Restriction mapping of a chlorobenzoate degradative plasmid and molecular cloning of the degradative genes

The genes for the degradation of 3-chlorobenzoic acid ( 3Cba ) are present in a 110-kb plasmid pAC27 . A circular map is established using the restriction endonucleases EcoRI, HindIII and Bg/II. The map is derived from the results obtained by partial restriction digestion, complete single and double restriction digestion and finally confirmed with hybridization of the digested fragments using different purified fragments as probes. The 3Cba degradative genes are found to be clustered in one region of the map (EcoRI fragment A) as judged by molecular cloning with a broad host range vector pLAFRI . A portion of the 3Cba degradative gene cluster appears to undergo ready recombination with the chromosome, even in a recA host, suggesting the probable transposable nature of such gene cluster.     

Macrophage activation downregulates the degradative capacity of the phagosome

The phagosome is key to most macrophage functions. It is the site of degradation of particulate material, of bacterial killing and the generation of peptides for antigen presentation. Despite its role at the fulcrum of the innate and acquired immune systems, little is known about the physiology of this organelle in activated macrophages. In this study, we utilize fluorometric techniques to characterize functional alterations in the lumenal environment of the maturing phagosome following stimulation of macrophages with interferon-gamma and/or lipopolysaccharide. In addition to modulating the kinetics of phagosomal acidification, activation results in a phagosome with diminished hydrolytic activities that varies markedly with the activation status of the cell. Differential levels of proteolytic, lipolytic and beta-galactosidase activities were observed in the phagosome but not in the total lysosomal extract, indicating selective delivery of enzymes to the developing phagosome. Despite the suppression of hydrolytic activities observed in early phagosomes, late phagosomes exhibit an enhanced and protracted accumulation of lysosomal cargo. The data are consistent with limiting proteolysis in the early phagosome to maximize epitope generation and antigen presentation while sequestering the degradative capacity in the late phagolysosome.     

Subcellular localization of the starch degradative and biosynthetic enzymes of spinach leaves

The subcellular localization of the starch biosynthetic and degradative enzymes of spinach leaves was carried out by measuring the distribution of the enzymes in a crude chloroplast pellet and soluble protein fraction, and by the separation on sucrose density gradients of intact organelles, chloroplasts, peroxisomes, and mitochondria of a protoplast lysate. ADP-Glucose pyrophosphorylase, starch synthase, and starch-branching enzymes are quantitatively associated with the chloroplasts. The starch degradative enzymes amylase, R-enzyme (debranching activity), phosphorylase, and D-enzyme (transglycosylase) are observed both in the chloroplast and soluble protein fractions, the bulk of the degradative enzyme activities reside in the latter fraction. Chromatography of a chloroplast extract on diethylaminoethyl-cellulose resolves the R- and D-enzymes from amylase and phosphorylase activities although the two latter enzyme activities coeluted. The digestion pattern of amylase with amylopectin as a substrate indicates an endolytic activity but displays properties unlike the typical α-amylase as isolated from endosperm tissue.     

Detection of pheromone biosynthetic and degradative enzymes in vitro

Highly sensitive, luminescent assays have been developed to measure enzyme activities involved in the metabolism of a major class of insect pheromones which includes fatty aldehydes, alcohols, and their acetate esters. These assays have been applied to measure the in vitro biosynthesis and degradation of the sex pheromone (trans:cis-11-tetradecenal, 96:4) of the eastern spruce budworm, Choristoneura fumiferana. Three activities were detected on analyses of extracts of the female moths: (a) an esterase that hydrolyzes both the cis and trans isomers of 11-tetradecenyl acetate, (b) an oxidase that converts fatty alcohols to aldehydes in the presence of O2, and (c) an NAD-dependent aldehyde dehydrogenase. The coupled luminescent response of bacterial luciferase to long chain aldehydes was used to measure rates of reaction as low as 0.1 pmol/min since only low amounts of material can be analyzed. Specific activities of these enzymes were higher in the pheromone producing gland than in other parts of the moth, implicating these enzymes, and the oxidase in particular, in the pathway of pheromone biosynthesis. The pathway was supported in vivo by demonstrating that topical application of 3H-labeled tetradecanyl acetate onto the insect gland resulted in the formation of [3H]tetradecanol and [3H]tetradecanoic acid, thus providing evidence that all three enzymes were functional in the living insects.     

Distribution in the environment of degradative capacitiesfor gasoline attenuation

A methodology allowing the detailed assessment ofthe capacities of microflorae to degrade gasoline inaerobic conditions has been developed. It consisted inthe determination of the degradation of a gasolinemodel mixture in liquid cultures in optimalconditions. The gasoline model mixture contained 23representative hydrocarbons of gasoline (GM23). Thekinetics and extent of biodegradation were evaluatedby continuous overall monitoring of CO₂production and final chromatographic analysis (usuallyafter about 30 days) of the consumption of eachhydrocarbon. The methodology was used with soil andwater samples from polluted and non polluted sites.The experimentation aimed at assessing thedistribution of the degradative capacities in theenvironment and the prospects for natural attenuationof gasoline. Nine microflorae were tested. The intrinsicbiodegradability (existence of mechanisms ofbiodegradation) appeared total for GM23 as shown bythe results obtained with several microflorae. Thedegradative capacities of microflorae from nonpolluted samples were high (total degradation rates atleast 85%). Incomplete degradation was observedessentially for trimethylalkanes(2,2,4-trimethylpentane and 2,3,4-trimethylpentane)and for cyclohexane. In several cases, samples frompolluted sites exhibited more extensive degradativecapacities, with total degradation of all hydrocarbonsbeing observed for three out of the six samples.     

Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

Phagocytes engulf unwanted particles into phagosomes that then fuse with lysosomes to degrade the enclosed particles. Ultimately, phagosomes must be recycled to help recover membrane resources that were consumed during phagocytosis and phagosome maturation, a process referred to as “phagosome resolution.” Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission. Here, we show that bacteria-containing phagolysosomes in macrophages undergo fragmentation through vesicle budding, tubulation, and constriction. Phagosome fragmentation requires cargo degradation, the actin and microtubule cytoskeletons, and clathrin. We provide evidence that lysosome reformation occurs during phagosome resolution since the majority of phagosome-derived vesicles displayed lysosomal properties. Importantly, we show that clathrin-dependent phagosome resolution is important to maintain the degradative capacity of macrophages challenged with two waves of phagocytosis. Overall, our work suggests that phagosome resolution contributes to lysosome recovery and to maintaining the degradative power of macrophages to handle multiple waves of phagocytosis.     

CIN85 regulates ubiquitination and degradative endosomal sorting of the EGF receptor

CIN85 has been demonstrated to interact with a number of proteins involved in endocytosis and intracellular sorting. However, the exact functional role of CIN85 in endocytosis remains unclear. We have investigated whether CIN85 plays a role in EGF-induced EGF receptor (EGFR) internalization, as previously suggested, or whether CIN85 is rather involved in endosomal sorting of the EGFR. When over-expressing a dominant negative interfering CIN85 mutant consisting of three SH3 domains only, we found that internalization of EGF was inhibited. However, when knocking down CIN85 by RNAi, the EGF–EGFR uptake appeared similar to in control cells. Furthermore, in CIN85 depleted cells, EGF-induced ubiquitination of the EGFR was decreased, and degradation of EGF–EGFR complexes was delayed. Our data further demonstrated that depletion of CIN85 increased the recycling of EGF, suggesting that CIN85 plays a role in endosomal sorting of the ubiquitinated EGFR. Our data also demonstrated that CIN85 was constitutively associated with Hrs, and this strengthens the hypothesis of a functional role of CIN85 in endosomal EGFR sorting.     

Physico-chemical and degradative properties of in-planta re-structured potato starch

Starch re-structured directly in potato tubers by antisense suppression of starch branching enzyme (SBE), granule bound starch synthase (GBSS) or glucan water dikinase (GWD) genes was studied with the aim at disclosing the effects on resulting physico-chemical and enzyme degradative properties. The starches were selected to provide a combined system with specific and extensive alterations in amylose and covalently esterified glucose-6-phosphate (G6P) contents. As an effect of the altered chemical composition of the starches their hydrothermal characteristics varied significantly. Despite of the extreme alterations in phosphate content, the amylose content had a major affect on swelling power, enthalpy for starch gelatinization and pasting parameters as assessed by Rapid Visco Analysis (RVA). However, a combined influence of the starch phosphate and long glucan chains as represented by high amylose or long amylopectin chain length was indicated by their positive correlation to the final viscosity and set back (RVA) demonstrating the formation of a highly hydrated and gel-forming system during re-structuring of the starch pastes. Clear inverse correlations between glucoamylase-catalyzed digestibility and amylopectin chain length and starch phosphate and lack of such correlation with amylose content indicates a combined structuring role of the phosphate groups and amylopectin chains on the starch glucan matrix.     

一株苯酚降解菌的鉴定及其降解特性

【目的】鉴定从某化工厂附近土样中分离到的一株耐高浓度苯酚的菌株T10,通过优化菌株的培养条件提高菌株对苯酚的降解率。【方法】根据菌株的形态、生理生化鉴定及16S rDNA测序分析确定其种属,以液体摇瓶培养菌株T10对苯酚的降解率为指标,对菌株的生长条件进行优化。【结果】菌株T10属恶臭假单胞菌(Pseudomonas putida)。添加葡萄糖、蛋白胨能有效缩短T10菌的生长周期,并使苯酚的降解率提高1.7倍。在菌体初始接种浓度为10%、温度为30°C、转速为180 r/min条件下,对初始苯酚浓度、pH和装液量的响应面优化结果如下:初始苯酚浓度3 000 mg/L、pH 7.5和装液量80 mL/250 mL,苯酚去除率最高可达到87.56%。【结论】T10菌能够耐受较高浓度的含酚废水,并且对苯酚有较强的降解能力,为下一步利用生物法处理含酚废水提供科学依据。     

Properties affecting transfer and expression of degradative plasmids for the purpose of bioremediation

Biodegradation - Plasmids, circular DNA that exist and replicate outside of the host chromosome, have been important in the spread of non-essential genes as well as the rapid evolution of...     

Rab4 affects both recycling and degradative endosomal trafficking

The small GTPases Rab4, Rab5 and Rab7 are endosomal proteins which play important roles in the regulation of various stages of endosomal trafficking. Rab4 and Rab5 have both been localized to early endosomes and have been shown to control recycling and endosomal fusion, respectively. Rab7, a marker of the late endosomal compartment, is involved in the regulation of the late endocytic pathway. Here, we compare the role of Rab4, Rab5 and Rab7 in early and late endosomal trafficking in HeLa cells monitoring ligand uptake, recycling and degradation. Expression of the Rab4 dominant negative mutant (Rab4AS22N) leads to a significant reduction in both recycling and degradation while, as expected, Rab7 mutants exclusively affect epidermal growth factor (EGF) and low density lipoprotein degradation. As also expected, expression of the dominant negative Rab5 mutant perturbs internalization kinetics and affects both recycling and degradation. Expression of Rab4WT and dominant positive mutant (Rab4AQ67L) changes dramatically the morphology of the transferrin compartment leading to the formation of membrane tubules. These transferrin positive tubules display swellings (varicosities) some of which are positive for early endosomal antigen-1 and contain EGF. We propose that the Rab4GTPase is important for the function of the early sorting endosomal compartment, affecting trafficking along both recycling and degradative pathways.     

Evolution of a degradative bacterial consortium during the enrichment of naphtha solvent

A microbial mixed culture able to degrade naphtha solvent, a model of hydrocarbon aromatic mixture, was isolated from a hydrocarbon-polluted soil. Composition of the population was monitored by phenotypic and molecular methods applied on soil DNA, on whole enrichment culture DNA, and on 85 isolated strains. Strains were characterized for their 16S rDNA restriction profiles and for their random amplified polymorphic DNA profiles. Catabolic capabilities were monitored by phenotypic traits and by PCR assays for the presence of the catabolic genes methyl mono-oxygenase ( xylA, M), catechol 2,3 dioxygenase (xylE) and toluene dioxygenase (todC1) of TOL and TOD pathways. Different haplotypes belonging to Pseudomonas putida, Ps. aureofaciens and Ps. aeruginosa were found to degrade aromatic compounds and naphtha solvent. The intrinsic catabolic activity of the microbial population of the polluted site was detected by PCR amplification of the xylE gene directly from soil DNA.     

Adhesive and degradative properties of human placental cytotrophoblast cells in vitro

Human fetal development depends on the embryo rapidly gaining access to the maternal circulation. The trophoblast cells that form the fetal portion of the human placenta have solved this problem by transiently exhibiting certain tumor-like properties. Thus, during early pregnancy fetal cytotrophoblast cells invade the uterus and its arterial network. This process peaks during the twelfth week of pregnancy and declines rapidly thereafter, suggesting that the highly specialized, invasive behavior of the cytotrophoblast cells is closely regulated. Since little is known about the actual mechanisms involved, we developed an isolation procedure for cytotrophoblasts from placentas of different gestational ages to study their adhesive and invasive properties in vitro. Cytotrophoblasts isolated from first, second, and third trimester human placentas were plated on the basement membrane-like extracellular matrix produced by the PF HR9 teratocarcinoma cell line. Cells from all trimesters expressed the calcium-dependent cell adhesion molecule cell-CAM 120/80 (E-cadherin) which, in the placenta, is specific for cytotrophoblasts. However, only the first trimester cytotrophoblast cells degraded the matrices on which they were cultured, leaving large gaps in the basement membrane substrates and releasing low molecular mass 3H-labeled matrix components into the medium. No similar degradative activity was observed when second or third trimester cytotrophoblast cells, first trimester human placental fibroblasts, or the human choriocarcinoma cell lines BeWo and JAR were cultured on radiolabeled matrices. To begin to understand the biochemical basis of this degradative behavior, the substrate gel technique was used to analyze the cell-associated and secreted proteinase activities expressed by early, mid, and late gestation cytotrophoblasts. Several gelatin-degrading proteinases were uniquely expressed by early gestation, invasive cytotrophoblasts, and all these activities could be abolished by inhibitors of metalloproteinases. By early second trimester, the time when cytotrophoblast invasion rapidly diminishes in vivo, the proteinase pattern of the cytotrophoblasts was identical to that of term, noninvasive cells. These results are the first evidence suggesting that specialized, temporally regulated metalloproteinases are involved in trophoblast invasion of the uterus. Since the cytotrophoblasts from first trimester and later gestation placentas maintain for several days the temporally regulated degradative behavior displayed in vivo, the short-term cytotrophoblast outgrowth culture system described here should be useful in studying some of the early events in human placen     

A degradative detour for mutant TP53

Accumulation of mutant TP53 proteins in cancer cells has been recognized as an important factor that promotes cancer progression and metastasis. Thus, strategies that promote the degradation of mutant TP53 might be beneficial for the treatment of cancers. In a recent issue of Genes & Development, we demonstrated that blocking macroautophagy under nutritional stress condition leads to the degradation of mutant TP53 through activating the chaperone-mediated autophagy (CMA) pathway in nonproliferating cancer cells. We propose CMA as a new degradative mechanism for mutant TP53 and the possibility of activating CMA as a new treatment for cancers with mutant TP53.     

Localisation of degradative enzymes in white-rot decay of lignocellulose

The use of immunogold-cytochemical labelling techniques in electron microscopy of wood infected by basidiomycete fungi has assisted in the elucidation of the localisation of enzymes which degrade lignocellulose. The use of specific immunocytochemical techniques is discussed with respect to the authenticity and accuracy of the methods, the use of adequate controls in the gold-labelling procedure, and the immunospecificity of the antibodies.Localisation of the lignin-degrading enzymes, lignin-peroxidase and laccase, has shown that these enzymes do not bind to wood cell walls unless the process of decay has already commenced. Similarly localisation of cellulases Endoglucanase II (EGII) and Cellobiohydrolase I (CBHI) has shown that these enzymes only bind to exposed ends of cellulose fibrils and to partially degraded areas of the wood cell wall. -Glucosidase is always immobilised within the extracellular polysaccharide layer surrounding fungal hyphae.This review postulates that there is regulation of the release sequence of these lignocellulolytic enzymes defining the spatial arrangement between the hyphae and the wood cell wall. This hypothesis is presented diagrammatically.     

联苯降解细菌的分离及其降解质粒某些特性的研究

:通过选择性富集培养 ,从芳烃污染的土壤中分离得到 16株以联苯作唯一碳源生长的细菌。初步鉴定 ,3株均为假单孢菌属(Psedomonas) 。都含有大小 2个质粒。质粒不稳定 ,易丢失。经苯甲酸钠和口丫啶橙的消除实验 ,结果表明 ,随着质粒丢失 ,菌株利用Bp生长的能力也丧失。酶活性分析表明 ,菌株只有邻苯二酚 2 ,3—氧化酶活性 ,没有邻苯二酚1 ,2—氧化酶活性。菌株对联苯的降解率 :bp2 菌为 93%、bp2 3菌为 81%、bp2 4 菌为 99%。降解过程中产生λmax331nm和λmax4 34nm2种中间代谢物。