Biochemical characterization of a factor released by macrophages.

Peritoneal macrophages from the mouse are able to release a factor, which inhibits the incorporation of [³H]thymidine into the DNA of lymphocytes. The biochemical characterization of this factor reveals that this factor is thymidine, a degradation product of cells dying in culture.     

一株苯系物降解真菌筛选及降解特性

采用逐量分批驯化的方法以污水处理厂污泥作为菌源,苯、甲苯、二甲苯为唯一碳源,驯化、分离、筛选能够有效降解苯系物的真菌,命名为B1。采用单因素以及正交实验方法并对真菌降解环境影响因素及降解效率进行了测定和研究。结果表明:真菌B1对苯系物降解的最佳条件为C:N=5:1,pH5,温度30℃,菌种接种量为5.5ml(50ml培养基)。采用GC对初始液相浓度0~90mg/L范围内的苯系物降解效果进行测定,未发现苯系物对真菌降解活性产生抑制作用。真菌对苯系物的降解效率为:甲苯>苯>二甲苯,最高降解效率分别达到87.39%,85.21%,81.47%。混合物降解效果略高于单一底物的降解效果。     

褐腐真菌产生的羟基自由基HO˙对纤维素作用的研究*

由褐腐真菌的典型菌株——密粘褶菌Gloeophyllum trabeum的胞外培养液中分离纯化得到一能还原Fe3+,产生羟基自由基HO˙的多肽组分(称作Gt因子)。 采用HO˙特异性的抑制剂硫脲,对Gt因子产生的HO˙在纤维素降解中的作用进行了对照研究,结果表明Gt因子及其产生的HO˙在纤维素降解中起着重要的作用,为褐腐菌HO˙氧化降解纤维素机制假说的确立提供了一些依据。     

Protein metabolism and survival of rat hepatocytes in early culture

Rat hepatocytes, cultured in a serum- and hormone-free medium on a substratum of adsorbed fibronectin, are in negative protein balance throughout the first 24 h of culture. The rate of protein loss at all times equals the difference between the high rate of protein degradation and the low rate of protein synthesis. A continuous decline in the rate of protein degradation gradually reduces the loss of protein, probably the result of medium conditioning as well as progressive culture deterioration. Inhibition of lysosomal protein degradation by amino acids and the protease inhibitor, leupeptin, reduces the protein loss considerably, but does not prevent cell death. The protein balance per se thus does not seem to be the limiting factor for the survival of cultured hepatocytes.     

Enhancement of Pyrroloquinoline Quinone Production and Polyvinyl Alcohol Degradation in Mixed Continuous Cultures of Pseudomonas putida VM15A and Pseudomonas sp. Strain VM15C with Mixed Carbon Sources

In a mixed continuous culture of Pseudomonas putida VM15A and Pseudomonas sp. strain VM15C with polyvinyl alcohol (PVA) as the sole source of carbon, growth of the PVA-degrading bacterium VM15C and, hence, PVA degradation were limited by the growth factor, pyrroloquinoline quinone, produced by VM15A. Feeding of a carbon source for VM15A, ethanol, with PVA enhanced pyrroloquinoline quinone production and caused increases in the VM15C population and PVA degradation in a mixed continuous culture. There was an optimum range for PVA degradation of the ethanol concentration, although pyrroloquinoline quinone concentrations in continuous mixed cultures increased with increasing ethanol concentration.     

Effect of pyocyanin on a crude-oil-degrading microbial community

Pseudomonas aeruginosa is an n-alkane degrader that is frequently isolated from petroleum-contaminated sites and produces factors that enhance its competitiveness and survival in many environments. In this study, one such factor, pyocyanin, has been detected in an oil-degrading culture containing P. aeruginosa and is a redox-active compound capable of inhibiting microbial growth. To examine the effects of pyocyanin further, an oil-degrading culture was grown with and without 9.5 microM pyocyanin and microbial community structure and oil degradation were monitored for 50 days. Denaturing gradient gel electrophoresis (DGGE) analysis of cultures revealed a decrease in the microbial community diversity in the pyocyanin-amended cultures compared to that of the unamended cultures. Two members of the microbial community in pure culture exhibited intermediate and high sensitivities to pyocyanin corresponding to intermediate and low levels of activity for the antioxidant enzymes catalase and superoxide dismutase, respectively. Another member of the community that remained constant in the DGGE gels over the 50-day culture incubation period exhibited no sensitivity to pyocyanin, corresponding to a high level of catalase and superoxide dismutase when examined in pure culture. Pyocyanin also affected the overall degradation of the crude oil. At 50 days, the culture without pyocyanin had decreased polycyclic aromatic hydrocarbons compared to the pyocyanin-amended culture, with a specific reduction in the degradation of dibenzothiophenes, naphthalenes, and C(29) and C(30) hopanes. This study demonstrated that pyocyanin influenced the diversity of the microbial community and suggests the importance of understanding how interspecies interactions influence the degradation capability of a microbial community.     

Mixed Continuous Cultures of Polyvinyl Alcohol-Utilizing Symbionts Pseudomonas putida VM15A and Pseudomonas sp. Strain VM15C

Stable mixed continuous cultures of Pseudomonas sp. strain VM15C and Pseudomonas putida VM15A, the former of which produced a polyvinyl alcohol (PVA)-degrading enzyme and the latter of which produced an essential growth factor for PVA utilization by strain VM15C, were established with PVA as the sole source of carbon and energy with chemostat cultivation. A high extent of PVA degradation was achieved at dilution rates of less than 0.030/h. The predominant strain in the cultures was the primary metabolizer of PVA, strain VM15C. The growth supporter, strain VM15A, existed as a minor population, although its population was maintained at an almost constant level throughout a dilution region in which the VM15C population decreased markedly as the dilution rate was raised. A crude growth factor which was prepared from a culture supernatant of strain VM15A and increased the specific growth rate of strain VM15C with PVA in an axenic batch culture was also effective for enhancing the VM15C population and PVA degradation in the mixed continuous culture at a high dilution rate (0.064/h). This indicated that the growth-limiting substrate for strain VM15C in the mixed continuous culture is the growth factor produced by strain VM15A.     

Function and mechanism of a low-molecular-weight peptide produced by Gloeophyllum trabeum in biodegradation of cellulose

A special low-molecular-weight peptide named Gt factor, was isolated and purified via HPLC from the culture extract of the brown-rot fungus Gloeophyllum trabeum. It had high-affinity Fe(3+)-chelating ability and could reduce Fe(3+) to Fe(2+). In the presence of O(2), it could produce hydroxyl radicals HO*. The effects of Gt factor on cellulose degradation suggested that Gt factor could disrupt inter- and intra- hydrogen bonds in cellulose chains by a HO*-involved mechanism. This resulted in depolymerization of cellulose chains, which produced more reducing and non-reducing ends, thus making cellulose accessible for further degradation. This pathway was quite different from the hydrolytic processes driven by cellulases, and Gt factor might play an important role in the early stage of cellulose depolymerization by brown-rot fungi.     

一株乙草胺降解菌的分离及其降解特性研究

【目的】分离获得一株能有效降解乙草胺的菌株,并研究其降解乙草胺的影响因素,为乙草胺生物修复提供微生物资源。【方法】通过富集培养和分离培养,从样品中筛选能以乙草胺为唯一碳氮源的菌株。通过划线培养获得单菌落,并采用革兰氏染色法和16S r RNA基因测序进行菌株的初步鉴定和系统分类。通过单因素试验研究初始乙草胺浓度、外加碳氮源对其降解乙草胺的影响,并基于正交设计进行优化。【结果】分离获得的一株菌为革兰氏阴性菌,初步确定为Pseudomonas sp.,能有效利用乙草胺进行生长。单因素试验证明在乙草胺初始浓度为10 mg/L时降解率最高;外加碳氮源能提高乙草胺降解率,其中葡萄糖和蛋白胨分别最为有效。正交设计表明在最优条件下,其对乙草胺降解率可以达到80.2%。【结论】菌株A-1能有效利用乙草胺进行生长,其降解乙草胺受多种因素影响。本研究将为利用进行该菌株进行乙草胺污染修复提供菌种资源。     

Suppression of the degradation of recombinant human apolipoprotein E by a protease inhibitor obtained from fetal bovine serum in serum-free culture

The recombinant human apolipoprotein E (Apo-E) produced by Chinese hamster ovary cells (CHO-322 cells) in serum free culture was degraded to 24K and 23K fragments that contained N-terminal amino acid. The degradation site of Apo-E to 24K fragment was between Arg180 and Leu181 and the C-terminal amino acid of 23K fragment was Gly169. In fetal bovine serum (FBS)-containing culture, the degradation was inhibited. However, in calf serum (CS) the inhibitory activity was not detected. Thus, we attempted the purification of the factor with this inhibitory activity from FBS. A protease inhibitor was purified to give a single peak from FBS by ammonium sulfate precipitation and combination of several column chromatographies. When this FBS-derived protease inhibitor (FBS-d-PI) was added to serum-free culture of CHO-322 cells, degradation of recombinant Apo-E to the 24K and 23K fragments was dose-dependently suppressed and accumulation of intact Apo-E in culture supernatant was observed. FBS-d-PI was found to be a glycoprotein with relative molecular size of 75K daltons under reducing condition, and 85K daltons under nonreducing condition by SDS-PAGE. A complex of FBS-d-PI and a cellular protease was also detected in culture supernatant by western blot analysis using mouse monoclonal antibodies against FBS-d-PI.     

Applicability of competitive and noncompetitive kinetics to the reductive dechlorination of chlorinated ethenes

Reductive dechlorination of chlorinated ethenes has typically been modeled using standard Michaelis-Menten kinetic equations, implying that each dechlorination step is catalyzed by a unique biological factor. An alternative kinetic model is based on the assumption that all steps are mediated by a single factor. These two options are considered in the context of chlorinated ethene degradation by a previously characterized anaerobic culture. Competitive kinetics afford better chi-squared and visual fits of the data set tested.     

Effect of Pyocyanin on a Crude-Oil-Degrading Microbial Community

Pseudomonas aeruginosa is an n-alkane degrader that is frequently isolated from petroleum-contaminated sites and produces factors that enhance its competitiveness and survival in many environments. In this study, one such factor, pyocyanin, has been detected in an oil-degrading culture containing P. aeruginosa and is a redox-active compound capable of inhibiting microbial growth. To examine the effects of pyocyanin further, an oil-degrading culture was grown with and without 9.5 μM pyocyanin and microbial community structure and oil degradation were monitored for 50 days. Denaturing gradient gel electrophoresis (DGGE) analysis of cultures revealed a decrease in the microbial community diversity in the pyocyanin-amended cultures compared to that of the unamended cultures. Two members of the microbial community in pure culture exhibited intermediate and high sensitivities to pyocyanin corresponding to intermediate and low levels of activity for the antioxidant enzymes catalase and superoxide dismutase, respectively. Another member of the community that remained constant in the DGGE gels over the 50-day culture incubation period exhibited no sensitivity to pyocyanin, corresponding to a high level of catalase and superoxide dismutase when examined in pure culture. Pyocyanin also affected the overall degradation of the crude oil. At 50 days, the culture without pyocyanin had decreased polycyclic aromatic hydrocarbons compared to the pyocyanin-amended culture, with a specific reduction in the degradation of dibenzothiophenes, naphthalenes, and C₂₉ and C₃₀ hopanes. This study demonstrated that pyocyanin influenced the diversity of the microbial community and suggests the importance of understanding how interspecies interactions influence the degradation capability of a microbial community.     

Localization of Polyvinyl Alcohol Oxidase Produced by a Bacterial Symbiont, Pseudomonas sp. Strain VM15C

An axenic culture of a polyvinyl alcohol (PVA)-degrading symbiont, Pseudomonas sp. strain VM15C, was established on PVA with a crude preparation of the growth factor (factor A) produced by the symbiotic partner Pseudomonas putida VM15A. An increase of factor A in the culture medium enhanced the cell-associated PVA oxidase activity as well as the growth rate, but decreased production of extracellular PVA oxidase. PVA oxidase in cells grown on PVA was present in the periplasmic space at a higher ratio than in cells grown on peptone. PVA degradation occurred rapidly with washed cells. PVA was also degraded by immobilized cells entrapped in agar gels.     

Inhibition by cycloheximide of degradation of cytochrome P-450 in primary cultures of adult rat liver parenchymal cells and in vivo

Degradation of cytochrome P-450 was studied in adult rat liver parenchymal cells in primary monolayer culture. In cells incubated in standard culture medium, the amount of cytochrome P-450 decreased at an accelerated rate relative to either the rate of degradation of total protein in the cells or the turnover of cytochrome P-450 in vivo. This change was succeeded by a spontaneous increase in the activity of haem oxygenase, an enzyme system that converts haem into bilirubin in vitro, measured in extracts from the cultured cells. This finding suggests that the rate of cytochrome P-450 breakdown may be controlled by factor(s) other than the activity of haem oxygenase. The decline in cytochrome P-450 and the subsequent increase in haem oxygenase activity was prevented by incubation of hepatocytes in medium containing an inhibitor of protein synthesis such as cycloheximide, puromycin, actinomycin D, or azaserine. The effect of cycloheximide appeared to be due to decreased breakdown of microsomal (14)C-labelled haem. By contrast, cycloheximide was without effect on the degradation of total protein, measured either in homogenates or in microsomal fractions prepared from the cultured cells. These results suggest that the conditions of cell culture stimulate selective degradation of cytochrome P-450 by a process that is inhibited by cycloheximide and hence may require protein synthesis. The findings in culture were verified in parallel studies of cytochrome P-450 degradation in vivo. After administration of bromobenzene, the degradation of the haem moiety of cytochrome P-450 was accelerated in vivo in a manner resembling that observed in cultured hepatocytes. Administration of cycloheximide to either bromobenzene-treated rats or to untreated rats decreased the degradation of the haem moiety of cytochrome P-450. However, the drug failed to affect degradation of haem not associated with cytochrome P-450, suggesting that cycloheximide is not a general inhibitor of haem oxidation in the liver. These findings confirm that the catabolism of hepatic cytochrome P-450 haem is controlled by similar cycloheximide-sensitive processes in the basal steady state in vivo, as stimulated by bromobenzene in vivo, or in hepatocytes under the conditions of cell culture. We conclude that the rate-limiting step in this process appears to require protein synthesis and precedes cleavage of the haem ring.     

Degradation of triphenyltin by a fluorescent pseudomonad

Triphenyltin (TPT)-degrading bacteria were screened by a simple technique using a post-column high-performance liquid chromatography using 3,3',4',7-tetrahydroxyflavone as a post-column reagent for determination of TPT and its metabolite, diphenyltin (DPT). An isolated strain, strain CNR15, was identified as Pseudomonas chlororaphis on the basis of its morphological and biochemical features. The incubation of strain CNR15 in a medium containing glycerol, succinate, and 130 microM TPT resulted in the rapid degradation of TPT and the accumulation of approximately 40 microM DPT as the only metabolite after 48 h. The culture supernatants of strain CNR15, grown with or without TPT, exhibited a TPT degradation activity, whereas the resting cells were not capable of degrading TPT. TPT was stoichiometrically degraded to DPT by the solid-phase extract of the culture supernatant, and benzene was detected as another degradation product. We found that the TPT degradation was catalyzed by low-molecular-mass substances (approximately 1,000 Da) in the extract, termed the TPT-degrading factor. The other fluorescent pseudomonads, P. chlororaphis ATCC 9446, Pseudomonas fluorescens ATCC 13525, and Pseudomonas aeruginosa ATCC 15692, also showed TPT degradation activity similar to strain CNR15 in the solid-phase extracts of their culture supernatants. These results suggest that the extracellular low-molecular-mass substance that is universally produced by the fluorescent pseudomonad could function as a potent catalyst to cometabolite TPT in the environment.     

携带大片段BIBAC克隆在不同农杆菌中的遗传稳定性研究

大片段克隆在农杆菌中的稳定性是利用可转化大片段载体进行农杆菌介导遗传转化的关键问题.选用插入片段分子质量分别为150kb和50kb的BIBAC克隆,测定了在3种农杆菌AGL-1,EHA105和LBA4404中的遗传稳定性.从第1、3、5次继代培养的农杆菌中抽提的质粒及酶切结果显示,由于农杆菌背景质粒的干扰,难以判断质粒的降解与否.将农杆菌质粒再转化到大肠杆菌宿主中,发现来自农杆菌AGL-1和EHA105的质粒出现了明显的降解,片段变小,而来自农杆菌LBA4404的质粒没有变化.结果表明,大片段BIBAC质粒在不同农杆菌菌株中的稳定性不同,在农杆菌LBA4404中比较稳定,适合用于遗传转化.     

Enhancement of oxidised low-density lipoprotein uptake by macrophages in response to macrophage migration inhibitory factor

We examined the expression of macrophage migration inhibitory factor (MIF) mRNA in murine macrophage cell line (RAW 264.7 cells) in response to oxidized low-density lipoprotein (oxLDL), and investigated the influence of MIF on the uptake and degradation of oxLDL by RAW 264.7 cells. MIF mRNA expression was markedly upregulated in the presence of oxLDL. Consistent with this, the MIF level of the culture medium was increased by stimulation with oxLDL in dose- and time-dependent manners. Next, we added recombinant rat MIF to the culture medium and examined its effects on the uptake of(125)I-labelled oxLDL. Pretreatment with MIF enhanced both the uptake and degradation of(125)I-oxLDL. Taken together, these results suggest that MIF released from macrophages in response to oxLDL stimulates oxLDL uptake and degradation in an autocrine and paracrine fashion, which potentially results in atherosclerosis.     

Inhibition of liver protein degradation in vivo by intensive treatment with cycloheximide.

The AA in experiments performed on male rats treated with 0,5 mCi/kg of cycloheximide have observed an inhibition of liver protein degradation and have suggested two mechanism: 1) cycloheximide prevents the acute proteolytic response induced in fibroblast culture by exposure to serum-deficient media and in rat livers after perfusion probably by inhibiting the lysosomal - autophagic system, without modifications in total activity of lysosome proteinases. 2) It may be that protein degradation rate and level of lysosomal proteinases reinterrelated; the latter probably being a factor controlling the overall cell protein turnover rate.     

Glucocorticoid regulation of chloroquine nonsensitive insulin degradation in cultured fetal rat hepatocytes

The influence of cortisol and other culture conditions on insulin degradation by the chloroquine-sensitive pathway and the chloroquine-nonsensitive pathway (CNP) was investigated in fetal rat hepatocytes during 3 days of culture. The proportions of the chloroquine nonsensitive release of 125I-insulin degradation products into the conditioned medium/h increased from the 1st to the 3rd day of culture, i.e. from 19 to 50% by cells grown in the presence of cortisol and from 17 to 82% by those grown in the absence of cortisol. Replacement of the conditioned medium with the respective fresh medium dramatically enhanced cellular insulin degradation by CNP, i.e. from 22 to 58%, and 19 to 85% in cells grown for 2 days in the presence and absence of cortisol, respectively. Thus, the conditioned medium contained some factor(s) that inhibited CNP. Therefore, we used the inhibited insulin and alpha-casein degradation by papain in vitro as an assay to investigate the nature of the putative anti-(insulin) protease. Cycloheximide completely prevented the appearance of anti-papain activity in the medium. Conditioned medium obtained from cells grown in the presence of cortisol contained about 2-fold more anti-papain activity than the medium that was obtained in the absence of the steroid. The release of anti-papain activity also declined with time from 1 to 3 days of culture and showed an inverse relationship with the magnitude of cellular insulin degradation by CNP. The inhibition of papain-mediated insulin degradation by the anti-(insulin) protease was noncompetitive. The anti-(insulin) protease was nondialyzable (up to the 10-kDa exclusion limit) and inactivated by heat treatment at 50 degrees C for 30 min. These results suggest that fetal hepatocytes synthesize and secrete a glucocorticoid-regulated heat-labile low molecular mass (less than 25 kDa) anti-(insulin) protease, which may contribute to the suppression of insulin degradation caused by the enzymes involved in CNP.     

Partial characterization of the macrophage factor that stimulates fibroblasts to produce collagenase and to degrade collagen

Rabbit bone marrow-derived macrophages in culture produce and release a soluble factor that activates collagenase secretion and collagen degradation by cultured skin fibroblasts from either rabbit, mouse or human origin. The factor is heat-labile and is inactivated by phenylglyoxal. After gel filtration, it is recovered in both an apparent high-Mr (67000-76000) and a low-Mr (9000-14000) form. Chromatography on cation exchangers suggests two molecular species with different charge properties. These characteristics are compatible with known properties of rabbit interleukin 1.