximately 9 (about 3.3 kb fragment) of the goat beta-casein gene as well as hLF minigene, neo gene inserted into them and tk gene ligated to the 3' end of the construct. The 9.4 kb goat genomic sequences as homologous arms were initially amplified by PCR with local goat tissue DNA. The expression vector was named pBC-tk-neo-hlf. Then the recombinant plasmid pBC-tk-neo-hlf containing hLF minigene was transfected into mice mammary tumor cell line C127 by liposome, cell clones were selected with G418. After proliferating, the transfected cells were induced with insulin, luteotropic hormone and hydrocortisone. The result of Western-blotting analysis showed that the transfected cells can secrete hLF protein, and the recombinant protein expressed in cultured cell supernatant has the similar molecular weight as the native protein. The expression level detected by ELISA was 0.21 microg/mL. This result indicated that the targeting vector could efficiently direct the expression of hLF in mammary cells,and it confirmed the validity of the constructed vector. At the same time, C127 cell line proved to be useful for evaluating the regulation of a foreign gene expression in mammary gland specific expression vector.

The DNA coding for the fusion protein of thromobopoietin mimetic peptide (TMP) and human IgG1 Fc fragment was amplified from recombinant plasmid pET28a/TMPFc, inserted into pPICZalphaA and transformed into Pichia pastoris using electroporation. The recombinants of correct phenotype were identified after screening on MDH and MMH culture medium. The fusion gene was verified with PCR and western blot. MTT method was used to test the activity of TMPFc in promoting the growth of Ba/ F3-mpl cell. The TMPFc with a 64 000 molecular weight was a secretary protein in the system and its expression amounted to 65% of the total protein in the medium supernatant. The TMPFc showed a promotive effect on the growth of Ba/F3-mpl in vitro. A significant portion of the secretary protein existed as dimer, which provided material for studying the dimer in future.

Human telomeric repeat binding factor 1(TRF1) contains one Myb-type DNA-binding repeat and an amino-terminal acidic domain. It can bind to the duplex array of TTAGGG repeats at chromosome ends and is shown to be important in preserving genomic stability, maintaining cell proliferative capacity, and blocking the activation of DNA-damage cell cycle checkpoints. Interestingly, the double strand DNA breaks sensor ATM interacts with and phosphorylates Pin2/TRF1 and inhibits its function after DNA damage. Are there some proteins else that can interact with TRF1 and influence its function? In order to analysis the interaction between TRF1 and other proteins, we must prepare the antiserum that can recognize the endogenous TRF1 of cell lysates. TRF1 cDNA was amplified using cDNA Library of HeLa cell by PCR and cloned into pUCm-T vector. Sequence analysis reveals identity to the GenBank report. The TRF1 cDNA was subcloned into expression vector pET-28c(+) and expressed in E. coli as a fusion protein of 65 kD. The recombinant TRF1 can express in the supernatant (about 12.3% in total protein) on the induction of 0.5 mmol/L IPTG at 37 degrees C for 3 hours. Western-blot analysis showed the recombinant protein can react with TRF1 polyclonal antibody sc-6165 (from Santa Cruz Company). His6-TRF1 was purified by Ni(2+) -NTA resin affinity chromatography made by ourselves and showed to be homogeneity in SDS-PAGE. Rabbits were immunized for four times to prepare polyclonal antibody. The unpurified antiserum can recognize the overexpressed TRF1 with myc-tag and the endogenous Pin2/TRF1 of cell lysate by Western-blot at 1:1000 dilution. At 1:400 dilution, the antiserum can interact with endogenous TRF1 by Immunofluorescence cell staining analysis. The endogenous TRF1 in different cell lines, such as HepG2, 803, MCF7 and HeLa, locates in the nucleus. The soluble expression TRF1 and preparation of its antibody lay the foundation to study it further.

Gamma-linolenic acid (GLA, C18:3delta6 ,9,12), an essential polyunsaturated fatty acid, plays an important role in hormone regulation and fatty acid metabolization. Delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme of the desaturation of linoleic acid (C18:2delta9,12) in the production of gamma-linolenic acid. A deficiency of GLA may have occurred when delta6-fatty acid desaturase activity decreases in aging, stress, diabetes, eczema, and some infections. To establish a new expression system for delta6-fatty acid desaturase gene in Pichia pastoris, which is an increasingly popular heterologous gene expression system, a gene encoding delta6-fatty acid desaturase from Mortieralla alpina was isolated by PCR amplification. The PCR product was then digested by EcoR I and Not I and subcloned into the intracellular expression vector pPIC3.5K to generate the recombinant vector pPIC3.5K-MA6. The resulting vector was linearized by Sac I and electroporated into P. pastoris SMD1168 (his- pep-) host cells. After electroporation, aliquots were spreaded on the MDS plates and incubated at 30 degrees C for three days until colonies appeared. Those transformants were subsequently screened for clones with high copy number by using the YPD plates containing G418. To identify the D6D constructs that were produced, chromosomal DNA of the transformants were prepared and used as template for PCR with the primer 5' AOX and 3' AOX. The PCR product of Mut+ recombinants was shown as a band of 1.38 kb of D6D gene and the product of 2.2 kb of AOX1 gene, while the product of Mut(s) transformants only was shown as a band of 1.38 kb of the D6D gene.To further confirm the transformants containing a functional D6D gene, the positive clones were selected and induced by methanol for expression. Those induced cultures were taken for analyses of the intracellular fatty acid composition by GC. The resultant chromatograms of fatty acid methyl esters showed that a novel peak was detected, which was not apparent in the case of control. Comparisons of the retention times of the newly yielded peaks with those of authentic standards have anticipated that the fatty acid is GLA. And this prospects was positively supported by definitive assignments of the compounds by GCMS analyses. Thus, the active delta6-fatty acid desaturase was expressed intracellularly in P. pastoris and gamma-linolenic acid reached 16.26% of the total fatty acid in recombinant P. pastoris strains. It was the first report about the expression of Mortieralla alpina D6D gene in P. pastoris.

The possibility of using a subunit or fragment of human chorionic gonadotropin (hCG) as an immunogen for birth control has been actively explored for many years. This protein homone is produced by the fertilized egg and is required for implantation of the blastocyst into the maternal uterus and the maitenance of pregnancy. In previous studies, several bio-synthesized hCG chimeric peptides (CP) that contain three linear B-cell epitopes (beta5, beta9 and beta8) of beta-hCG subunit together with various foreign 'promiscuous' T-cell epitopes were constructed and expressed as potential new hCG vaccine immunogens. In order to detect antibodies to each of the individual B-cell epitopes present in the animal antiserum raised against the hCG CPs, we decided to construct three recombinant proteins, each contains a single target B-cell epitope (betaE) of beta-hCG. Two sets of DNA fragments were chemically synthesized encoding the beta5, beta9 and beta8 epitopes (betaE) 45 approximately 52, 113 approximately 116 or 133 approximately 144 of beta-hCG subunit and were inserted into the downstream of streptavidin (Stv) gene in pTSA18 separately, with or without an extra TAA codon at the 3'-terminals of the genes. SDS-PAGE analysis revealed that only Stv-betaE (-beta5, -beta9 or -beta8) fusion genes set with the TAA codon can be expressed in E. coli BL21 (DE3) pLysS strain at high level after 1mM IPTG induction for 4 hours. Additionally, these fusion proteins can all be recognized by specific polyclonal antiserum (RS-4157) generated upon immunization with the loop peptide 38 approximately 57 of beta-hCG, monoclonal antibody (mAb) FB12 to beta9 epitope and mAb OT3A that specially recognizes reporter sequence 133 approximately 139 of beta8 epitope 137 approximately 144. Each of the proteins can be purified to 95% relative homogeneity using an improved method of preparative gel polyacrylamide gel electrophoresis. The yields were 5 mg per 1 L culture. The three target Stv-betaE fusion proteins will be useful in determining the immunogenicity of designed hCG CPs and hCG vaccines, including hCG DNA vaccines.

Auxin-responsive elements (AuxRE) interact with a new class of plant-specific transcription factors, auxin response factors (ARFs). Some of ARFs have been shown to repress or activate expression of genes with an AuxRE promotor element. In Arabidopsis, ARFs play important roles in early embryo development and vascular strand formation (ARF5), floral patterning (ARF3) and photo- and gravitropic responses (ARF7). Two cut surfaces (distal and proximal) of mango (Mangifera indica L. var. Zi-Hua) cotyledon showed different patterns of adventitious root formation, with only the proximal cut surface, but not the distal one, could be induced to form the roots. Thus, the mango cotyledon is a good system for studying adventitious root formation. A cDNA fragment homologous to the Arabidopsis auxin response factor-like protein and relates to adventitious root formation from the cut sections were isolated using suppressive subtractive hybridization (SSH). Two cDNA clones, designated as MiARF1 (mango auxin response factor 1 gene, GenBank accession number AY255705) and MiARF2 (mango auxin response factor 2 gene, GenBank accession number is AY300808), were identified by 3'RACE. MiARF1, 3 272bp long, contains an open reading frame (ORF) of 2 523bp, 5'UTR of 285bp and 3'UTR of 464bp, MiARF2, 1 474bp long, contains an ORF of 981bp, 5' UTR of 285bp and 3'UTR of 208bp. The deduced MiARF1 and MiARF2 are homologues of auxin response factor (ARF) family of transcriptional regulators, and show high similarity to ARF of Arabidopsis in conserved domains. The motifs of MiARF1 EL-WHACAGPL in DBD (DNA binding domain) and GDDPW in IV domain are identical to that of ARF-like protein of Arabidopsis. MiARF2 is identical to MiARF1 in a large part of DBD, but lacks a carboxyl-terminal domain containing conserved motifs III and IV. Virtual Northern blot showed that the expression of MiARF2 was high in rooting tissue of cultured cotyledon sections but low in non-rooting tissue, and the MiARF1 was expressed both in the rooting and non-rooting tissues. We suggest that the MiARF2 is related to adventitious root formation of mango cotyledon section.

Induced by 42 degrees C, the recombinant engineering bacterial pBV/cpa408 was highly expressed. After having been pelleted by 80% (NH4)2 SO4 and dialysised, the expressed protein was isolated and purified by the gel filtration choromatography. Then according to an amount of 1.0 mg/kg, the Kunming Mice (body weighted 18g) were immuned with the purified protein by intraperitoneal inoculation. One week after the first enhanced immunization, the Kunming Mice were attacked with an amount of 1.0MLD alpha-toxin, in which the eight mice immuned all survive and the control group all died. During the period of immunization, the titre of the mouse's serum antibody was measured by ELISA. One week after the first immunization, the titre of the mice's serum antibody was 1:800, but that of one week after the first enhanced immunization reached to 1:6400.

Serum used widely in mammalian cell culture is also a potential source of bacterial, mycoplasmal and viral contaminations. In addition, the complex biological components in serum make harder the subsequent product recovery process. High cost, high batch variation and potential source limitation are among the other shortcomings. So serum-free or even protein-free medium are preferable for recombinant protein production. However, without serum to provide essential components such as hormones, growth factors and binding proteins, cells are easy to die. In this study, CHO-dhfr- cells were genetically engineered to make them adapted to IMEM, a protein-free medium, and resistant to apoptosis. The genes in choice are insulin-like factor (Igf-1), Bcl-2 and cyclin E. Bcl-2 is a mitochondrial membrane-integrated protein. It can block the release of cytochrome c by maintaining the integrity of mitochondrial membrane, and thus inhibit apoptosis. Igf-1 is similar both in structure and function to insulin, a growth factor added to serum-free medium to promote cell growth and is the only protein component in many currently used serum-free media. cyclin E is a cell cycle protein expressed continuously in G1 phase. When cyclin E accumulates to certain amount, cell cycle was driven to S phase. So cyclin E is a proliferation-promoting protein. By co-express Igf-1/Bcl-2 or Bcl-2/ cyclin E in CHO-dhfr- cells with a dicistronic expression vector, we constructed two cell lines: CHO-IB and CHO-BC. The high expression of each protein was confirmed by Western blot and flow cytometry. Apoptosis was analyzed by flow cytometry and DNA ladder detection, and the two cell lines were both found much more resistant to apoptosis induced by withdrawal of serum or addition of actinomycin D than the CHO-dhfr- parent cell. Cell proliferation assay by MTT method showed that the two cell lines proliferated much faster than CHO-dhfr- in IMDM medium without serum. Continuously culture assay proved that the two cell lines grow very well in IMEM protein-free medium supplemented with fibronectin and vitronectin to ease adherence. When compared to CHO-dhfr-, the two cell lines exhibited much more viable cell numbers and faster growth rate.

High-level expression of phytase with high specific activity is an effective way to improve phytase fermentation potency and reduce its production cost. The gene appA encoding Escherchia coli phytase AppA with high specific activity was modified and artificially synthesized according to the bias in codon choice of the high expression gene in Pichia pastoris without changing the amino acid sequence of the AppA. The modified gene, appA-m, was inserted in the Pichia pastoris expression vector pPIC9, then introduced into the host Pichia pastoris by electroporation. The Pichia pastoris recombinants for phytase overexpression were screened by enzyme activity analysis and SDS-PAGE. The result of Southern blotting analysis of the recombinant yeast indicated that only one copy of the appA-m gene was integrated into the genome of Pichia pastoris. The result of Northern analysis of the recombinant yeast showed that the modified gene was effectively transcribed. SDS-PAGE analysis of the phytase expressed in Pichia pastoris revealed that the phytase was overexpressed and secreted into the medium supernatant. There are three phytase proteins with apparent molecular weight in approximately 50kD, 52kD and 54kD respectively in the media, which are larger in the size than the native phytase from E. coli. The results of N-terminal sequecing and deglycosylation of the expressed phytase in Pichia pastoris proved that the expressed phytase were glycosylated protein with different glycosylation degree. The expressed phytase Pichia pastoris shared similar pH and temperature optima to those of the natural phytase from E. coli and had highly resistant to pepsin digestion. In 5-L fermentor, after induced by 0.5% methanol for 120 h, the expression level of phytase protein was 2.5 mg/mL, and the phytase activity (fermentation potency) exceeded 7.5 x 10(6) IU/mL, which was the highest among those of all kinds of recombinant strains reported now.

Hepatitis E is a main cause of acute viral hepatitis in developing countries where it occurs as sporadic cases and in epidemics form. The causative agent, hepatitis E virus, is transmitted primarily by the fecal-oral route. The approximately 7.5 kb positive-sense single-strand RNA genome includes three open reading frames (ORFs), one of which (ORF2) is postulated to encode the major viral capsid protein (pORF2) of 660 amino acid residues. We earlier showed that a bacterially expressed peptide, designated as NE2, located from amino acid residues 394 to 606 of ORF2, was found to aggregate into homodimer to at least hexamer. To understand the interface domains within this peptide vital for dimerization and formation of major neutralizing epitopes, NE2 protein underwent terminal-truncated and site-directed mutation. The hydrophobic region, ORF2 aa597-aa602 (AVAVLA), played a key role in oligomerization. Any amino acid residue of this region replaced with glutamic acid residue, the peptide can not refold as homodimer and/or oligomer. The immunoreactivities of these mutant peptides, blotted with anti-HEV neutralizing monoclonal antibody (8C11) and convalescent human sera, show associated to the formation of homodimer. The intermolecular contact region on homodimer was investigated by chemical cross-linking of two site-directed cysteines. When the alanine on aa597 site mutated with cysteine, two different homodimers were found in SDS-PAGE analysis. One (42kD) can be disassociated with 8mol/L urea, which is postulated to form by virtue of hydrophobic interaction, and the other (60kD) falls apart with the reductant DTT present. The exact conformation, generating the cross-linking reaction of cysteines, was further investigated by induced-oxidation on monomer and hydrophobic homodimer of A597C protein with GSH/GSSG. And the results revealed, it is the conformation of hydrophobic homodimer that induces the disulfide bond come into being, instead of the one of monomer. So the aa597 site was verified to be located on interface domain of hydrophobically interacting homodimeric complex. To evaluate the biological significance of hydrophobicity of interface domain, we searched natural variations as to the region on all available databases with NCBI blast program. All variations on these amino acid residues kept higher hydrophobicity, which suggests that the hydrophobic domain is critical for the assemblage and propagation of HEV. NE2 N-terminal deletions up to aa458 had no effect on dimerization and took no exact part in formation of major neutralizing epitopes, but the fragment may act as helper for the formation of major neutralizing epitopes on NE2. Interestingly, the C-terminus aa605-aa660 of ORF2 can also act as helper instead of the N-terminus of NE2. This study suggests an interface domain of NE2 might be vital for HEV capsomer assembly and formation of major neutralizing epitopes. These results may offer clues to the rational design of recombinant anti-HEV vaccine.

Human beta2-microglobulin (beta2m) is the light chain of major histocompatibility complex (MHC) class I molecule. High-yield production of this protein is a prerequisite to the preparation of MHC I tetramer. The present study aims to obtain recombinant human beta2m expressed in Escherichia coli (E. coli), for the purpose of preparing MHC class I tetramers. For cloning of human beta2m gene, a pair of specific primers was designed based on the published sequence of this gene and the cDNA of full coding region for beta2m precursor was obtained by RT-PCR from the total RNA of human leukocytes. The amplified cDNA was subsequently cloned and its sequence was confirmed by DNA sequencing analysis (the sequence has been deposited in GenBank with accession number of AY187687). The prokaryotic expression vector containing a gene encoding mature beta2m was constructed by inserting the DNA fragment, which was generated by PCR reaction with the cloned beta2m gene as template, into an IPTG-inducible expression vector pET-3c plasmid. The first eight codons for N terminal amino acid residues of beta2m were optimized for its expression in E. coli. The complete sequence of beta2 m gene in the expression vector was verified by DNA sequencing analysis. High-yield expression of beta2m was achieved in E. coli transformed with the expression vector, and most of the recombinant beta2m existed in the inclusion body after IPTG induction. The inclusion body was washed extensively and beta2m in the inclusion body was solublized with 8 mol/L urea. The beta2m was refolded by dialysis and purified by ion-exchange chromatography (Q-Sepharose). Western blotting assay indicated that the polyclonal antibody against human native beta2m could react specifically with the recombinant protein. The purified protein appeared as a single band on both SDS-PAGE and Western blotting, indicating that it was chemical and antigenic pure. This work establishes a convenient approach for renaturation and purification of large quantity of recombinant beta2m which is identical to the native protein without any tags fused except for a methionine residue at the amino terminus. This provides the basis for the preparation of MHC tetramers.

Supplement effects of ions, sugars, and amino acids on the thermostability of liquefying type alpha-amylase from Bacillus subtilis were examined. The addition of 1 mmol/L Ca2+ or about 50 mmol/L Na+ remarkably stimulated the thermostability of this enzyme among ions examined. The thermostability of the enzyme was enhanced and reduced by the extrinsic addition of 50 mmol/L acidic amino acid such as glutamic acid and alkaline amino acid of the concentrations of sugars from 0 to 1000 mmol/L the thermostability of alpha-amylase increased almost such as arginine, respectively. With the increases linearly. By the co-existence of Na+ or K+ with some amino acids or sugars the thermostability of this enzyme was fairly increased. The changes in the fluorescence intensity of alpha-amylase were examined as a function of the incubation temperature on the enzyme, which showed a good agreement with those of residual activities.

The biosensor based on optical imaging ellipsometry, can be used to detect directly, without labeling, the surface concentration of biomolecules on solid surface. The feasibility of using protein A to immobilize antibody on the silicon surface of the imaging ellipsometry biosensor was investigated in this study. The results showed that the anti-IgG immobilized by the protein A on silicon surface could bind effectively human IgG, and the human IgG immobilized on silicon surface by protein A bound more polyclonal antibody molecules than that immobilized on silicon surface directly, suggesting that protein A might block the surface to prevent the absorption of human IgG on surface directly, which might compromise its native configuration. The silicon surface modified with protein A is expected to be used to immobilize a variety of antibodies, as protein A can bind selectively the Fc regions of many mammalian IgG. The combination of imaging ellipsometry and the protein A surface modification has the potential to be developed into immunoassays of high sensitivity.

The gene encoding the nucleocapsid (N) protein of vesicular stomatitis virus (VSV-NJ) was subcloned from pMD-VN5, and inserted into pBAD/Thio TOPO vector. The recombinant plasmid was identified by restriction analysis and PCR. It was sequenced to confirm the correct sequences and the correct junctional orientations of the inserted N gene. The results of SDS-PAGE and Western immunoblotting revealed that the N protein was expressed in Escherichia coli LGM194 in a high level and the recombinant fusion protein, which contained a N-terminal HP-Thioredoxin and a C-terminal polyhistidine tag. It had a molecular mass of approximately 63.5 kD and immunologically reactive activity. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of vesicular stomatitis using 186 serum samples from experimentally infected goats and guinea-pigs with VSV-NJ and VSV-IN, and from field origin and reference serum samples. The sensitivity and specificity of the ELISA were compared with those of the standard microtiter serum neutralization (MTSN) tests. The ELISA and MTSN test results were highly correlated for detection of VSV antibodies. The ELISA was as sensitive as the SN assay in detecting positive serum to VSV. The correlation between SN titers and ELISA titers was statistically significant. These data suggest that the recombinant fusion N protein of VSV could be used as a recombinant test antigen for the serodiagnosis of vesicular stomatitis. The ELISA based on the reconmbinant nucleocapsid protein may offer the best combination of rapidity, sensitivity, simplicity, economy, and laboratory biosafety of any of the methods yet developed for VSV serodiagnosis. This study lay on foundation for the development of the diagnosis methods in serology for VSV.

To study the effects of c9, t11-conjugated linoleic acid (c9, t11-CLA) on the critical enzyme (COX-2) and its product - prostaglandin E2 (PGE2) of linoleic acid metabolism path in human gastric adenocarcinoma cell line (SGC-7901).

To investigate the expression of Epstein-Barr Virus (EBV) latent membrane protein 2A (LMP2A) in tumor tissues of nasopharyngeal cancer (NPC) and discuss the possibility (feasibility) that LMP2A was selected (to select <of selecting> LMP2A ) as a target antigen in immunologic treatment of NPC.

To study the expression of a specific inhibitor of endogenous carbon monoxide (CO) generating enzyme Heme oxygenase-1 (HO-1) in allergic rhinitis guinea pigs.

IMGT, the international ImMunoGeneTics information system((R))(http://imgt.cines.fr) created in 1989, by the Laboratoire d'ImmunoGénétique Moléculaire (LIGM), Université Montpellier II and CNRS, Montpellier, France, is a high quality integrated information system, secialized in immunoglobulins (IG), T cell receptors (TR), major histocompatibility complex of human and other vertebrates and related proteins of the immune system that belong to the IgSF and Mhc superfamilies. IMGT/LIGM-DB, the first and the largest IMGT database, manages more than 92,000 IG and TR nucleotide sequences from human and 150 other vertebrate species in May 2005. IMGT/LIGM-DB provides expertly annotated sequences and standardized knowledge based on IMGT-ONTOLOGY, the first ontology for immunogenetics and immunoinformatics. The strategy developed by IMGT, for the IG and TR nucleotide sequence annotation, involves two different approaches that depend on the nature of the sequences, genomic DNA (gDNA) or complementary DNA (cDNA).

The field of bioinformatics has exploded over the past decade. Hopes have run high for the impact on preventive, diagnostic, and therapeutic capabilities of genomics and proteomics. As time has progressed, so has our understanding of this field. Although the mapping of the human genome will certainly have an impact on health care, it is a complex web to unweave. Addressing simpler "Single Nucleotide Polymorphisms" (SNPs) is not new, however, the complexity and importance of polygenic disorders and the greater role of the far more complex field of proteomics has become more clear. Proteomics operates much closer to the actual cellular level of human structure and proteins are very sensitive markers of health. Because the proteome, however, is so much more complex than the genome, and changes with time and environmental factors, mapping it and using the data in direct care delivery is even harder than for the genome. For these reasons of complexity, the expected utopia of a single gene chip or protein chip capable of analyzing an individual's genetic make-up and producing a cornucopia of useful diagnostic information appears still a distant hope. When, and if, this happens, perhaps a genetic profile of each individual will be stored with their medical record; however, in the mean time, this type of information is unlikely to prove highly useful on a broad scale. To address the more complex "polygenic" diseases and those related to protein variations, other tools will be developed in the shorter term. "Top-down" analysis of populations and diseases is likely to produce earlier wins in this area. Detailed computer-generated models will map a wide array of human and environmental factors that indicate the presence of a disease or the relative impact of a particular treatment. These models may point to an underlying genomic or proteomic cause, for which genomic or proteomic testing or therapies could then be applied for confirmation and/or treatment. These types of diagnostic and therapeutic requirements are most likely to be introduced into clinical practice through traditional forms of clinical practice guidelines and clinical decision support tools. The opportunities created by bioinformatics are enormous, however, many challenges and a great deal of additional research lay ahead before this research bears fruit widely at the care delivery level.

All decision models use some form of language to describe domain elements and their interactions. The terminology is often specific and even unique to the algorithm and is a choice of designers. Nevertheless the domain elements and concepts of any decision problem are almost never unique and are used and reused in many other decision problems. The same is true about the information about those elements in the context of different decision problems. Put together, the information about any given element forms our knowledge about the element and if stored properly in a knowledgebase, can be used and reused as necessary without the need for duplication.In this paper we discuss creation of an ontology using UMLS vocabulary and semantic network that provides an abstract understanding of elements (or objects) in the problem domain. Based on this ontology, a knowledgebase will be constructed that provides further information about the object in relation to another object or objects as described in the semantic links.A knowledgebase structured as such will have the benefit of problem-independence. It can be expanded as needed to include other objects that are used in a different series of problems and therefore, will have a one to many mapping between knowledgebase and decision models. Updating the knowledgebase will update the decision models seamlessly and maintenance will be less of an issue across decision models and within the knowledgebase. We are using this approach in building Bayesian decision models using Bayesian networks; however, this approach is not limited to Bayesian networks and has been and can be used for other decision making purposes.

The diagnostic variability in pathology, widely reported in the literature, is partly due to the use of different classification systems by pathologists. The descriptions of morphological characteristics on the same image within different classification systems can be considered as different points of view of pathologists. Our aim is to represent the points of view of the experts in pathology during image interpretation and to propose a method ological and technical solution in order to implement interoperability between these points of view. According to the hybrid ontology approach, we developed a system in three stages consisting in 1) the representation of the various points of view in local ontologies 2) the realization of a shared vocabulary and the development of a mapping tool used to allow the matching of local ontologies and shared vocabulary 3) the development of a transcoding algorithm for the translation of a case description from one point of view to another. A first evaluation of the transcoding algorithm was conducted for 33 cases of breast pathology. Our results show that the pathologists generally produce descriptions of the cases which do not follow rigorously the interpretation rules corresponding to the point of view they assert to adopt. While most of the concepts of local ontologies can be transcoded from a local ontology to another one (varying from 62.5 % to 100% according to the local ontology), the transcoding of a description which is valid according to a certain point of view, often results in a description which is not rigorously in accordance with the new point of view. These results underline the differences of interpretation rules existing in the different points of view.

The last two decades have seen considerable efforts directed towards making electronic health records interoperable through improvements in medical ontologies, terminologies and coding systems. Unfortunately, these efforts have been hampered by a number of influential ideas inherited from the work of Eugen Wüster, the father of terminology standardization and the founder of ISO TC 37. We here survey Wüster's ideas - which see terminology work as being focused on the classification of concepts in people's minds - and we argue that they serve still as the basis for a series of influential confusions. We argue further that an ontology based unambiguously, not on concepts, but on the classification of entities in reality can, by removing these confusions, make a vital contribution to ensuring the interoperability of coding systems and healthcare records in the future.

Pathologies and acts are classified in thesauri to help physicians to code their activity. In practice, the use of thesauri is not sufficient to reduce variability in coding and thesauri do not fit computer processing. We think the automation of the coding task requires a conceptual modelling of medical items: an ontology. Our objective is to help pneumologists code acts and diagnoses with a software that represents medical knowledge by an ontology of the concerned specialty. The main research hypothesis is to apply natural language processing tools to corpora to develop the resources needed to build the ontology. In this paper, our objective is twofold: we have to build the ontology of pneumology and we want to develop a methodology for the knowledge engineer to build various types of medical ontologies based on terminology extraction from texts.

The authors present a formal representation of ICD10 based on GALEN CRM. The goal of the work is to create a coding support tool for coding clinical diagnoses to ICD10. The formal representation of the first two chapters of ICD10 has been almost completed. The paper presents the main aspects of the modelling, and the experienced problems. The constructed ontology has been converted to OWL, and a test system has been implemented in Prolog to verify the feasibility of the approach. The system successfully identified diseases in medical records from gastrointestinal oncology. The classifier module is still under development.

How can information technology be used to model and handle clinical knowledge in everyday work so that clinicians can more systematically learn from collected clinical data? Ontology is a crucial element in answering this question. Based on nearly ten years of clinical experience, fundamental requirements of an ontology for oral medicine are presented. The use of the proposed W3C standards RDF and OWL for the design and implementation of an ontology for oral medicine is then described and discussed. The reported work contributes to knowledge representation in oral medicine by presenting the pioneering work towards an ontology for oral medicine using RDF/OWL, thereby testing the latter on a new domain.

The presentation assess the usability of the ontology platform protégé integrated with the terminology reasoning tool RACER to represent different terminology systems as the CEN European standard EN 1828 which is a categorical structure and the extensive French coding system CCAM supported by a GALEN representation. We present the 2 systems and some results showing the easiness to test the consistence of the ontology or of instances of terminology systems.This type of software tool which is accessible as open source could support a convergent "reference terminology representation" approach. Based on a formal representation development and allowing diversity in linguistic expressiveness of end users this approach can associate shared knowledge acquisition in the public domain and competing systems, software developers and researchers.

This paper discusses available definitions of population and criticise them against some basic rules of ontology. None of the found definition satisfies the requirement of an ontology that supports building consistent public heath databases. Most definitions define population on territorial bases or as reproductive communities. The author argues that populations are systems (its members must be in connection to each other) and individuals forming the population must share a common resource. Proper usage of the definitions may help to build consistent ontology for public health indicators and consistent databases.

BCAA granules (a mixture of branched-chain amino acids) have been used to reverse the hypoalbuminemia of decompensated liver cirrhotic patients in Japan. Our previous studies showed that BCAA promoted albumin secretion through the mTOR signal transduction pathway in rat primary hepatocyte culture [Ijichi C, Matsumura T, Tsuji T, Eto Y. Branched-chain amino acids promote albumin synthesis in rat primary hepatocytes through the mTOR signal transduction system. Biochem Biophys Res Commun 2003;303:59-64]. However, the mTOR-activating effect of BCAA in the experimental cirrhotic animals presenting with hypoalbuminemia has not yet been examined. The purpose of this study is to assess whether oral administration of BCAA induces mTOR activity in the livers of normal rats and CCl(4)-induced cirrhotic rats (CCl(4) rats). Biochemical analysis of liver extracts isolated from several rats showed that oral administration of BCAA (0.75g/kg body weight (BW)) induced phosphorylation of 4E-BP1 and stimulated the enzymatic activity of p70 S6K. Both of these molecules act downstream of mTOR. From the results, we conclude that orally administrated BCAA augments albumin synthesis in the liver, not only by supplementation of material substrates for protein synthesis, but also by induction of an mTOR signal that is critical for translational initiation. Furthermore, we conclude that induction of mTOR signaling is one of the major pharmacological mechanisms by which BCAA granules reverse the hypoalbuminemia of cirrhotic patients.

The goals of this short review are to familiarize readers with the stargazer mouse and to outline the major functional defects associated with this mutant. The roles of the stargazin protein in calcium channel function and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor trafficking are discussed; focus is placed on studies regarding the thalamus, whence absence seizures potentially originate, and the cerebellum, which is associated with the ataxic phenotype. Finally, two additional alleles of stargazer, waggler and stargazer 3Jackson (3J), illustrate the value of an allelic series for understanding stargazin function.

To detect protein expression of MMPs and TIMPs in various salivary gland neoplasms and to investigate their roles in invasion and metastasis of the malignant salivary gland tumors.

To study the expression and significance of p65 which is an important subtype of nuclear transcription factor kappaB (NF-kappaB) and its inhibitory protein IkappaBalpha in malignant transformation of hamster buccal mucosa.

To provide evidence for illustrating the molecular mechanism of nickel carcinogenesis, and to identify the differential expression of protein in crystalline NiS-induced neoplastic transformation of human bronchial epithelial cell by proteomics technology.

To investigate whether or not adaptive response of hPARP-1 protein normal and deficient cells is induced by low dose of hydroquinone (HQ), and to analyze the relationship between the adaptive response and micronuclei formation, and cell cycle alteration in human embryo lung fibroblasts (HLF), so as to elucidate the mechanism of adaptive response.

To investigate the value of urinary S100B protein and lactate/creatinine ratio determination in early identification of neonatal hypoxic-ischemic encephalopathy (HIE).

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear transcription factor that comprises the primary molecular target for thiazolidinedione (TZD) insulin-sensitizing drugs. Whilst expressed in many tissues in humans, its abundant expression in adipose tissue is believed to be the focal point through which TZDs regulate genes involved in glucose and lipid metabolism and via which these agents ultimately improve the hyperglycemia of type 2 diabetes. However, TZDs exhibit many additional properties, not least an array of effects which suggest a broad attack on the inflammatory process. Thus, TZDs have been shown to reduce plasma levels of the chemokine, monocyte chemotactic protein-1 (MCP-1), the anti-fibrinolytic protein, plasminogen activator inhibitor-1 (PAI-1), the endothelial cell adhesion molecules, e-selectin and inter-cellular adhesion molecule-1 (ICAM-1), the leucocyte-activating molecule, CD40L, and the tissue-remodeling enzyme, matrix metalloproteinase-9 (MMP-9). Further tangible evidence of a reduction by TZDs of systemic inflammation in patients with the classical metabolic syndrome stems from falls in the white blood cell count, P-selectin-positive platelets and in the acute-phase inflammatory proteins, C-reactive protein, serum amyloid A and fibrinogen. At the tissue level, TZDs improve vascular endothelial function, and reduce the rate of progression of intimal-medial thickening of the carotid artery and the microalbuminuria of type 2 diabetes. Further, TZDs have been shown to be efficacious in inflammatory diseases as wide-ranging as psoriasis, ulcerative colitis and non-alcoholic steatohepatitis (NASH). In the case of the latter, a broad spectrum of TZD-related properties is visible. Here, these drugs improve insulin sensitivity for glucose metabolism, reduce hyperinsulinemia, hepatic steatosis, inflammation and fibrosis, and lower the circulating levels of liver transaminases (ALT, AST), alkaline phosphatase and gamma glutamyl transferase. These effects in humans are also well-supported by investigative animal and in vitro studies. The ameliorative effects on liver fibrosis are of particular interest since they suggest that TZDs are able to activate a program of corrective tissue-remodeling. The basis for this action may be partly an ability to inhibit matrix protein secretion by hepatic stellate cells. An analogous action has also been seen in kidney mesangial cells. In conclusion, TZDs are important new drugs, presently indicated for the treatment of type 2 diabetes but with a spectrum of properties which suggests their potential for treating a number of degenerative inflammatory diseases, including NASH. However, full-scale, long-term clinical trials are needed with TZDs to test their potential to treat NASH, not least because of the (hepatotoxic) legacy of the prototype TZD, troglitazone, but also in view of the escalating burden of liver disease which is accompanying the increasing global prevalence of clinical obesity and type 2 diabetes.

Non-alcoholic steatohepatitis (NASH) is one of the most common liver disorders. This is highly prevalent in obese and diabetic subjects. Persons with central obesity are at particular risk. Other clinical predictors are age more than 40-50 years and hyperlipidemias, but none of these factors is invariable for causation of NASH. Other reported associations are, celiac disease, Wilson's Disease and few other metabolic diseases. Drugs, particularly amiodarone, tamoxifen, nucleoside analogues and methotrxate have also been linked to NASH. The disease is evenly distributed in both sexes but advanced disease is more common in women. Ethnic variation exists and African Americans are less affected than Hispanic Americans. Specific clinical features of NASH are infrequent. Patients usually come to clinical attention by elevated liver enzymes found on routine evaluation but on history, about two third of patients will admit to have mild fatigue and about half will report right upper quadrant pain. Rarely, patient may present with a complication of cirrhosis. Physical examination may reveal hepatomegaly and splenomegaly. Research in last few years has stressed that development of steatosis, stetohepatitis, fibrosis with subsequent cirrhosis are most probably the result of insulin resistance. Therefore, clinical features may reflect existence of insulin resistance. Obesity, particularly central obesity is most important of these. Patients may have sleep apnea syndrome. Hypertension and manifestations of diabetes mellitus like polyuria, polydypsia, and neurological deficits may occur. Patients may have varying combination of obesity, diabetes, hyperlipidemia, hypertension and impaired fibrinolysis (syndrome X). Children with insulin resistance may show acanthosis nigricance. Patients with polycystic ovary syndrome, which consists of insulin resistance, diabetes, obesity, hirsutism, oligo or polymenorrha and hyperlipidemia may have NASH. Other rare manifestations of insulin resistance, which can be seen in patients of NASH are lipomatosis, lipoatrophy/lipodystrophy and panniculitis. Most other rare conditions known to cause NASH like peroxisomal diseases, mitochondialpathies, Weber-Christian disease, Mauriac syndrome, Madelung's lipomatosis and abetaliopprotenemia also have insulin resistance. This is believed that primary defect underlying insulin resistance is impairment in postreceptor pathways (through tyrosine kinase activity) of insulin action. Primary defect in insulin receptors appear uncommon. This results in down regulation of insulin receptor substance 1 (IRS-1) signaling by excess free fatty acids. In muscle, activated IRS-1 promotes translocation of glucose transporter protein 4 (GLUT4) to cell membrane. As a result, monocyte glucose uptake by GLUT4 increases glucose disposal from blood and reduced need for insulin. PKC-0 is a likely candidate as serine kinase in muscle regulated by fatty acids that can impair the activation of IRS-1. Insulin resistance is usually evaluated by fasting insulin levels, Quantitative Insulin Check Index (QUICKI) and Homeostasis Model Assessment of Insulin Resistance (HOMA), C-peptid/insulin ratio oral glucose tolerance test and hyper insulinemic euglycemic clamp. The clamp technique is considered the gold standard.

Since non-alchoholic steatohepatitis (NASH) is often accompanied with metabolic syndrome comprising obesity, type-2 diabetes and hypertension, it is hypothesized that adipocytokines, insulin resistance and autonomic nervous system play crucial roles in disease progression of NASH. On the other hand, hepatic stellate cells (HSCs) have been shown to produce leptin when they get activated during hepatic fibrogenesis. Therefore, we investigated the role of leptin in fibrogenesis in the liver. Xenobiotics-induced liver fibrosis was extremely diminished in ob/ob mice and Zucker (fa/fa) rats, an inborn leptin- and leptin receptor (Ob-R)-deficient animal, respectively. Further, leptin increased transforming growth factor (TGF)-beta mRNA in isolated sinusoidal endothelial cells and Kupffer cells, suggesting that leptin promotes hepatic fibrogenesis through up-regulation of TGF-beta in the liver. Moreover, leptin augmented PDGF-dependent proliferation of HSCs by enhancing downstream intracellular signaling pathways via mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K)/Akt. Taken together, it is postulated that leptin acts as a profibrogenic cytokine in sinusoidal microenvironment. These findings indicate that leptin is one of the key regulators for inflammation and progression of fibrosis in various chronic liver diseases including NASH.

Citrin, encoded by SLC25A13, is a liver-type mitochondrial aspartate-glutamate carrier (AGC), of which deficiency, in autosomal recessive trait, causes neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2). NICCD patients have jaundice, hypoproteinemia, hypoglycemia, galactosemia, growth retardation, fatty liver and multiple aminoacidemia including citrulline, methionine, threonine and tyrosine. Some of the neonates who have experienced NICCD suffer from severe CTLN2 more than 10 years or several decades later. In CTLN2, neuropsychotic symptoms such as disorientation, aberrant behavior, coma and death are observed. Laboratory findings reveal hyperammonemia, citrullinemia, fatty liver and liver-specific decrease in a urea cycle enzyme, argininosuccinate synthetase (ASS). In some cases, hyperlipidemia, pancreatitis and hepatoma are accompanied with CTLN2. Citrin as a liver-type AGC plays a role in supplying aspartate to the cytosol for urea, protein and nucleotide synthesis by exchanging mitochondrial aspartate for cytosolic glutamate and proton, and transporting cytosolic NADH reducing equivalent to mitochondria as a member of malate aspartate shuttle essential for aerobic glycolysis. AGC is also important for gluconeogenesis from lactate. Although it is difficult to explain pathogenesis of the symptoms such as cholestasis in NICCD and liver-specific decrease of ASS protein in CTLN2 from the functions of the AGC, some are understandable by the loss of citrin functions. Many CTLN2 patients have been treated with a low protein and high carbohydrate diet and glycerol at the hyperammonemic coma. We argue that those treatments may result in fatty liver, hyperlipidemia, hyperammonemia and even death due to loss of the citrin functions. Loss of citrin first cause deficiency of aspartate in the cytosol, which results in an increase in cytosolic NADH/NAD(+) ratio and then activation of fatty acid synthesis pathway to compensate the aberrant ratio. This follows inhibition of fatty acid oxidation. The peculiar fondness for food of CTLN2 patients who like protein and dislike carbohydrate and sweets may be related to their metabolic requirements.

To study the variations of p38 mitogen-activated protein kinase (p38MAPK) activity on lipopolysaccharide (LPS)-induced inflammation of nasal epithelial cells and its significance in vitro.

To isolate and identify a new protein involved in drug-resistance from culture supernatants of isoniazid (INH)-resistant Mycobacterium tuberculosis.

Effect of enviroment temperature on near infrared spectroscopic quantitative analysis was studied. The temperature correction model was calibrated with 45 wheat samples at different environment temperaturs and with the temperature as an external variable. The constant temperature model was calibated with 45 wheat samples at the same temperature. The predicted results of two models for the protein contents of wheat samples at different temperatures were compared. The results showed that the mean standard error of prediction (SEP) of the temperature correction model was 0.333, but the SEP of constant temperature (22 degrees C) model increased as the temperature difference enlarged, and the SEP is up to 0.602 when using this model at 4 degrees C. It was suggested that the temperature correctional model improves the analysis precision.

A resonance light scattering method for the determination of trace proteins was developed. In the presence of Triton X-100, proteins reacted with Titan yellow to form a combination product, resulting a significant enhancement of resonance light scattering (RLS). The deltaI(RLS) was directly proportional to the concentration of protein in the range of 0.03-0.9 microg x mL(-1), with the detection limit 17.7 ng x mL(-1) for BSA. This method was applied to the determination of the proteins in synthetic and human serum samples, and compared to the CBB method, with satisfactory results.