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The up-regulation of MUC1 protein is associated with malignant phenotype of cancer. We investigated the significance of KL-6, one of the MUC1 antigens, as a tumor marker in hepatitis C virus positive hepatocellular carcinoma (HCC). Serum KL-6 was determined in 203 patients with chronic hepatitis (CH), 47 patients with liver cirrhosis (LC) and 78 patients with HCC. KL-6 was higher in HCC compared to non-HCC (p=0.0005) and was higher in patients with multiple HCC nodules compared to a single nodule (p=0.02). There was no correlation between KL-6 and existent tumor markers for HCC such as alpha-fetoprotein, lens culinaris agglutinin-reactive alpha-fetoprotein or des-gamma-carboxyprothrombin. In the prospective analysis, the cumulative incidence of HCC was significantly greater in CH and LC patients with high initial KL-6 (above 400U/ml) compared to the others (p=0.02). Moreover, in the prospective observation of 25 patients whose HCC was completely cured by radiofrequency ablation therapy, the cumulative incidence of distant recurrences was significantly greater in patients with high initial KL-6 compared to the others (p=0.005). These results suggest that serum KL-6 could be a novel tumor marker in the diagnosis and the prediction of prognosis of HCC that may have additive value to the existent markers.
Insulin resistance, the impaired action of insulin, has been linked to many important consequences, including Type 2 diabetes, hypertension, dyslipidemia, acanthosis nigricans and polycystic ovarian syndrome. Although there are some genetic causes for insulin resistance, the most common cause is an excess of nutrition a condition called "Nutrient Toxicity". Both excess glucose and excess fat can cause insulin resistance in muscle and fat tissues and excess fat can cause insulin resistance in the liver. High fat feeding and fat infusion rapidly lead to the development of insulin resistance caused by impairment in glucose transport. Other studies have shown defects in insulin signaling possibly secondary to activation of Protein Kinase C resulting from the accumulation of active fatty acyl CoA's. Glucose toxicity has been studied both in vivo and in vitro. In vivo it has been shown that rats over-expressing the gluconeogenic enzyme Phosphoenol Pyruvate Carboxykinase (PEPCK) develop insulin resistance in fat and muscle tissues and some features of the metabolic syndrome including mild obesity and dyslipidemia. Excess glucose entry in fat cells results in increased flux through the hexosamine biosynthesis pathway leading to activation of protein kinase C and impairment of glucose transport. Obesity resulting from excess nutrient intake can also cause insulin resistance by an increase in the production of agents that impair insulin action such as TNFalpha and resistin and a decrease in the production of an insulin sensitizing compound adiponectin. Both glucose and free fatty acids acutely stimulate insulin secretion but chronic exposure to high levels of either nutrient leads to impairment of beta cell function. The combination of insulin resistance and beta cell failure leads to diabetes. Nutrient toxicity is thus the driving cause of the diabetes epidemic that is being recorded around the world.
To explore the expression of protein kinase C (PKC) and the regulatory effect of nerve growth factor (NGF) in the lung and the visceral sensory afferent system (C(7)-T(5) spinal ganglia and the corresponding posterior horn of the spinal cord) of asthmatic guinea pigs.
To study the changes of pulmonary surfactant associated protein A (SPA) of lung tissue in acute pulmonary embolism (PE).
Phosphatase and tensin homolog (PTEN) is a multifunctional phosphatase whose substrate is phosphatidylinositol-3,4,5-triphosphate (PIP3), and it is also a ubiquitously expressed tumor suppressor gene that down-regulates phosphatidylinositol-3-kinases (PI3Ks). Although there are a few reports about PTEN related to hepatocellular carcinoma, the role of PTEN in the liver remains unclear. Therefore, to clarify the role of PTEN in the liver, we generated and analyzed hepatocyte-specific Pten-deficient mice (Pten-deficient mice). The liver of 40-week-old Pten-deficient mice revealed macrovesicular steatosis, ballooning hepatocytes, lobular inflammatory cell infiltration, and perisinusoidal fibrosis that are characteristic of human nonalcoholic steatohepatitis (NASH). By 80 weeks of age, 100% of Pten-deficient livers showed adenomas and 66% had hepatocellular carcinomas. Thus, PTEN is important for the prevention of adipogenic and tumorigenic transformation, and Pten-deficient mice are a novel model for NASH and hepatocellular carcinoma. Our results suggest that the controlled blocking of molecules acting downstream of PI3K might provide significant therapeutic benefit to patients predisposed to NASH and hepatocellular carcinoma.
The technology of cloning involves transplanting a diploid nucleus into a mature oocyte cytoplast. The cytoplast is then activated to initiate the first cell cycle of development as a nuclear transplant embryo. Initiation and regulation of events during the first cell cycle are, therefore, critical for proper reprogramming of the donor nucleus and development as a cloned embryo. Activation is normally induced by the sperm and is mediated by a series of intracellular free calcium ([Ca(2+)](i)) oscillations that last for several hours. Although it is not known precisely how the sperm induces activation, current evidence favors the delivery, by the sperm, of a soluble protein factor that causes the production of IP3. IP3 acts to open a Ca(2+) channel in the endoplasmic reticulum and release Ca(2+) into the cytosol. A variety of methods have been used to duplicate or replace the sperm-induced [Ca(2+)](i) increase to cause activation in nuclear transplant embryos. It has been found that treatments that cause a single transient [Ca(2+)](i) activate some oocytes with the level of activation increasing as the oocyte ages. Attempts have been made to extend the period of time over which [Ca(2+)](i) oscillations occur. This has been successful in increasing activation rates of less mature oocytes but the techniques are still cumbersome. An alternative method, that has been very successful, is the combination of a treatment that elevates [Ca(2+)](i) and a treatment that maintains low levels of maturation promoting factor for several hours after the initial [Ca(2+)](i) elevation. The sperm also contributes the centrosome that organizes microtubules during the first cell cycle. One current hypothesis for regulation of sperm centrosomal activity consists of a dephosphorylation of sperm connecting piece proteins following sperm entry into the oocyte and activation of the oocyte. Dephosphorylation of these proteins results in the disassembly of the connecting piece and assembly of a functional centrosome. In nuclear transfer, centrosomal components are contributed by the donor cell. If the cell is fused to the cytoplast before centriole replication then a single aster forms. If the cell is fused after centriole replication then two asters form. In either case and even in parthenogenetic oocytes, which do not have centrioles, the first cell cycle progresses to metaphase. However, progress is slow and some defects are observed in the assembly of chromosomes into a metaphase plate.
High concentration of corticosterone leads to morphological and functional impairments in hippocampus, ranging from a reversible atrophy of pyramidal CA3 apical dendrites to the impairment of long-term potentiation (LTP) and hippocampus-dependent learning and memory. Glutamate and N-methyl-D-aspartate (NMDA) receptor play an important role in this effect. Because of the importance of brain-derived neurotrophic factor (BDNF) in the functions of the hippocampal neurons, alteration of the expression of BDNF is thought to be involved in the corticosterone effect on the hippocampus. To determine whether change in BDNF in the hippocampus is involved in the corticosterone effect, we injected corticosterone (2 mg/kg, s.c.) to Sprague-Dawley rats and measured the mRNA, proBDNF and mature BDNF protein levels in the hippocampus. We also measured the phosphorylation level of the transcription factor cAMP response element binding protein (CREB). Furthermore, we intraperitoneally injected NMDA receptor antagonist MK801 (0.1 mg/kg) 30 min before corticosterone administration to investigate whether and how MK801 affected the regulation of BDNF gene expression by corticosterone. Our results showed that 3 h after single s.c. injection of corticsterone, the expression of BDNF mRNA, proBDNF and mature BDNF protein decreased significantly (P<0.01). MK801 promoted the downregulation of BDNF gene expression in the rat hippocampus by corticosterone. We also found that either applying corticosterone or co-applying corticosterone with MK801 downregulated the phosphoration level of CREB, the latter (corticosterone plus MK801) being more effective (P<0.05). Taken together, our results indicate that corticosterone downregulates BDNF gene expression in the rat hippocampus through CREB pathway and that blockade of NMDA receptor enhances this effect of corticosterone in reducing BDNF expression.
The present study aimed to investigate the role of spinal p38 mitogen-activated protein kinase (p38 MAPK) activation in chronic constriction injury (CCI) of the sciatic nerve induced neuropathic pain. CCI model was produced by loosely ligating the left sciatic nerve proximal to the sciatica's trifurcation with 4-0 silk thread in male Sprague-Dawley rat. SB203580, a specific inhibitor of the p38 MAPK, was intrathecally administered on day 5 post-CCI. Thermal and mechanical nociceptive thresholds were assessed with the paw withdrawal lantency (PWL) to radiant heat and the paw withdrawal threshold (PWT) to von Frey filaments respectively. The protein levels of the phosphorylated p38 MAPK (p-p38 MAPK) and phosphorylated cAMP response element binding protein (pCREB) were assessed by Western blot analysis. The results showed that CCI significantly increased the expressions of cytosolic and nuclear p-p38 MAPK in the spinal cord. Intrathecal administration of SB203580 dose-dependently reversed the established mechanical allodynia and thermal hyperalgesia induced by CCI. Correlated with behavior results, SB203580 dose-dependently inhibited the CCI-induced increase of the expressions of cytosolic and nuclear p-p38 MAPK and nuclear pCREB in the spinal cord. Taken together, these findings suggest that the activation of p38 MAPK pathway contributes to the development of neuropathic pain induced by CCI, and that the function of p-p38 MAPK may partly be accomplished via the CREB-dependent gene expression.
Extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase (MAPK), transduces a broad range of extracellular stimuli into diverse intracellular responses. It has been reported that ERK is involved in the modulation of nociceptive information and central sensitization produced by intense noxious stimuli or peripheral tissue inflammation. Our previous studies showed that the spinal neurons sensitization was involved in morphine withdrawal response. This study was to investigate the role of the spinal ERK in morphine dependence and naloxone-precipitated withdrawal response. To set up morphine-dependent model, rats were subcutaneously injected with morphine (twice a day, for 5 d). The dose of morphine was 10 mg/kg on the first day and was increased by 10 mg/kg each day. On day 6, 4 h after the injection of morphine (50 mg/kg), morphine withdrawal syndrome was precipitated by an injection of naloxone (4 mg/kg, i.p.). Using anti-phospho-ERK (pERK) antibody, the time course of pERK expression was detected by Western blot. U0126, a mitogen-activated protein kinase kinase (MEK) inhibitor, or phosphorothioate-modified antisense oligonucleotides (ODN) was intrathecally injected 30 min or 36, 24 and 12 h before naloxone-precipitated withdrawal. The scores of morphine withdrawal symptom and morphine withdrawal-induced allodynia were observed. One hour after naloxone-precipitated withdrawal, pERK expression in the spinal dorsal horn was assessed by immunohistochemical analysis and Western blot was used to detect the expression of cytosolic and nuclear fraction of pERK in the rat spinal cord. The results showed that the expression of cytosolic and nuclear fraction of pERK, not non-phospho-ERK, in the spinal cord was gradually increased following the injection of morphine. When morphine withdrawal was precipitated with naloxone, the expression of the spinal pERK further increased. Intrathecal administration of U0126 or antisense ODN against ERK decreased the scores of morphine withdrawal, attenuated morphine withdrawal-induced allodynia and also inhibited the increase of pERK expression in the spinal cord of morphine withdrawal rats. These results suggest that activation of the spinal ERK is involved in morphine-dependent and naloxone-precipitated withdrawal response.
The aim of this study was to investigate the effects of angiotensin II (Ang II) on extracellular signal-regulated protein kinase (ERK) signaling pathway in cultured vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. VSMCs from SHR and WKY rats were treated with 1x10(-7) mmol/L Ang II for 24 h in the absence or presence of 30 min of pre-treatment of valsartan (1x10(-5) mmol/L) or PD98059 (1x10(-5)mmol/L), selective inhibitor of ERKs- dependent pathways, when they were cultured in 20% calf serum medium. VSMCs of SHR and WKY cultured in serum-free medium were used as control groups. Among the different treatments, VSMCs from the SHR and WKY were devided into four groups: (1) control, (2) Ang II, (3) Ang II + valsartan, (4) Ang II + PD98059. ERK activity in VSMCs was measured by immuno-precipitation. Proteins of total ERK (t-ERK), phosphorylated-ERK (p-ERK) and mitogen-activated protein kinases phosphatase-1 (MKP-1) in VSMCs were detected by Western blot. MKP-1 mRNA in VSMCs was measured by RT-PCR. In VSMCs from WKY or SHR rats, ERK activity, p-ERK, MKP-1 and MKP-1 mRNA in Ang II group were higher than those in control group (P<0.05). In both SHRs and WKYs, there were no significant differences in ERK activity, p-ERK, MKP-1 and MKP-1 mRNA among the control group, Ang II + valsartan group and Ang II + PD98059 group. ERK activity, p-ERK, MKP-1 and MKP-1 mRNA in SHRs were significantly higher than those in WKYs with same treatments (P<0.01). There was no significant difference in t-ERK among different groups and no difference in t-ERK between SHRs and WKYs (P>0.05). Our results show that Ang II activates VSMCs ERK signaling pathways via Ang II type 1 (AT(1)) receptors. Ang II increased ERK activity and p-ERK, but not t-ERK, accompanied by an increase in MKP-1 mRNA expression and protein. Among the different treatments, ERK activity and p-ERK were higher in SHR than in WKY. Valsartan and PD98059 blocked Ang II-stimulated ERK activation. These results suggest that ERK signaling pathway plays an important role in the pathogenesis of hypertension. The effect of Ang II on SHR and WKY VSMCs' ERK signaling pathway may be mediated by AT(1) receptors, enhancing ERK activity and the amount of p-ERK, and then increasing MKP-1 mRNA and its expression.
Resveratrol (trans-3, 4', 5-trihydroxy stilbene), a phytoalexin found in grape skins and red wine, has been reported to have a wide range of biological and pharmacological properties. It has been speculated that resveratrol may have cardioprotective activity. The objective of our study was to investigate the effects of resveratrol on intracellular calcium concentration ([Ca(2+)](i)) in rat ventricular myocytes. [Ca(2+)](i) was detected by laser scanning confocal microscopy. The results showed that resveratrol (15~60 mumol/L) reduced [Ca(2+)](i) in normal and Ca(2+)-free Tyrode's solution in a concentration-dependent manner. The effects of resveratrol on [Ca(2+)](i) in normal Tyrode's solution was partially inhibited by pretreatment with sodium orthovanadate (Na3VO4, 1.0 mmol/L, P<0.01), an inhibitor of protein tyrosine phosphatase, or L-type Ca(2+) channel agonist Bay K8644 (10 mumol/L, P<0.05), but could not be antagonized by NO synthase inhibitor L-NAME (1.0 mmol/L). Resveratrol also markedly inhibited the ryanodine-induced [Ca(2+)](i) increase in Ca(2+)-free Tyrode's solution (P<0.01). When Ca(2+) waves were produced by increasing extracellular Ca(2+) concentration from 1 to 10 mmol/L, resveratrol (60 mumol/L) could reduce the velocity and duration of propagating waves, and block the propagating waves of elevated [Ca(2+)](i). These results suggest that resveratrol may reduce the [Ca(2+)](i) in isolated rat ventricular myocytes. The inhibition of voltage-dependent Ca(2+) channel and tyrosine kinase, and alleviation of Ca(2+) release from sarcoplasmic reticulum (SR) are possibly involved in the effects of resveratrol on rat ventricular myocytes. These findings could help explain the protective activity of resveratrol against cardiovascular disease.
15-hydroxyeicosatetraenoic acid (15-HETE) plays an important role in hypoxia-induced pulmonary vasoconstriction. Release of nitric oxide (NO) is apparently decreased and activity of endothelial nitric oxide synthase (eNOS) is impaired in chronic hypoxia. However, little is known whether 15-HETE contributes to eNOS/NO pathway in the constriction induced by 15-HETE. We examined the response of rat pulmonary artery (PA) rings to 15-HETE, the production of NO, total eNOS expression and the phosphorylation of eNOS in bovine pulmonary artery endothelial cells (BPAECs) stimulated by 15-HETE. Rat PA rings were divided into three groups: endothelium intact group, endothelium denuded group, and nitro-L-arginine methyl ester (L-NAME, 0.1 mmol/L, an inhibitor of eNOS) group. Constrictions to 15-HETE were significantly enhanced in endothelium denuded group and L-NAME group (both P< 0.05 vs endothelium intact group, n= 9); BPAECs were incubated in different conditions to test nitrite production by Greiss method. Nitrite production was significantly reduced by 1 mumol/L 15-HETE (P<0.05), and increased by the lipoxygenase inhibitors, 10 mumol/L cinnamyl 3,4- dihydroxy-[alpha] -cyanocinnamate (CDC, P< 0.05) and 0.1 mmol/L nordihydroguiairetic acid (NDGA, P< 0.01 ); Western blot analysis of extracts from BPAECs incubated with 15-HETE in different time was carried out to test total eNOS expression, and the expression was changed unobviously. Immunoprecipitation (IP) and Western blot analysis of cell extracts from BPAECs treated with 2 mumol/L 15-HETE in different length of time were accomplished, using phospo-eNOS-threonine 495 (Thr495, an inhibitory site) antibody for IP, and eNOS or 15-lipoxygenase (15-LO) antibodies for Western blot. 15-HETE depressed eNOS activity by increasing the levels of phospho-eNOS-Thr 495. The data suggest that eNOS/NO pathway is involved in PA constrictions induced by 15-HETE and that 15-HETE depresses eNOS activity by phosphorylation in Thr495 site. The protein interaction between phospho-eNOS (Thr495) and 15-LO is discovered for the first time.
About 20 - 50% individuals with intrauterine growth retardation (IUGR) could not achieve catch-up growth and remain small in size till adulthood. There are few reports on the relation between intestinal development and body catch-up growth of IUGR. Studies showed that early "nutritional programming" would results in long-term effects on the body growth and organic function, and gastrointestinal development is closely related to the body development as well. The authors aimed to study the effect of early nutritional interventions on serum IGF1, IGFBP3, intestinal development and catch-up growth of pups with IUGR by using diets with different protein and caloric levels during the first four weeks of life.
Insulin resistance, obesity, diabetes, dyslipidemia and nonalcoholic fatty liver are components of the metabolic syndrome, a disease complex that is increasing at epidemic rates in westernized countries. Although proinflammatory cytokines have been suggested to contribute to the development of these disorders, the molecular mechanism of the development of this syndrome is poorly understood. In this study, we show that expression of suppressor of cytokine signaling SOCS-1 and SOCS-3 is increased in livers of obese insulin-resistant animals, and that adenoviral-mediated overexpression of SOCS-1 or SOCS-3 in liver causes insulin resistance through down-regulation of tyrosine phosphorylation of insulin receptor substrate (IRS) proteins. Moreover, the increased SOCS-1 and SOCS-3 also cause a prominent up-regulation of the key regulator of fatty acid synthesis in liver, sterol regulatory element binding protein (SREBP)-1. Conversely, inhibition of SOCS-1 and SOCS-3 in livers of obese diabetic db/db mice by antisense treatment modestly improves insulin sensitivity, but completely normalizes the increased expression of SREBP-1. The latter leads to dramatic amelioration of hepatic steatosis and hypertriglyceridemia. Promoter activity analysis reveals that expression of SOCS-1 or SOCS-3 with SOCS-3 being more potent enhances SREBP-1c expression, while it is inhibited by expression of STAT3. This STAT3-mediated inhibition of SREBP-1c expression is antagonized by co-expression of SOCS proteins. Moreover, db/db mice display decreased STAT3 phosphorylation in liver that is normalized by antisense treatment of SOCS proteins. These data suggest that obese subjects in the persistent inflammatory states, such as elevated circulating tumor necrosis factor-alpha, may have down-regulated STAT3-mediated signaling by increased SOCS proteins, leading to up-regulation of SREBP-1c expression and increased fatty acid synthesis in liver. Thus, SOCS proteins play an important role in pathogenesis of the metabolic syndrome by concordantly modulating cytokine signaling and insulin signaling.
Electron transfer (ET) reactions in systems involving proteins require an oriented interaction between electron donor and acceptor in order to accommodate their respective redox centres in optimal orientation for efficient ET. Such type of reactions are critical for the maintenance of the physiological functions of living organisms, since they are implicated in vital actions, as is, for example, in the photosynthetic ET chain that leads to NADPH reduction. In this particular case, a small redox protein ET chain is responsible for ET from Photosystem I (PS I) to NADP(+). In this system the enzyme responsible for NADP(+) reduction is ferredoxin-NADP(+) reductase (FNR), a FAD-containing NADP(+) dependent reductase. In order to produce such reduction, this enzyme receives electrons from a [2Fe-2S] plant-type ferredoxin (Fd), which is previously reduced by PS I. Moreover, in the case of some algae and cyanobacteria, an FMN-dependent protein, flavodoxin (Fld), has been shown to replace Fd in this function. The processes of interaction and ET between FNR and all of its substrates involved in the photosynthetic ET chain, namely Fd, Fld and NADP(+)/H have been extensively investigated in recent years using a large number of techniques, including the introduction of site-specific mutations in combination with kinetic and structural studies of the produced mutants. The present manuscript summarises the information so far reported for an efficient interaction between FNR and its substrates, compares such information with that revealed by other systems for which the FNR structure is a prototype and, finally, discusses the implications of the processes of association in ET between FNR and its substrates.
Plants can change the size of their light harvesting complexes in response to growth at different light intensities. Although these changes are small compared to those observed in algae, their conservation in many plant species suggest they play an important role in photoacclimation. A polyclonal antibody to the C-terminus of the Arabidopsis thaliana chlorophyllide a oxygenase (CAO) protein was used to determine if CAO protein levels change under three conditions which perturb chlorophyll levels. These conditions were: (1) transfer to shaded light intensity; (2) limited chlorophyll synthesis, and (3) during photoinhibition. Transfer of wild-type plants from moderate to shaded light intensity resulted in a slight reduction in the Chl a/b ratio, and increases in both CAO and Lhcb1 mRNA levels as well as CAO protein levels. CAO protein levels were also measured in the cch1 mutant, a P642L missense mutation in the H subunit of Mg-chelatase. This mutant has reduced total Chl levels and an increased Chl a/b ratio when transferred to moderate light intensity. After transfer to moderate light intensity, CAO mRNA levels decreased in the cch1 mutant, and a concomitant decrease in CAO protein levels was also observed. Measurements of tetrapyrrole intermediates suggested that decreased Chl synthesis in the cch1 mutant was not a result of increased feedback inhibition at higher light intensity. When wild-type plants were exposed to photoinhibitory light intensity for 3 h, total Chl levels decreased and both CAO mRNA and CAO protein levels were also reduced. These results indicate that CAO protein levels correlate with CAO mRNA levels, and suggest that changes in Chl b levels in vascular plants, are regulated, in part, at the CAO mRNA level.
A protein was isolated from membranes of the green sulfur bacterium Chlorobium tepidum. This protein was characterized by gel electrophoresis, gel filtration, analytical ultracentrifugation and amino acid sequencing. The molecular weight of the purified protein was shown to be 26 kDa by SDS-PAGE. HPLC gelfiltration, SDS-PAGE and analytical ultracentrifugation are consistent with the presence of a homogenous protein in the preparations. Amino acid analysis was obtained from the isolated protein after fragmentation with Lys-C, trypsin and cyanogen bromide. The cleavage pattern resulting from these treatments combined with Edman sequencing yield a sequence allowing the identification of an integral membrane agglutinin in Chl. tepidum.
Mass mapping analysis based on cyanylation (CN) of the protein and CN-induced cleavage indicates that all three cysteine residues in the insertion into the light-activated pea leaf chloroplast fructose bisphosphatase (E.C. 3.1.3.11) are able to participate in disulfide bond formation. There is a major peak in the mass spectrum of the cleavage products indicating that Cys173 forms a disulfide bond with Cys153, consistent with the structure of the oxidized enzyme in PDB files 1d9q and 1dcu, and a minor peak indicating that Cys173 forms an alternate disulfide bond with Cys178. The Cys173-Cys178 disulfide bond was not apparent in the available crystal structures.
The Del1 mutant of the green alga Chlamydomonas reinhardtii with a defined deletion in the chloroplast encoded psbA gene is unable to grow photoautotrophically. We show here that this mutant can be transformed with PCR-generated psbA fragments of varying length to yield photosynthetically growing colonies. PCR fragments need not be purified but can be directly precipitated from the amplification reaction onto tungsten particles, allowing fast and efficient mutagenesis experiments. Flanking regions bordering the deletion breakpoints have been systematically shortened from both sides. The shortest fragment giving rise to significant numbers of transformants contains about 50 bp upstream and 120 bp downstream of the deletion breakpoint. This technique greatly simplifies comprehensive structure-function analyses of the D1 protein in Chlamydomonas, but could perhaps be adapted to other chloroplast genes in this or other organisms as well.
To study the effect of selenium supplementation on myelin basic protein (MBP) mRNA expression in cerebrum of high-iodine intake filial mice.
The SK-N-SH human neuroblastoma cells were treated with acetylcholine (Ach) and atropine (Atro), the proliferation activity of cells was detected by CCK-8 method and the cell cycle distribution was determined by flow cytometry. The changes of the mRNA and protein of mAchR1 and c-fos were analysed by fluorescence quantitative PCR, western blot and flow cytometry indirect-immunofluorescence. Results showed 1 mmol/L Ach could significantly stimulate the proliferation of SK-N-SH cells and 1 mmol/L Atro could prevent SK-N-SH cells from S phase growing into G2/M phase; 1 mmol/L Ach and 1 mmol/L Atro had feedback adjustment on the protein level of mAchR1, but had no significant effect on mRNA level of mAchR1. 1 mmol/L Ach increased the both levels of mRNA and protein of Fos obviously, this effect was reversed by Atro. All phenomena clue us cholinergic receptors joining the events of cell proliferation.
The ubiquitin/proteasome system is regarded as a major pathway of proteolysis in eukaryotic cells, in which the proteasome acts as primary protease for its function of degrading substrate proteins to short peptides. In the present paper, cytological, statistical studies and Fourier transform infrared (FTIR) analysis on the effects of MG132, an inhibitor of proteasome, on the pollen germination and tube growth of Pecea wilsonii were carried out in an artificial experimental system. It is showed that MG132 significantly reduced the germination rate and tube growth. Furthermore, MG132 treatment lead to vacuolization occurred both in tube cytoplasm and generative cell. While DMSO and non-proteasome inhibitor E-64 do not have similar effects. FTIR analysis revealed that MG132 treatment markedly reduced the contents of wall-bound proteins and pectin at the apex of tube. Those findings provided evidence that by inhibiting the activity of proteasme, MG132 strongly affects pollen germination and tube growth of P. wilsonii, and that UPP plays an important role in organization and maintaining polarized growth of pollen tube. Inhibition of UPP will induce apoptosis of pollen tube.
Several studies have demonstrated the potential of olfactory ensheathing cells (OECs) for the repair of central and peripheral nerve injury. In this work, ensheathing cells that express the receptor gene coding for enhanced green fluorescent protein (eGFP) from adult mice were isolated and purified, and then, its biological characters were examined in vitro. The work was based on combinations of fluorescence confocal, phase contrast, cell proliferation assay and immunolabeling identification. The results showed that (1) two major morphologically and immunohistochemically distinct types of cells were present after 15 days in the primary cultures of adult transgenic mice olfactory nerves and glomerular layers of the olfactory bulb. One cell type was bipolar or multipolar OECs and strained positively for P75 low affinity neurotrophic receptor (P75N), S100, and glial fibrillary acidic protein(GFAP). The other type was fibroblasts with flat or endothelial-like shape, and reacted with antibody against Thy1.1. (2) A simple, inexpensive method for purifying ensheathing cells, in which various harvested cell types showed different rates of attachment to the uncoated culture ware, was developed. This technique neither binds any antibodies nor requires any costly equipment, and yields a large number of highly purified cells. (3) In sequential observations over 22 days in culture, the population of purified cells was maintained and continued to proliferate for longer. This experiment not only supported and advanced the ensheathing cell research but also offered ideal materials of in vivo transplantation for the repair of CNS injury.
Polystyrene/poly (methyl methacrylate) (PS/PMMA) was studied after the thin films were prepared on glass substrate by spin-coating from THF. Raman spectroscopy combined with microscopy was used to obtain information on the morphology and structure of the thin films. From the relative intensities of the peaks around 1 604 and 1 585 cm(-1) due to stretching of benzene rings, and 1 728 cm(-1) due to stretching of C=O for PS and PMMA respectively, the authors could define the composition of the domains in the sea-island-like phase-separated structure in the microscopic image. Furthermore, the structure evolution was followed by Raman spectroscopy during the in-situ annealing of PS/PMMA (30/70) blend thin films at 210 degrees C. And the effect of SERS on the PS thin films was also discussed.
The structure and characteristics of CdTe thin films are dependent on the working atmosphere states in close-spaced sublimation. In the present paper, CdTe polycrystalline thin films were deposited by CSS in mixture atmosphere of argon and oxygen. The physical mechanism of CSS was analyzed, and the temperature distribution in CSS system was measured. The dependence of preliminary nucleus creation on the atmosphere states (involving component and pressure) was studied. Transparencies were measured and optic energy gaps were calculated. The results show that: (1) The CdTe films deposited in different atmospheres are cubic structure. With increasing oxygen concentration, a increases and reaches the maximum at 6% oxygen concentration, then reduces, and increases again after passing the point at 12% oxygen concentration. Among them, the sample depositing at 9% oxygen concentration is the best. The optic energy gaps are 1.50-1.51 eV for all CdTe films. (2) The samples depositing at different pressures at 9% oxygen concentration are all cubical structure of CdTe, and the diffraction peaks of CdS and SnO2:F still appear. With the gas pressure increasing, the crystal size of CdTe minishes, the transparency of the thin film goes down, and the absorption side shifts to the short-wave direction. (3) The polycrystalline thin films with high quality deposit in 4 minutes under the depositing condition that the substrate temperature is 550 degrees C, and source temperature is 620 degrees C at 9% oxygen concentration.
The interaction and absorption spectral behavior of o-chlorophenylfluorone (o-CPF)-Mo(VI) and protein are studied in OP microemulsion medium. Under the optimum condition, the molar absorptivity of o-CPF-Mo(VI)-BSA system at 532 nm is 6.12 x 10(6) L x mol(-1) x cm(-1), and Beer's law is obeyed in the range of 0-14 microg x mL(-1) of BSA. Moreover, the binding number between the complex and BSA determined by molar ratio and slope ratio methods is 91. The mechanism of interaction of BSA and o-CPF-Mo (VI) is also discussed. OP microemulsion media introduced into the protein determination increased markedly the sensitivity of the system. The result of experiment indicates that the method possesses a high sensitivity as well as a high selectivity. It can be used to determine protein in human urine with satisfactory results.
The temperature-dependent Henry's Law Constant for hydrogen peroxide was measured. The gas phase of hydrogen peroxide from the vapor saturator collected in a cryogenic trap was analyzed by a spectrophotometric determination, based on the oxidative decoloration of BPR (bromopryogallol red) reaction with hydrogen peroxide under the catalysis of hemin. At 10 degrees C - 35 degrees C, the relationship between Henry's Law constant K(H) (mol x L(-1) x atm(-1)) of hydrogen peroxide and temperature T (K) can be expressed as ln K(H) = a/T - b, where a = 7 269+/-22, and b = 13.26+/-0.08. The standard heat of hydrogen peroxide aqueous solution is 60.43+/-0.18 kJ x K(-1) x mol(-1).
The localization of activity of glucose oxidase (GOD) on the pussian blue modified electrode by SiO2 sol-gel was studied by X-ray microanalysis. Glucose and Ce(NO3)3 served as substrate and capture respectively. Substrate was catalysed by immobilized GOD toproduce H2O2, and the H2O2 was captured by Ce(NO3)3 to form precipitate. The precipitation deposited the active site of immobilized GOD on the enzyme electrode. The X-ray microanalysis showed that the distribution of active enzyme on the surface of the enzyme electrode was uniform, and the GOD immobilized maintained high activity. The X-ray analysis exhibited that the properties of the enzyme electrode depended on the distribution of enzyme activity on its surface from the view of microstructure, and the developed GOD electrode was of high sensitivity and excellent stability. The results agreed with the results of the electrochemical study.
Tyrosylprotein sulfotransferase (TPST), the enzyme responsible for the sulfation of tyrosine residues, has been identified and characterized in submandibular salivary glands previously (William et al. Arch Biochem Biophys 338: 90-96). Tyrosylprotein sulfotransferase catalyses the sulfation of a variety of secretory and membrane proteins and is believed to be present only in the cell. In the present study, this enzyme was identified for the first time in human saliva. Analysis of human saliva and parotid saliva for the presence of tyrosylprotein sulfotransferase revealed tyrosine sulfating activity displayed by both whole saliva and parotid saliva at pH optimum of 6.8. In contrast to tyrosylprotein sulfotransferase isolated from submandibular salivary glands, salivary enzyme does not require the presence of Triton X-100, NaF and 5'AMP for maximal activity. Similar to the submandibular TPST, the enzyme from saliva also required MnCl(2) for its activity. Maximum TPST activity was observed at 20 mM MnCl(2). The enzyme from saliva was immunoprecipitated and purified by immunoaffinity column using anti-TPST antibody. Affinity purified salivary TPST showed a single band of 50-54 kDa. This study is the first report characterizing a tyrosylprotein sulfotransferase in a secretory fluid.
To compare the expression difference of transforming growth factor beta1 (TGF-beta1), matrix metalloproteinases1,7,9 (MMP-1,7,9 ) and tissue inhibitors of matrix metalloproteinases-1 (TIMP-1) protein among the chronic rhinosinusitis tissues, nasal polyps tissues and normal nasal mucosa tissues.
To investigate the clinical implication and the expression of CD44v6 and MMP-9 protein in laryngeal squamous cell carcinoma (LSCC).
Hepatic stellate cells (HSC) play an essential role in fibrogenesis. Many stimuli cause HSC to activate, lose their Vitamin A and produce collagen. It is unclear whether Vitamin A loss causes activation, potentiates it or is simply an event in the cascade of activation changes. We determine if exogenous retinoids prevent the activation of freshly isolated rat HSC activated by plating on plastic. We also determine if retinoids: (1) reverse HSC activation; (2) maintain/restore HSC intracellular retinoid levels; (3) maintain expression of HSC nuclear receptors for retinoic acid (RAR) in HSC that are becoming activated or are chronically activated. Markers of activation in freshly isolated HSC were decreased by either retinol or retinoic acid without increases in HSC retinoid concentration. mRNA levels for RAR-alpha, RAR-beta and RAR-gamma, the nuclear receptors for retinoic acid, decreased during activation of freshly isolated HSC even with retinoid supplementation. RAR-alpha, RAR-beta and RAR-gamma mRNA and RAR-beta protein was undetectable in chronically activated HSC and remained absent after retinoic acid supplementation. Activation markers in chronically activated HSC were only slightly decreased after retinoid exposure. We conclude that exposure of HSC to extracellular retinoids diminishes some markers of activation but does not prevent HSC activation.
Progressive muscular dystrophy (PMD) is characterized by muscle fiber necrosis, regeneration, and endomysial fibrosis. Although absence of dystrophin and subsarcolemmic protein has been known as the cause of muscle fiber degeneration, pathogenesis of interstitial fibrosis is still unknown. The aim of this study was to investigate the role of connective tissue growth factor (CTGF) in PMD and its relationship with muscular fibrosis.
Cyclooxygenase (COX) is the obligate, rate-limiting enzyme for the conversion of arachidonic acid into prostaglandins, which mediate mitogenesis, apoptosis, angiogenesis, blood flow, secondary injury, and inflammation. COX is consist of 2 subtypes: COX-1 and COX-2. In recent years, there are a number of lines of evidence that COX-1 and COX-2 play a important in role brain injuries.
The purpose of this study was to evaluate the effects of the angiotensin-converting enzyme (ACE) inhibitor enalapril and diuretic indapamide on the peripheral blood pressure and the central blood pressure in Chinese patients with essential hypertension.
To investigate the protein and mRNA expression patterns of apoptosis-related genes, together with evidence of apoptosis, in relation to experimental autoimmune inner ear disease (AIED).
This study examined the effects of three different cellular stresses on oocyte maturation in meiotically arrested mouse oocytes. Cumulus-cell enclosed oocytes (CEO) or denuded oocytes (DO) from immature, eCG-primed mice were cultured for 17-18 h in dbcAMP-containing medium plus increasing concentrations of the metabolic poison, sodium arsenite, or the free radical-generating agent, menadione. Alternatively, oocytes were exposed to osmotic stress by pulsing with sorbitol and returned to control inhibitory conditions for the duration of culture. Arsenite and menadione each dose-dependently induced germinal vesicle breakdown (GVB) in both DO and CEO. DO, but not CEO, pulsed for 60 min with 500 mM sorbitol were stimulated to resume maturation. The lack of effect in CEO suggests that the cumulus cells may be playing a protective role in osmotic stress-induced GVB. The AMP-activated protein kinase (PRKA; formerly known as AMPK) inhibitors, compound C and araA, completely blocked the meiosis-stimulating effects of all the tested stresses. Western blots showed that acetyl-CoA carboxylase, an important substrate of PRKA, was phosphorylated before GVB, supporting a role for PRKA in stress-induced maturation. Together, these data show that a variety of stresses stimulate GVB in meiotically arrested mouse oocytes in vitro and suggest that this effect is mediated through activation of PRKA.
Hypertriglyceridemia and dietary lipids have been suggested to modulate the severity of alcoholic liver disease and the progression to alcoholic cirrhosis (AC). The intestinal fatty acid binding protein (IFABP) is the main transporter of dietary fatty acids into the enterocyte and has a genetic polymorphism, FABP2 A54T that has been associated with hypertriglyceridemia. We determined the frequency of the FABP2 gene polymorphism using PCR-RFLP and measured serum triglycerides, HDL, LDL, total lipids and cholesterol in 67 patients with AC and in 124 unrelated healthy individuals. Frequencies of genotypes and alleles were similar between the two groups. The healthy subjects, who were homozygous for the Thr54 genotype had significantly higher mean triglyceride serum concentrations than those homozygous for the Ala54 genotype (P<0.05). However, AC patients who were homozygous for the Thr54 genotype, had lower mean triglyceride serum concentrations (P<0.01), and had a significantly longer period of continued alcohol abuse prior to the diagnosis of liver cirrhosis compared to the AC patients homozygous for the Ala54 genotype (P<0.05). Our data suggests that the polymorphism Thr54 of the FABP2 gene is associated with a later onset of AC in the lower economic status Mexican population studied.
Capacitation of mammalian sperm, including alterations in flagellar motility, is presumably modulated by chemical signals encountered in the female reproductive tract. This work investigates signaling pathways for adenosine and catecholamine agonists that stimulate sperm kinetic activity. We show that 2-chloro-2'-deoxyadenosine and isoproterenol robustly accelerate flagellar beat frequency with EC50s near 10 and 0.05 microM, respectively. The several-fold acceleration is maximal by 60 sec. Although extracellular Ca2+ is required for agonist action on the flagellar beat, agonist treatment does not elevate sperm cytosolic [Ca2+] but does increase cAMP content. Acceleration does not require the conventional transmembrane adenylyl cyclase ADCY3, since it persists in sperm of ADCY3 knockout mice and in wild-type sperm in the presence of the inhibitors of conventional adenylyl cyclases SQ-22536, MDL-12330A, or 2', 5'-dideoxyadenosine. In contrast, the acceleration by these agents is absent in sperm that lack the predominant atypical adenylyl cyclase, SACY. Responses to these agonists are also absent in sperm from mice lacking the sperm-specific Calpha2 catalytic subunit of protein kinase A (PRKACA). Agonist responses also are strongly suppressed in wild-type sperm by the protein kinase inhibitor H-89. These results show that adenosine and catecholamine analogs activate sperm motility by mechanisms that require extracellular Ca2+, the atypical sperm adenylyl cyclase, cAMP, and protein kinase A.
To investigate the role of nuclear encoded genes in mitochondrial function during oocyte maturation and early embryogenesis we examined the expression pattern and function of the cytochrome oxidase (Cox) subunits, Cox5a, 5b, and 6b1 during oocyte maturation and early embryo development. Transcription of Cox5a, 5b, or 6b1 was observed in oocytes and during early development; their expression levels were abundant in mature oocytes (MII) and zygotes (1C), and lowest at the 2-cell stage (2C), gradually increasing from 4-cell to blastocyst stage. Immunocytochemical studies revealed that COX5A, 5B, or 6B1 proteins were expressed in all blastomeres of the blastocyst. Silencing of mRNA expression by RNA interference (siRNA) did not inhibit oocyte maturation or developmental events up to the morula and blastocyst stages, but disrupted mitochondrial distribution. Significantly higher apoptosis and lower cell numbers were observed in siRNA-treated blastocysts. Real time RT-PCR revealed that silencing of Cox5a, 5b, or 6b1 did not alter mRNA levels of Bcl-xL (Bcl2l1), but increased transcription levels of proapoptotic genes, Bax and caspase 3 (Casp3). Furthermore, mRNA and protein levels of E-cadherin (CDH1) were decreased in siRNA microinjected blastocysts. These results suggest that gene expression of the Cox subunits, Cox5a, 5b, and 6b1 is not required for embryo developmental events up to the blastocyst stage. The loss of these genes leads to mitochondrial dysfunction that results in apoptosis of the blastocyst stage embryos.
To study the effect of heat shock protein 70 (HSP70) on activation of nuclear factor-KappaB (NF-KappaB) and degradation of inhibitor KappaB-alpha protein (I-Kappa B alpha) in brain edema induced by Pertussis bacilli infection in rats.
To investigate changes and functions of glucocorticoid (GR) and heat shock protein 70 (HSP70) at the level of cellular receptor in liver in hemorrhagic shock after trauma.
Macrophages are essential in cleaning up apoptotic debris during follicular atresia. However, the key factors of this process are still unclear. In the present study, we evaluated CD44 mRNA, CD44 protein, and CD44 antigen glycosylation on macrophages during follicular atresia in the pig. Atresia was classified into five stages: stage I, healthy follicles; stage II, early atretic follicles having apoptotic granulosa cells with an unclear basement membrane; stage III, progressing atretic follicles having apoptotic granulosa cells completely diffused from the basement membrane; stage IV, late atretic follicles with increasing lysosomal activity; and stage V, disintegrated atretic follicles having collapsed theca cells and strong lysosomal activity. Immunohistological analysis showed that macrophages expressing CD44 invaded the inside of stage III follicles, accompanied by a collapse of basement membrane. Semiquantitative RT-PCR showed that only mRNA of the CD44 standard isoform (CD44s) was present in inner cells of follicles, and not any CD44 variant isoform (CD44v) mRNAs. The amount of CD44s mRNA was increased at stage III. Western blot and lectin blot analyses showed that CD44 was markedly expressed at stage III and glycosylated with polylactosamine at the same time. After macrophages invaded atretic follicles at stages III-V, the CD44 expressed on macrophages was glycosylated with polylactosamine. The lysosomal activity began to increase at stage IV, and reached the highest level at stage V. Increased CD44s protein and posttranslational modification of CD44 with polylactosamine on macrophages from stage III could be involved in the cleaning up apoptotic granulosa cells.
Previously we reported that testicular germ cells undergo FAS-mediated apoptosis after exposure of mice to the Sertoli cell toxicant mono-(2-ethylhexyl) phthalate (MEHP) and that this process is partially dependent on the TRP53 protein (p53). Recent reports have suggested that TRP53 may influence the ubiquitinylation and consequent proteosomal degradation of a negative regulator of FAS, CFLAR (L) (c-FLIP [L]), in human colon cancer cells. To further characterize the relationship between CFLAR and TRP53, we used the transformed germ cell line GC-2spd (ts), which harbors a temperature-sensitive Trp53 mutation that allows for TRP53 activation at 32 degrees C. We report here that GC-2 cells expressed a 10-fold increase in basal cell membrane FAS levels and an increased sensitivity to FAS agonistic antibody (JO2)-triggered apoptosis only when they were maintained at the permissive TRP53 temperature. After JO2 exposure, CFLAR (L) protein levels were enhanced only at the nonpermissive TRP53 temperature (37 degrees C) while real-time PCR results indicated an absence of Cflar (L) mRNA changes in GC-2 cells regardless of the temperature. Furthermore, transfection of GC-2 cells at 37 degrees C with siRNA against Cflar resulted in reduction of CFLAR (L) protein levels and increased sensitivity to JO2-mediated apoptosis. The CFLAR (L) protein was also more strongly ubiquitinylated in response to JO2 treatment at the permissive TRP53 temperature. Taken together, these data suggest that the TRP53 protein influences the sensitivity of GC-2 cells to undergo FAS-mediated apoptosis by modulating the expression of FAS on their cell membranes and subsequently influencing the degradation of the antiapoptotic protein CFLAR (L).
To observe effects of acupuncture and electroacupuncture on antioxidant enzyme and Ca2+(-) ATPase activities and Ca2+ content in mitochondria of skeletal muscle cells in rats of acute swimming exercise and explore the mechanism of acupuncture in increasing sport ability.
Understanding the composition of gene lists that result from high-throughput experiments requires elaborate processing of gene annotation lists. In this article we present GOAT (Gene Ontology Analysis Tool), a tool based on the statistical software 'R' for analysing Gene Ontologytrade mark (GO) term enrichment in gene lists. Given a gene list, GOAT calculates the enrichment and statistical significance of every GO term and generates graphical presentations of significantly enriched terms. GOAT works for any organism with a genome-scale GO annotation and allows easy updates of ontologies and annotations.
We report the self-assembly and subsequent oligomerization of organic molecules based on terthiophenes bearing a terminal alkyne moiety. Molecules with thioacetate and phosphonic acid functional groups were synthesized, enabling molecular self-assembly on metal (Au and Pd) and metal oxide [Al(2)O(3), HfO(2), and indium tin oxide (ITO)] surfaces, respectively. The molecules were assembled from solution and then oligomerized using either 2,5-norbornadiene-rhodium(I) chloride dimer or UV light. UV-vis and infrared absorption spectroscopies and electrochemical techniques show that the molecules assemble to form dense monolayers on the substrate surfaces and oligomerize under the action of a catalyst or UV light.
The Derjaguin-Landau-Verwey-Overbeek (DLVO) interaction forces between a chemically heterogeneous substrate and a spherical particle approaching the surface are calculated using a simple model. This model decomposes the heterogeneous substrate into nanoscale subunits and applies a pairwise summation of forces between each subunit and the approaching particle to determine the net DLVO force. This approach leads to a three-dimensional description of the forces arising from the chemical heterogeneity of the surface. In this three-dimensional realm, we observe the emergence of a substantial lateral force at specific separation distances from the substrate. More specifically, the lateral forces become significantly larger than the normal forces at separation distances between those of the repulsive barrier and the secondary minimum of the DLVO interaction energy curve. These lateral forces are most pronounced at high electrolyte concentrations, particularly at biological salt concentrations of approximately 0.1 M. Furthermore, the lateral forces are found to be significantly higher when the particle is near the edge of a heterogeneous region of the substrate. On the basis of the evidence of this study, and depending on the characteristics of the system, both the physical roughness and the chemical heterogeneity of a surface can significantly affect how a particle will interact with it.
In this study, we perform molecular dynamics simulations of adhesive contact and friction between alkylsilane Si(OH)(3)(CX(2))(10)CX(3) and alkoxylsilane Si(OH)(2)(CX(2))(10)CX(3) (where X = H or F) self-assembled monolayers (SAMs) on an amorphous silica substrate. The alkylsilane SAMs are primarily hydrogen-bonded or physisorbed to the surface. The alkoxylsilane SAMs are covalently bonded or chemisorbed to the surface. Previously, we studied the chemisorbed systems. In this work, we study the physisorbed systems and compare the tribological properties with the chemisorbed systems. Furthermore, we examine how water at the interface of the SAMs and substrate affects the tribological properties of the physisorbed systems. When less than a third of a monolayer is present, very little difference in the microscopic friction coefficient mu or shear stresses is observed. For increasing amounts of water, the values of mu and the shear stresses decrease; this effect is somewhat more pronounced for fluorocarbon alkylsilane SAMs than for the hydrocarbon SAMs. The observed decrease in friction is a consequence of a slip plane that occurs in the water as the amount of water is increased. We studied the frictional behavior using relative shear velocities ranging from v = 2 cm/s to 2 m/s. Similar to previously reported results for alkoxylsilane SAMs, the values of the measured stress and mu for the alkylsilane SAM systems decrease monotonically with v.
Photoinitiated chemical vapor deposition (piCVD) is an evolutionary CVD technique for depositing polymeric thin films in one step without using any solvents. The technique requires no pre- or post-treatment and uses a volatile photoinitiator to initiate free-radical polymerization of gaseous monomers under UV irradiation. Glycidyl methacrylate (GMA) was used as a test monomer for its ability to undergo free-radical polymerization, and 2,2'-azobis(2-methylpropane) (ABMP) was used as the photoinitiator, as it is known to produce radicals when excited by photons. GMA and ABMP vapors were fed into a vacuum chamber in which film growth was observed on a substrate exposed to UV irradiation. The resulting poly(glycidyl methacrylate) (PGMA) thin films were comprised of linear chains and had high structural resemblance to conventionally polymerized PGMA, as shown by the high solubility in tetrahydrofuran and the infrared and X-ray photoelectron spectroscopy measurements. The introduction of ABMP into the vacuum chamber significantly increased growth rates. The maximum growth rate achieved was approximately 140 nm/min and represents a 7-fold enhancement over the case without ABMP. The molecular weight was found to increase with increasing monomer-to-initiator (M/I) feed ratio, and the polydispersity indexes (PDIs) of the samples were between 1.8 and 2.2, lower than the values obtained in conventional batch polymerization but in agreement with the theoretical expressions developed for low-conversion solution-phase polymerization, which are applicable to continuous processes such as piCVD. Molecular-weight distributions can be narrowed by filtering out wavelengths shorter than 300 nm, which induce branching and/or cross-linking. The strong dependence of the molecular weight on the M/I ratio, the rate enhancement due to the use of a radical photoinitiator, the good agreement between the experimental, and the theoretical PDIs provide evidence of a free-radical mechanism in piCVD. The clear films obtained in this work had number-average molecular weights between 12 500 and 97 000 g/mol. The similarities in growth conditions, growth rates, and molecular weights between the initiated CVD, a previously reported thermal process able to synthesize a wide range of polymers, and the piCVD of PGMA suggest that piCVD can also be used to produce those polymers and potentially others whose monomers undergo free-radical mechanisms. This paper serves as an introduction to the technique by demonstrating piCVD's ability in synthesizing high-molecular-weight PGMA thin films with narrow molecular-weight distributions from vapors of GMA and ABMP in a single, dry step under UV irradiation.
Substrate hydration is demonstrated to be crucial to film quality during self-assembled (SA) film deposition of tridecafluoro-1,1,2,2,-tetrahydrooctyltrichlorosilane (FOTS) from the vapor phase. The surface hydration was studied by thermogravimetric analysis, and a model was developed to predict the conditions necessary to desorb all of the water adsorbed on a fused silica surface without significantly altering the concentration of the surface hydroxyl groups. The nature of the SA film was investigated as a function of the degree of rehydration of the dehydrated silica surface. The wettability and microstructure of the SA films were examined by water contact angle, ellipsometry, X-ray photoelectron spectroscopy, and atomic force microscopy. There is an optimum degree of substrate hydration, on the order of 1-1.2 monolayers of adsorbed water, required to produce a dense, durable and uniform FOTS film with high water repellency and a smooth surface.
Hydrophilic modification of ultrafiltration membranes was achieved through blending of Pluronic F127 with poly(ether sulfone) (PES). The chemical composition and morphology changes of the membrane surface were confirmed by water contact angle, X-ray photoelectron spectroscopy, scanning electron microscopy, and protein adsorption measurements. The decreased static water contact angle with an increase in the Pluronic F127 content indicated an increase of surface hydrophilicity. XPS analysis revealed enrichment of PEO segments of Pluronic F127 at the membrane surface. The apparent protein adsorption amount decreased significantly from 56.2 to 0 microg/cm(2) when the Pluronic F127 content varied from 0% to 10.5%, which indicated that the blend membrane had an excellent ability to resist protein adsorption. The ultrafiltration experiments revealed that the Pluronic F127 content had little influence on the protein rejection ratio and pure water flux. Most importantly, at a high Pluronic F127 content membrane fouling, especially irreversible fouling, has been remarkably reduced. The flux recoveries of blend membranes reached as high as 90% after periodic cleaning in three cycles.
To characterize the sites on the protein surface that are involved in the adsorption to silica nanoparticles and the subsequent rearrangements of the protein/nanoparticle interaction, a novel approach has been used. After incubation of protein with silica nanoparticles for 2 or 16 h, the protein was cleaved with trypsin and the peptide fragments were analyzed with mass spectrometry. The nanoparticle surface area was in 16-fold excess over available protein surface to minimize the probability that the initial binding would be affected by other protein molecules. When the fragment patterns obtained in the presence and absence of silica nanoparticles were compared, we were able to characterize the protein fragments that interact with the surface. This approach has allowed us to identify the initial binding sites on the protein structure and the rearrangement of the binding sites that occur upon prolonged incubation with the surface.
We report a simple modification of poly(dimethylsiloxane) (PDMS) surfaces with poly(ethylene glycol) (PEG) through the adsorption of a graft copolymer, poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) from aqueous solution. In this approach, the PDMS surface was treated with oxygen plasma, followed by immersion into aqueous solution containing PLL-g-PEG copolymers. Due to the hydroxyl/carboxylic groups generated on the PDMS surface after oxygen plasma, the polycationic PLL backbone is attracted to the negatively charged surface and PEG side chains exhibit an extended structure. The PEG/aqueous interface generated in this way revealed a near-perfect resistance to nonspecific protein adsorption as monitored by means of optical waveguide lightmode spectroscopy (OWLS) and fluorescence microscopy.
Cell-cell adhesion is a dynamic process requiring recruitment, binding, and reorganization of signaling proteins in the plane of the plasma membrane. Here, we describe a new system for investigating how this lateral mobility influences cadherin-based cell signaling. This model is based on tethering of a GPI-modified E-cadherin protein (hEFG) to a supported lipid bilayer. In this report, membrane microfluidics and micropatterning techniques are used to adopt this tethered protein system for studies with the anchorage-dependent cells. As directly formed from proteoliposomes, hEFG exhibits a diffusion coefficient of 0.6 +/- 0.3 microm(2)/s and mobile fraction of 30-60%. Lateral structuring of the supported lipid bilayer is used to isolate mobile proteins from this mixed mobile/immobile population, and should be widely applicable to other proteins. MCF-7 cells seeded onto hEFG-containing bilayers recognize and cluster this protein, but do not exhibit cell spreading required for survival. By micropatterning small anchors into the supported lipid bilayer, we have achieved cell spreading across the bilayer surface and concurrent interaction with mobile hEFG protein. Together, these techniques will allow more detailed analysis of the cellular dynamics involved in cadherin-dependent adhesion events.
Signaling mechanisms coordinating uterine angiogenesis and tissue remodeling during decidualization are not completely understood. Prostanoid signaling is thought to play a functionally important role in each of these events. In the present study, we demonstrate that the subfamily of G-protein-coupled receptors that binds and becomes activated by the terminal signaling lipid in the sphingolipid pathway, sphingosine-1-phosphate (S1P), were expressed during uterine decidualization. Three of the five known S1P receptors, termed endothelial differentiation genes (Edg; Edg1, Edg3, and Edg5) were upregulated in the uterine deciduum from Day of Pregnancy (DOP) 4.5 to 7.5, while Edg6 and Edg8 expression remained unchanged. Consistent with angiogenesis in general during decidualization, we believe EDG1 and EDG5 to be regulated by the embryo because no microvascular expression for these receptors was observed in oil-induced deciduomas. Observed expression of EDG1 and EDG5 showed a similar expression pattern to that previously reported for prostaglandin-endoperoxide synthase 2 (PTGS2), transitioning from the sublumenal stromal compartment in the antimesometrial pole (DOP 5) to the microvasculature of the mesometrial pole (DOP 7). Furthermore, these two receptors colocalized with PTGS2 at three additional sites at the maternal:fetal interface throughout pregnancy. Treatment of cultured predecidualized stromal cells with S1P resulted in upregulation of Ptgs2 mRNA and PTGS2 protein, but not the downstream enzyme prostacyclin synthase. These combined results suggest the existence of a link between the sphingolipid and prostanoid signaling pathways in uterine physiology, and that, based on their expression pattern, S1P receptors function to coordinate uterine mesometrial angiogenesis during the implantation phase of early gestation.
Mammalian immature oocytes contain large nuclei referred to as germinal vesicles (GVs). The translocation of maturation/M-phase promoting factor (MPF) into GVs just before the activation of MPF has been reported in several species. To examine whether the GV is required for MPF activation in mammalian oocytes, porcine immature oocytes were enucleated and their MPF activity and CCNB (also known as cyclin B) levels were investigated. The activation of MPF at the start of maturation was detected at normal levels in enucleated oocytes, whereas reactivation to induce the second meiosis was not observed. Although protein synthesis was found to be normal both qualitatively and quantitatively, even in the absence of the nucleus, CCNB1 did not sufficiently accumulate in the enucleated oocytes. The defects in the enucleated oocytes were reversed by the injection of GV material into the enucleated oocytes. Furthermore, the inhibition of CCNB1 degradation revealed drastic accumulation of CCNB1, indicating active synthesis of CCNB1 in enucleated oocytes. The mitogen-activated protein kinase cascade remained unaffected by enucleation. These results indicate that GV is not required for the activation of MPF during the first meiosis, but that it is required for the second meiosis because of its promotion of CCNB1 accumulation.
Interferon-gamma (IFNG) induces apoptotic cell death in bovine luteal cells, but the pathway(s) involved in this process are not well defined. Evidence supporting the involvement of an IFNG-inducible enzymatic pathway that degrades tryptophan in IFNG-induced death of bovine luteal cells is presented in this study. The IFNG-inducible enzyme indoleamine 2,3-dioxygenase (INDO) catalyzes the first step in a metabolic pathway that degrades tryptophan. In the first experiment, RT-PCR revealed the presence of INDO mRNA in luteal cells treated with IFNG, but not in untreated cells. To determine whether INDO participates in IFNG-induced death of bovine luteal cells, an experiment was performed to test the effect of 1-methyl-D-tryptophan (1-MT), an inhibitor of INDO, on IFNG-induced DNA fragmentation in luteal cells. Single-cell gel electrophoresis and microscopic image analysis revealed that 1-MT inhibited DNA fragmentation induced by IFNG. To determine whether supplementation of cell cultures with additional tryptophan could also protect luteal cells from IFNG-induced DNA fragmentation, luteal cells were cultured in the presence of IFNG, and L-tryptophan was added to cultures to achieve final concentrations that were 5-, 10-, or 25-fold higher than the concentration of L-tryptophan found in nonsupplemented culture medium. Supplementation of IFNG-treated luteal cell cultures with elevated concentrations of tryptophan also prevented IFNG-induced DNA fragmentation. We conclude that INDO participates in IFNG-induced death of bovine luteal cells, through a mechanism that involves degradation of tryptophan, thereby reducing tryptophan concentrations to a point insufficient to meet luteal cells needs.
To explore the factors influencing the differentiation of fibroblasts into chondrocyte phenotype induced by a growth factor, cartilage-derived morphogenetic protein 1 (CDMP1).
To investigate the effects of mitogen-activated protein kinase phosphatase-1 (MKP-1) on the interaction between hypoxia-inducible factor (HIF)-1alpha and coactivator p300.
To investigate the relationship between the polymorphism of microsomal triglyceride transfer protein gene (MTP-493G/T) and the level of serum lipids in Guangxi Heiyi Zhuang population.
To investigate the effect of protein kinase C (PKC) on the activity of hexokinase (HK) in rat peritoneal mesothelium cell (PMC) and the functional consequences in glucose uptake.
To explore the effect of multidrug resistance-associated protein (MRP) antisense RNA mediated by recombinant adenoviruses on reversal of the multidrug resistance in human drug-resistant hepatocellular carcinoma (HCC) cells.
A novel chemiluminescence method for the determination of uric acid based on its inhibitory effect on the catalysis of Co2+ for the oxidation of luminol by H2O2 is described. The proposed method is simple, very sensitive and inexpensive. The decrease of cheniluminescence intensity is linearly correlated with uric acid concentration. The calibration graph is linear over the range of 1.0 x 10(-10) - 7.0 x 10(-6) mol x L(-1) and the detection limit is 1.1 x 10(-11) mol x L(-1). The relative standard deviation (RSD) for 5.0 x 10(-8) is 1.9% (n=4). The method has been successfully applied to the determination of uric acid in human urine and serum samples.
This paper introduces the principle and method with which the model about the quantitative analysis of Fourier transformation near infrared (NIR) spectroscopy by MAXR regression procedure can be established. In this way, the authors have selected the wave length information by Matlab language design programming in order to establish the quantitative analysis models with near infrared spectroscopy. Taking sixty-six wheat samples as experiment materials, quantitative analysis models to determine protein content are established with thirty-three samples. The relative coefficient are 0.977 1 and 0.976 5 respectively and the standard error are 0.335 and 0.340 between the predication result of the two models which include respectively two or three wave length information and Kjeldahl's value for the protein content of the another thirty-three wheat samples. When selecting the wave length information, the MAXR regression procedure can establish the optimum regression models which contain 1 or 2...or k wavelength information respectively. MAXR regression procedure is a useful method when selecting the optimum wavelength information because of its shorter computation time, and the method not only can carefully select the essential wavelength information to establish NIR spectroscopy quantitative analysis models of resisting multicollinearity information disturbance, but also to establish the work for selecting optimum wavelength information which can direct to design the special NIR analysis instrument for analyzing specific component in the special samples.
This article studied on the food safety of HPT protein (Hygromycin B Phosphotransferase), a kind of plant selectable marker gene expression product, which mainly included the quantity and quality detection of HPT in the SCK transgenic rice plants, the possible dietary intakes and its stability in the simulative digestive system.
Neonatal thymus graft and thymus calf extract (TME) in vivo treatment exert similar corrective actions on different mouse age-related alterations. The aim of the present paper is to investigate whether a vegetal extract, wheat sprout extract (WESPRE), could mimic the thymus action on recovering age-related alterations and if this extract can cure an age-associated pathology, the cataract in dogs. Present experiments were carried out by using WESPRE and TME in vivo in old mice to check their ability to recover the altered DNA synthesis in hepatocyte primary cultures. Old mice treated with WESPRE and TME showed a recovery of hepatocyte DNA synthesis levels when compared with the old untreated ones. The increase of DNA and protein contents observed in aged animals is reduced by WESPRE treatments to levels observed in young mice hepatocytes. We measured also WESPRE phosphorylation activity by endogenous kinase: it was from 10 to 40 times higher with respect to wheat seeds. Old dogs were orally treated for a month and the lens opacity analysed before and after the treatment. Results showed a reduction from 25 to 40% of lens opacity. The efficacy of wheat sprouts in the recovery of age-related alterations and in treating age-associated pathologies could be due to the contemporary presence of small regulatory acid peptides, a remarkable level of highly energetic phosphoric radicals and antioxidant molecules, peculiarities that may be, to some extent, related to the aging process regulation.
A 3204-bp full-length cDNA of bovine NALP9 was cloned and its genomic organization was analyzed. The 2988-bp open reading frame covers 9 exons and encodes a deduced protein of 996 amino acids containing Pyrin, Nacht and leucine-rich repeat domains like the human NALP gene family members. Mapping with the WGRH5000 panel and fluorescence in situ hybridization assigned NALP9 in close vicinity to BM2078 (LOD score 25.71; distance 0.0 cR5000) on bovine chromosome 18, BTA18q25-q26, within a previously identified QTL region for reproductive traits flanked by the bovine marker BM2078 and TGLA227. BAC contig analysis revealed that NALP9, NALP8, and NALP5 map in this QTL region. Temporospatial expression of these members of the NALP gene family was monitored. Among the adult tissues examined, transcripts of NALP8 and NALP9 were detected exclusively in testis and ovary, whereas transcripts of the NALP5 gene are limited to the ovary. The transcripts of NALP9, NALP8, and NALP5 were detected in oocytes before and after in vitro maturation and with a gradual decline from 2-cell to 8-cell stage, suggesting no reactivation at the time of bovine maternal to embryonic transition. Assignment to a QTL region for reproductive traits and preferential expression of NALP9, NALP8, and NALP5 in oocyte, germinal lineage, and gonad cells may suggest their functional relevance to reproduction and possible contribution to phenotypic variation.
Intestinal tract, which produces more than fifty kinds of gut peptides, is regarded as the largest endocrine organ. With regard to the gut peptides, a number of studies were focused on their structure, function and the roles in some diseases. The changes in output or distribution of gut peptides in the intestinal tract during development have been largely unknown. This study was aimed to investigate the changes of somatostatin (SST) and somatostatin receptor 2 (SSTR2) in small intestinal and hepatic tissues during the development of macaque. The tissue samples of small intestine, liver or blood samples from peripheral and portal vein of 4 macaques in 6-month fetus, 2-day neonate, 45-day neonate and adult were obtained after anesthetization. The concentrations of SST in blood or tissues of macaques were measured by radioimmunoassay. The distributions of SST in small intestinal or hepatic tissues were visualized by immunohistochemical staining. The expression of SSTR2 was detected by in situ hybridization. SST concentration of intestinal tissue in 6-month-old macaque was (27.3+/-16.6) ng /mg protein and light positive staining of SST was localized in mucosal crypts but negative in muscle layer. The intestinal concentration of SST increased gradually with macaque development and reached to the peak [(120.1+/-35.3) ng /mg protein] in adult. It was significantly higher than that in fetus (P<0.01). Strong positive staining of SST was found in both mucosal crypts and myenteric nerve plexus of adult animal. SSTR2 was obviously expressed in intestinal epithelium of fetus but its expression was greatly reduced in epithelium and was shifted to mucosal crypts when grown to adult. Negative staining of SSTR2 in muscle layer of fetal or neonatal macaque turned to be positive in myenteric nerve plexus of adult. The levels of SST or SSTR2 in liver decreased gradually during development. SST concentrations of small intestinal tissue kept significantly higher than those of hepatic tissues in the macaque developing stages. SST levels of portal vein were also maintained significantly higher than those of peripheral blood in the macaque developing stages. In conclusion, the level of SST and expression of SSTR2 in mucosal crypt increased gradually with macaque development. SST from intestinal tract was quickly degraded in portal vein before entering into liver. SST positive myenteric nerve plexus was visualized only in mature macaque.
This study was designed to evaluate the role of bcl-2 transcriptional regulation induced by calmodulin I (CaM I) in pressure overload rat hypertrophic hearts. The model of hypertensive Sprague-Dawley rat was established by abdominal aortic constriction. The hearts were collected four weeks after abdominal aortic constriction. Velocity and isopyknic gradient centrifugation was employed to fractionate rat myocardial nuclei. Western blot analysis revealed a marked increase in phosphorylated cAMP response-element binding protein (pCREB) of cardiac hypertrophy group compared with that in control group (P<0.05), while the protein level of cAMP response-element binding protein (CREB) was constant (P>0.05). Immunohistochemistry results showed a significant increase of CaM I protein in cardiac hypertrophy group relative to the control group (P<0.05). Nuclear run off transcription assay displayed a significant increase in bcl-2 mRNA treated with trifluoperazne compared with non-drug treatment (P<0.05). The results obtained suggest that the transcription of bcl-2 is possibly regulated by CaM I hypertrophic rat hearts, and CREB phosphorylation seems to be a minor factor in bcl-2 transcriptional regulation.
In this paper, we studied the relationship between the prostaglandin F(2alpha) (PGF(2alpha))-induced cardiac hypertrophy and calcineurin (CaN) signal transduction pathway in vivo and in vitro. Male Sprague-Dawley rats were given a single i.p. injection with monocrotaline (MCT) (60 mg/kg) and then given orally with celecoxib (20 mg/kg) or vehicle once a day for 14 d before (from d 1 to d 14) or after (from d 15 to d 28) right ventricular hypertrophy (RVH) was formed. Body weight (BW), right ventricular weight (RV), left ventricular with septum weight (LV), as well as lung weight were determined. RVH index (RVHI=RV/LV), RV/BW, and lung weight/BW were calculated and histological changes were observed with transmission electron microscope. PGF(2alpha) level, atrial natriuretic peptide (ANP) and CaN mRNA expressions, expression of CaN and its downstream effectors, NFAT(3) and GATA(4) protein were assayed by EIA kit, RT-PCR, and Western blotting, respectively. The cardiomyocyte hypertrophy in primary culture induced by PGF(2alpha) (0.1 micromol/L) was evaluated by measuring the cell diameter, protein content, and ANP mRNA as well as CaN mRNA expressions. It was found that 14 d or 28 d after MCT was given, the RVHI, RV/BW, and lung weight/BW were significantly increased by 47%, 53% and 118%, and by 64%, 94% and 156%, respectively; at the same time PGF(2alpha) levels in RV tissue were increased by 44% and by 51% with increasing RVHI, and elevated expressions of ANP and CaN mRNA, as well as CaN, NFAT(3) and GATA(4) proteins in a positive correlation manner. Furthermore, some histological injuries were found in RV tissue. Celecoxib, a cyclooxygenase inhibitor, obviously blunted the elevation of RVHI, RV/BW, and lung weight/BW no matter it was given before or after RVH. In vitro experiments showed that 0.1 micromol/L PGF(2alpha) significantly increased the cardiomyocyte diameter and protein content, and promoted ANP and CaN mRNA expressions, which was blocked by cyclosporin A, a CaN inhibitor. Our results indicate that PGF(2alpha) may be involved in cardiac hypertrophy induced by MCT in rats through CaN signal transduction pathway.
Roscovitine is a specific inhibitor of cyclin-dependent kinases (cdks) cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35. The studies on the enzyme inhibitory properties and cellular effects of roscovitine revealed that it arrests cells in G(2)/M and G(1)/S phase, inhibits the proliferation of mammalian cells and induces cell death. However, the characteristics of cell death and exact mechanism by which this cdk inhibitor kills transformed cells are unknown. We previously investigated that the roscovitine induces apoptotic death of mitotic PC12 cells. The present study was to identify whether the roscovitine-induced death is related with the specific elements of caspases in pathway of apoptosis. The morphological data of caspase-3 immunofluorocytochemistry double staining with hoechst 33342 indicated that apoptotic nuclei were identified as nuclei with chromatin condensation and nuclear fragmentation, and that caspase-3 active p17 subunit co-existed in PC12 cells treated with roscovitine 50 micromol/L for 4 h. The number of the caspase-3 positive cells increased significantly to about 42%, as compared with the normal control (P<0.001). The data of MTT assay showed that the number of viable cells treated by roscovitine (50 micromol/L) alone for 12 h was 29.03%, of the untreated controls. Both a broad-spectrum caspase inhibitor Z-VAD-FMK (50 mumol/L) and a specific caspase-3 inhibitor Z-DEVD-FMK (100 micromol/L) increased viable PC12 cells to 45.16%, (Z-DEVD-FMK) and 58.06%, (Z-VAD-FMK), respectively, in the presence of roscovitine. Non-erythroid a-spectrin is a cytoskeleted protein that is a substrate of caspase-3 cysteine proteases. To confirm the activity of caspase-3 that produced in roscovitine (50 micromol/L for 12 h)-induced PC12 cell death, activated caspase-3 specific 120 kDa spectrin breakdown products (SBDP) were detected by Western bloting using the mouse anti-non-erythroid a-spectrin monoclonal antibody. The mean relative density of bands corresponding to caspase-3 specific SBDP levels were significantly increased in the cytosolic fractions treated with roscovitine, as compared to the normal control (P<0.001). These results indicate that caspase signals, especially caspase-3 signal are necessary for the progression of proliferating PC12 cell apoptotic death evoked by roscovintine.
The intestinal microflora of deep-sea amphipods, in enrichment culture employing starch, urea, and N-acetyl-d-glucosamine and when examined under simulated in situ conditions, exhibited growth rates and substrate conversion approximately equal to, or greater than, atmospheric controls during short-term incubation. These observations are significant since these microorganisms may play an important role in biodegradation in the deep sea.
The rates of growth and substrate uptake in a biological film continuous-flow reactor were studied. The experiments were performed with high fluid velocities to bring the reactor operation to the reaction-controlled regime, thus avoiding external diffusional resistances. The glucose uptake experiments were performed with small film thicknesses so that full substrate penetration within the entire film thickness could be obtained. In this way, the catalyst effectiveness factor was 1.0 and the observed rate was the true, or intrinsic, rate. The results of the experiments indicate that both the intrinsic rate of substrate uptake and the rate of film growth are independent of the substrate concentration remaining in the reactor (zero-order reactions). However, the value of the initial substrate concentration when the film is in the early stages of growth defines the magnitude of both the rate of uptake and growth. This effect of the initial substrate concentration follows a saturation-function pattern.
The disadvantages associated with the supplementation of noncomposted nutrients to mushroom compost at spawning were largely overcome by encapsulating microdroplets of vegetable oil within a protein coat that was denatured with formaldehyde. Increases in mushroom yield of 60% were obtained. Delayed nutrient release was indicated by prolonged stimulation of yields beyond the first few flushes.
Decomposition of C-labeled lignin to CO(2) by the lignin-decomposing fungi Phanerochaete chrysosporium and Coriolus versicolor required a growth substrate such as cellulose or glucose. Growth with lignin as sole carbon addition to an otherwise complete medium was negligible.
The mechanism responsible for an increased rate of acid production when yogurt starter cultures are grown in milk treated with lactase enzyme was investigated by studying carbohydrate utilization and acid development by a pure culture of Streptococcus thermophilus and a mixed yogurt starter culture consisting of S. thermophilus and Lactobacillus bulgaricus. In milk containing glucose, galactose, and lactose, glucose and lactose (but not free galactose) were fermented. Fermentation of lactose in control milk was accompanied by the release of free galactose, with the result that carbohydrate utilization was less efficient than in treated milk. This phenomenon also occurred when lactose was fermented by S. thermophilus in broth culture. Carbohydrate utilization by the mixed yogurt culture was more rapid when the lactose in milk was partially prehydrolyzed. Our results suggest that the more rapid acid development that took place when a mixed yogurt starter culture was grown in milk containing prehydrolyzed lactose was the result of a more rapid and efficient utilization of carbohydrate by S. thermophilus when free glucose in addition to lactose was available for fermentation. The evidence presented also suggests that uptake and utilization of glucose and lactose by S. thermophilus are different in broth and milk cultures.
Using selective media containing galactitol, over 130 Enterobacteriaceae have been isolated from paper mill process waters collected from different localities. These bacteria were extensively characterized and tested for acetylene-reducing (nitrogen-fixing) activity under anaerobic conditions. High activity was found in representatives of Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter cloacae, Erwinia herbicola, Citrobacter freundii, Citrobacter intermedius, and Escherichia coli. Under argon, nitrogenase synthesis was generally not repressed by 5 mM l-glutamate, l-aspartate, l-leucine or Casamino Acids (0.5 g/liter). In many strains, both the specific activities (nanomoles of C(2)H(4) per minute per milligram of protein) and the activities (nanomoles of C(2)H(4) per minute) had considerably declined after 24 h. In three selected strains, activity in intact cells grown under nitrogen was unaffected by the presence during assay of 10 mM l-amino acids or ammonium acetate. All of the strains examined were tolerant towards inactivation of nitrogen-fixing activity by 1.8% (vol/vol) oxygen during assay, and inactivation by up to 10% oxygen was partly reversible. Representatives of the six taxa synthesized nitrogenase in stirred aerobic cultures, though the protein concentrations attained were lower than under anaerobic conditions. It seems reasonable to suggest that under natural conditions, nitrogen fixation is able to contribute significantly to the nitrogen economy of the cells.
Group N streptococci, which have the lactose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) and phospho-beta-d-galactosidase (beta-Pgal), grew rapidly on lactose and converted more than 90% of the sugar to l-lactate. In contrast, Streptococcus lactis 7962, which does not have a beta-Pgal, grew slowly on lactose and converted only 15% of the sugar to l-lactate. With glucose and galactose, this strain had growth rates and fermentation patterns similar to those of other S. lactis strains, suggesting that the rapid and homolactic fermentation of lactose that is characteristic of group N streptococci is dependent upon a functional PEP-dependent PTS and the presence of beta-Pgal. Seventeen strains of group N streptococci were examined for the activator specificities of pyruvate kinase and lactate dehydrogenase. The properties of each enzyme from all the strains, including S. lactis 7962, were similar. Pyruvate kinase had a broad activator specificity, whereas activation of lactate dehydrogenase was specific for ketohexose diphosphate. All intermediates of lactose metabolism from the hexose phosphates to the triose phosphates activated pyruvate kinase. No activation was obtained with adenosine 5'-monophosphate. K and Mg were required for pyruvate kinase activity but could be replaced by NH(4) and Mn, respectively. Lactate dehydrogenase was activated equally by fructose-1,6-diphosphate and tagatose-1,6-diphosphate, the activation characteristics being pH dependent. The roles of pyruvate kinase and lactate dehydrogenase in the regulation of lactose fermentation by group N streptococci are discussed.
Tyrosine phenol-lyase was purified 32-fold from Aeromonas phenologenes ATCC 29063, the organism that produces phenol in refrigerated haddock. The purification procedure included ammonium sulfate fractionation, protamine sulfate treatment, and column chromatography with Sephadex G-200, diethyl-aminoethyl-cellulose, and hydroxyapatite. The enzyme was found to be thermally inactivated at temperatures above 40 degrees C. The optimum pH of the enzyme was found to be pH 8.5. The Michaelis constants for l-tyrosine and pyridoxal phosphate were 2.3 x 10 M and 3.2 x 10 M, respectively. The molecular weight of tyrosine phenol-lyase was found by gel filtration and electrophoresis to be approximately 380,000.
The nutritional characteristics of an alga (Microcystis sp.) that occurs naturally in a Guatemalan lake were determined. The sun-dried material proved to have a high protein content (55.6%) and to be a possible good source of calcium and phosphorus (1, 169.1 and 633.4 mg/100 mg, respectively). Amino acid analysis showed that total sulfur amino acids were the most deficient ones, giving a protein score of 42 to the material. The in vitro protein digestibility of the material was 69.5%. Biological trials demonstrated that when the material was offered as the only protein source, very low consumption and a high mortality rate were obtained whether or not the diet was supplemented with 0.4% dl-methionine. However, when the material supplied 25% of the total protein of a corn-algae diet, the protein quality of the cereal was significantly improved (P < 0.05).
The cellulolytic activity of rumen contents was assayed by measuring losses in weight and tensile strength of cotton yarn incubated in rumen contents in the presence of dietary additives (barley, tallow) and at different pH values. The addition of barley depressed cellulolysis and the titer of filter paper-degrading bacteria only if the pH was allowed to fall. Lowering the pH from 7.0 to 6.0 by addition of HCl almost completely inhibited attack of cotton and greatly reduced the titer of filter paper-degrading bacteria. The layering of tallow on cotton inhibited attack of cotton but did not decrease the titer of filter paper-degrading bacteria. The results are discussed with special reference to the importance of the study of cellulosic substrates, which require a known enzyme or mixture of enzymes for attack.
Asparaginase was found in the soluble fraction of cells of Azotobacter vinelandii, and its activity remained the same during growth of the organism in a nitrogen-free medium. The specific activity and the yield of A. vinelandii increased twofold in the presence of ammonium sulfate. Within limits, the temperature (30 to 37 degrees C) and pH (6.5 to 8.0) of the medium showed little effect on the levels of enzyme activity. The enzyme was purified to near homogeneity by standard methods of enzyme purification, including affinity chromatography, and had optimum activity at pH 8.6 and 48 degrees C. The approximate molecular weight was 84,000. The apparent K(m) value for the substrate was 1.1 x 10 M. Metal ions or sulfhydryl reagents were not required for enzyme activity. Cu, Zn, and Hg showed concentration-dependent inhibition, whereas amino and keto acids had no effect on the enzyme activity. Asparaginase was stable when incubated with rat serum and ascites fluid. The enzyme had no effect on the membrane of sheep erythrocytes and did not inhibit the incorporation of radioactive precursors into deoxyribonucleic acid, ribonucleic acid, and protein in Yoshida ascites sarcoma cells. Asparaginase activity was not detected in the tumor cells.
A stable variant of Methylomonas methanolica, with a higher temperature optimum for growth, was obtained after mutagenic treatment and selection. The mutant strain M13V has an optimum growth temperature of 35 to 37 degrees C and a maximum at 43 degrees C, as compared with 30 and 40 degrees C for the wild strain. Strain M13V and M. methanolica have similar basic characteristics and cell composition. An extracellular polysaccharide is produced by both strains, but this property is more pronounced in strain M13V. In strain M13V the production is favored by high temperature, low growth rate, and oxygen limitation. In continuous culture of strain M13V, the polysaccharide production was partly growth associated and partly independent of the growth rate. The extracellular polysaccharide acted as a flocculating agent. A relationship between polysaccharide concentration and sedimentation rate was found. Biomass production from strain M13V is most effective at 35 degrees C with respect to both growth rate and substrate utilization. It was found that the yield coefficient for methanol was independent of the dilution rate, whereas the yield coefficient for oxygen increased and the production coefficient for carbon dioxide decreased at increasing dilution rates. These results are discussed in connection with the polysaccharide production.
A device for sampling at depths of up to 6,000 m is described in which 3 liters of seawater is concentrated over a Nucleopore filter to about 13 ml and retrieved under in situ pressure and temperature. Subsamples can be withdrawn into transfer units that are equipped with individual gas accumulators for preventing loss of pressure during prolonged periods of storage. Transfer of samples or sample portions into sterile medium contained in pre-pressurized incubation vessels and continued subsampling therefrom permit time course experiments for the study of natural populations of deep-sea microorganisms in the absence of decompression. A test experiment with a water sample from a depth of 2,600 m supplemented with radioactively labeled Casamino Acids showed reduced rates of substrate incorporation and respiration as compared with data from a decompressed control. The barotolerance observed in this study was characterized by reduced, rather than equal, activities recorded at elevated pressures as compared with 1-atm controls.
Cyst walls of Acanthamoeba rhysodes, A. palestinensis, A. castellanii, and one other strain of Acanthamoeba contain 36 to 45% protein and 20 to 34% carbohydrate. More than half of the protein in the walls of A. palestinensis, A. castellanii and Acanthamoeba sp. is accessible to and hydrolyzed by protease, and 67 to 69% of the carbohydrate of A. palestinensis and A. rhysodes walls is hydrolyzed by cellulase. The extent of hydrolysis of walls of the other amoebae by these enzymes is appreciably less, and chitinase and beta-1,3-glucanase have no detectable effect. Protease solubilizes 10% or less of the weight of intact cysts, and no solubilization is observed with cellulase. Walls of A. palestinensis are extensively degraded in soil, the activity is less with A. rhysodes, and little attack on the other amoebae occurs. When added to soil, the protozoa excyst and grow for short periods, the trophozoites then die, and chiefly cysts persist thereafter.
Methanogenic bacteria, which are presently identified on the basis of cell morphology and substrate conversion to CH(4), can be differentiated from nonmethanogens and identified in pure or mixed culture on the basis of their autofluorescence under ultraviolet illumination.
The isofunctional enzymes of catechol 1,2-dioxygenase from species of Acinetobacter, Pseudomonas, Nocardia, Alcaligenes, and Corynebacterium oxidize 3-methylcatechol according to both the intradiol and extradiol cleavage patterns. However, the enzyme preparations from Brevibacterium and Arthrobacter have only the intradiol cleavage activity. Comparison of substrate specificity among these isofunctional dioxygenases shows striking differences in the oxidation of 3-methylcatechol, 4-methylcatechol and pyrogallol.
Indonesian tapé ketan is a fermentation in which a mold, Amylomyces rouxii Calmette (Chlamydomucor oryzae Went and Prinsen Geerligs), in combination with one or more yeasts such as Endomycopsis burtonii converts steamed rice to a sweet-sour, slightly alcoholic paste. A study was made to determine the biochemical changes that occur in the substrate during fermentation. It was found that the product was ready for consumption after fermentation at 30 degrees C for 36 to 48 h. A. rouxii used about 30% of the total rice solids, resulting in a crude protein of 12% in 96 h, whereas the combination of the mold with E. burtonii reduced total solids by 50% in 192 h, causing crude protein to increase to 16.5%. Soluble solids increased from 5 to about 67% in 36 h and decreased to 12% at 192 h with A. rouxii alone, whereas soluble solids fell to about 8% at 192 h in the fermentation with both the mold and the yeast. The mold, by itself, reduced the starch content of the rice from 78 to 10% in 48 h and to less than 2% in 144 h. The mold plus yeast reduced the starch content to about 18% in 48 h; however the "starch" content did not fall below 6% even at 192 h, presumably because the yeast was producing glycogen, which was determined along with the residual starch. With both the mold and the mold plus yeast fermentations, reducing sugars increased from less than 1% to approximately 5% in 24 h and reached maximum concentration, 16 to 17%, between 36 and 48 h. A. rouxii by itself produced a maximum of about 5.6% (vol/vol) ethanol at 96 h. The highest concentration of ethanol (8%, vol/vol) was produced by the mold plus E. burtonii at 144 h. The mold by itself reduced the starting pH from 6.3 to about 4.0 in 48 h. The combination of the mold and yeast reduced the pH to 4.1 in 144 h. The mold increased total acidity to approximately 6.2 meq of H per 100 ml, and the combination of the mold and yeast increased the total acidity to 7.8 meq of H per 100 ml in 192 h. At 48 h there was practically no difference in the volatile acidity (0.20) for the combined fermentation compared with 0.26 meq of H per 100 ml for the mold fermentation. The mold and at least one species of yeast were required to develop the rich aroma and flavor of typical Indonesian tapé.
Earlier studies have shown that the activity of the estuarine detrital microflora measured by various enzyme activities, muramic acid and adenosine 5'-triphosphate (ATP) content, heterotrophic potentials, and respiratory activities correlates with the incorporation of C and P into the microbial lipids. In this study, these lipids were reproducibly fractionated into neutral lipid, glycolipid, and phospholipid classes. Distinct differences between the active microflora of oak leaves, sweet gum leaves, and pine needles were evidenced both in the rate of lipid synthesis and in the proportions of neutral lipids, glycolipids, and phospholipids. Successional changes in the microflora of leaves incubated in a semitropical estuary, previously suggested by ATP-to-muramic acid ratios and scanning electron micrography, were reflected in changes in the proportions of C in major lipid classes when analyzed from the same type of detritus. Short incubation times with C gave lipid compositions rich in phospholipids that are typical for the faster-growing bacterial populations; longer incubation with C gave lipid compositions richer in neutral and glycolipids, more characteristic of slower-growing eukaryotes or morphologically more complex prokaryotes. The metabolism of the lipids of the estuarine detrital microflora was examined by a pulse-chase experiment with C. Glycolipids lost C at a rate equal to the loss of C of the slow component of muramic acid. Individual phospholipids lost C from their backbone glycerol esters at different rates.
Baked-bean waste was found to be a favorable substrate for amylase production by Aspergillus foetidus NRRL 337. Under optimum conditions, the yields of alpha-amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3) were 47 and 226 U, respectively, per ml of the waste fermented.
Laboratory experiments were conducted to validate theoretical predictions describing a dialysis continuous process for the fermentation of whey lactose to ammonium lactate, in which the fermentor contents are poised at a constant pH by adding ammonia solution and dialyzed through a membrane against water. Dried sweet-cheese whey was rehydrated to contain 230 mg of lactose per ml, supplemented with 8 mg of yeast extract per ml, charged into a 5-liter fermentor without sterilization, adjusted in pH (5.3) and temperature (44 degrees C), and inoculated with Lactobacillus bulgaricus. The fermentor and dialysate circuits were connected, and steady-state conditions were established. A series of such conditions was managed nonaseptically for 94 days to study the process and to demonstrate efficiency and productivity. As time progressed, the fermentation remained homofermentative and increased in conversion efficiency, although membrane fouling necessitated dialyzer cleaning about every 4 weeks. With a retention time of 19 h, 97% of the substrate was converted into products. Relative to nondialysis continuous or batch processes for the fermentation, the dialysis continuous process enabled the use of more concentrated substrate, was more efficient in the rate of substrate conversion, and additionally produced a second effluent of less concentrated but purer ammonium lactate.
Multiple biochemical assays of microbial mass and activities were applied to the estuarine detrital microbiota colonizing morphologically similar polyvinyl chloride needles and needles from slash pine (Pinus elliottii). Biodegradable pine needles consistently showed 2- to 10-fold higher values of extractable adenosine 5'-triphosphate, rates of oxygen utilization, activities of alkaline phosphatase and phosphodiesterase, and the mucopeptide cell wall component muramic acid than did the polyvinyl chloride needles, during a 14-week incubation in a semitropical estuary. The higher activities by the microbiota of the biodegradable substrate correlated with estimates of the microbial density from scanning electron microscopy. The microbial community associated with the nondegradable substrate showed minimal activity of beta-d-galactosidase, beta-d-glucosidase, and alpha-d-mannosidase in contrast to the biota of the degradable substrate, which showed 10- to 100-fold higher activities of these glycoesterases. These enzymes logically could be involved in catabolism of the carbohydrate polymers of the detritus. Assuming equivalent rates of predation, a surface that is also a utilizable substrate supports a three- to fivefold more active microbial population.
Growth rates and final cell yields of a polychlorinated biphenyl (PCB)-sensitive pseudomonad isolated from the open ocean were reduced in a dose-dependent manner by 10 to 100 mug of Aroclor 1254 per liter, a commercial mixture of PCB isomers added to its culture medium. Effects on growth rates were detected within 1 h (approximately one doubling time) of treatment. By 4 h posttreatment, the amounts of deoxyribonucleic acid and ribonucleic acid per cell in exponentially growing populations treated with sublethal doses of Aroclor were detectably lower than in appropriate controls. Corresponding cell protein values were slightly higher than in controls. Selective degradation of cell proteins or nucleic acids was not detected in cells whose growth was totally suppressed for 4 h by PCBs. Cells whose growth rate was inhibited 20 to 50% by Aroclor synthesized protein at normal rates for periods in excess of 5 h from the time the chlorinated hydrocarbons were added. In contrast, rates per cell of adenine uptake and adenine incorporation into deoxyribonucleic acid and total nucleic acids by the cells treated with PCBs were significantly lower than in control cells. Intracellular adenine pools of cells whose growth was inhibited to 20% of the control rate by PCBs were 30% smaller and appeared to require a longer interval to equilibrate than those of untreated cells. This may indicate impaired transport and/or efflux of this nucleic acid precursor through the membrane of affected cells. Inhibition of nucleic acid synthesis in this sensitive bacterium by PCBs could explain the observed inhibitory effects of the chlorinated hydrocarbons on its growth.
The fluorescence of a fermentation culture was studied for its application as an estimator of biomass concentration. The measurement was obtained by irradiating the culture with ultraviolet light (366 nm) through a glass window and detecting fluorescent light at the window surface at 460 nm. It was estimated that over one-half of the fluorescent material was intercellular reduced nicotinamide adenine dinucleotide, with the remainder being reduced nicotinamide adenine dinucleotide phosphate and other unidentified intercellular and extracellular fluorophores. The culture fluorescence was found to be a function of biomass concentration, together with environmental factors, which presumably act at the cellular metabolic level to modify intercellular reduced nicotinamide adenine dinucleotide pools (e.g., dissolved oxygen tension, energy substrate concentration, and inhibitors). When these environmental conditions were controlled, a linear relationship was obtained between the log of the biomass concentration and the log of the fluorescence. Under these conditions, this relationship has considerable potential as a method to provide real-time biomass concentration estimates for process control and optimization since the fluorescence data is obtained on line. When environmental conditions are variable, the fluorescence data may be a sensitive index of overall culture activity because of its dependence on intercellular reduced nicotinamide adenine dinucleotide reserves and metabolic rates. This index may provide information about the period of maximum specific productivity for a specific microbial product.
Filtrates from microbial heterotrophic activity assay experiments in which tritiated glucose was used as a radioactively labeled substrate were collected, and their radioactivities were determined. These filtrates were subsequently evaporated to dryness to remove tritiated water generated by metabolism of the labeled glucose; the residue was suspended to original volume with distilled water, and the tritium levels were again assayed. In the water samples assayed, the amount of tritium label retained by the microorganism was about 75% of that respired.
The treatment of a hardwood sawdust with 1% NaOH solution at 121 degrees C dissolved 19.7% of the dry matter, mainly hemicellulose and lignin. Fermentation of the treated solids by Chaetomium cellulolyticum for 48 h gave a product containing 12.5% crude protein (total N x 6.25) on a dry weight basis. The in vitro rumen digestibility of the 48-h fermentation product was 30%, compared to 24% for the alkali-treated but unfermented sawdust. Growth was independent of sawdust particle size in the range 40 to 100 mesh. Fermentation of the pretreatment liquor gave a product containing up to 50% crude protein (dry weight basis) with an in vitro rumen digestibility of 65 to 76%. Approximately 6.7 g of crude protein was obtained from the treated solids and 2.2 g from the pretreatment liquor per 100 g of sawdust treated. The product from the pretreatment liquor fermentation has potential as a high-protein animal feed supplement but could not be produced economically without an outlet for the relatively indigestible product from the solids fermentation. Growth on the pretreatment liquor was strongly pH dependent; there was a considerable increase in the lag phase when the pH was lowered from 7.5 to 5.2. This effect appears to be due to an inhibitor whose toxicity is reduced at high pH.
It is now possible to obtain accurate total counts of the bacteria of natural waters with the use of acridine orange staining and epifluorescence microscopy. This approach can be coupled to highly sensitive measurements of heterotrophic activity using radioisotopes. To accomplish this, three variations of a "specific activity index" are suggested, based on different approaches to measuring heterotrophic activity with radiolabeled organic solutes. The denominator of each index is the direct count of bacteria from a given natural sample. Three numerators are presented, each of which has been shown to vary directly with heterotrophic bacterial activity: V(max), turnover rate, and direct uptake (at high substrate concentrations). Each approach is illustrated with data from estuarine and coastal waters of northeastern Massachusetts. The data show major differences in specific activity that accompany such habitat differences as distances within or offshore from an estuary and vertical location in the water column. These and other data suggest that specific activity is a valid indicator of the physiological state and metabolic role of the bacteria. Some evidence is presented in support of the hypothesis that the natural bacteria are adapted to conditions of nutrient starvation by becoming "dormant," existing for an unknown period of time in a reversible physiological state that reflects the availability of organic nutrients.
Five species of rumen bacteria with overlapping substrate fermentative capabilities were tested for substrate preferences and evidence of catabolite regulatory mechanisms. All five bacteria showed evidence of some type of catabolite regulatory mechanism. In the six-substrate test system that was used, utilization of every substrate was inhibited by another substrate in at least one of the bacteria. Inhibited versus noninhibited substrate data suggest that the five bacteria have different strategies of substrate utilization and thus occupy separate niches in the rumen. The significance of these observations to understanding the rumen ecosystem is discussed.
The kinetic parameters K(m), V(max), T(t) (turnover time), and v (natural velocity) were determined for H(2) and acetate conversion to methane by Wintergreen Lake sediment, using short-term (a few hours) methods and incubation temperatures of 10 to 14 degrees C. Estimates of the Michaelis-Menten constant, K(m), for both the consumption of hydrogen and the conversion of hydrogen to methane by sediment microflora averaged about 0.024 mumol g of dry sediment. The maximal velocity, V(max), averaged 4.8 mumol of H(2) g h for hydrogen consumption and 0.64 mumol of CH(4) g h for the conversion of hydrogen to methane during the winter. Estimated natural rates of hydrogen consumption and hydrogen conversion to methane could be calculated from the Michaelis-Menten equation and estimates of K(m), V(max), and the in situ dissolved-hydrogen concentration. These results indicate that methane may not be the only fate of hydrogen in the sediment. Among several potential hydrogen donors tested, only formate stimulated the rate of sediment methanogenesis. Formate conversion to methane was so rapid that an accurate estimate of kinetic parameters was not possible. Kinetic experiments using [2-C]acetate and sediments collected in the summer indicated that acetate was being converted to methane at or near the maximal rate. A minimum natural rate of acetate conversion to methane was estimated to be about 110 nmol of CH(4) g h, which was 66% of the V(max) (163 nmol of CH(4) g h). A 15-min preincubation of sediment with 5.0 x 10 atm of hydrogen had a pronounced effect on the kinetic parameters for the conversion of acetate to methane. The acetate pool size, expressed as the term K(m) + S(n) (S(n) is in situ substrate concentration), decreased by 37% and T(t) decreased by 43%. The V(max) remained relatively constant. A preincubation with hydrogen also caused a 37% decrease in the amount of labeled carbon dioxide produced from the metabolism of [U-C]valine by sediment heterotrophs.