Novel transmembrane lipases of alpha/beta hydrolase fold

Processing of exogenous glycerol esters is an initial step in energy derivation for many bacterial cells. Lipid-rich environments settled by a variety of organisms exert strong evolutionary pressure for establishing enzymatic pathways involved in lipid metabolism. However, a certain number of enzymes involved in this process remain unknown since they do not share detectable sequence similarity with any known protein domains. Using distant homology detection and fold recognition we predict that bacterial transmembrane proteins belonging to the uncharacterized domain of unknown function 2319 (DUF2319) family possess the alpha/beta hydrolase fold domain together with the catalytic triad critical for hydrolysis. A detailed analysis of sequence/structure features and genomic context indicates that DUF2319 proteins may be involved in lipid metabolism. Therefore, these enzymes are likely to serve as extracellular lipases.     

PcrA/UvrD/Rep DNA helicases in bacterial genomes

Helicases, which utilize the energy liberated by the hydrolysis of nucleotides to unwind nucleic acids, are involved in many aspects of nucleic acid metabolism. Various DNA helicases from the PcrA/UvrD/Rep subfamily are essential for the survival of different pathogenic bacteria and we have recently shown that they can be inhibited with small synthetic molecules. Altogether this suggests that these enzymes are potential new drug targets. Since little is known about the presence of these enzymes in bacterial genomes, 99 bacterial genomes were analyzed in the present study. This analysis reveals which and how many of these enzymes are found in bacteria, but more important, it identifies several of these enzymes as potential drug target candidates. In addition, this work identifies several proteins, called here PURL, that have a high homology with the PcrA/UvrD/Rep proteins and that may form an additional group in this helicase subfamily.     

Assay of bacterial and fungal activity in diesel contaminated soil using a 14C-glucose utilization method

Changes in the relative metabolism of soil bacteria and fungi following contamination with diesel were assessed using a modified substrate-induced respiration (SIR) method including selective antibiotic inhibition. ¹⁴CO₂ release from radiolabelled glucose was used as an indication of population activity. In a Sandy Gley Soil with no history of contamination, the population activity shifted from 38 ± 4% (bacterial): 62 ± 4% (fungal) to 73 ± 4% (bacterial): 27 ± 4% (fungal) after treatment with diesel.     

帕尼培南/倍他米隆治疗重症感染30例临床疗效及安全性分析

目的探讨帕尼培南/倍他米隆治疗重症感染临床疗效及安全性。方法 30例临床诊断为重症感染的患者静脉应用帕尼培南/倍他米隆1.0 g/8 h,观察患者临床症状、体征转归及实验室指标变化。结果应用帕尼培南/倍他米隆治疗重症感染30例,痊愈15例,显效10例,死亡1例,总有效率达83.3%。细菌培养结果阳性16例,培养阳性率为53.3%,细菌清除率为88.8%,不良反应率为6.7%。结论帕尼培南/倍他米隆治疗重症感染有较好的临床疗效,不良反应率低。     

The functions and biosynthesis of the c-type cytochromes of bacterial respiratory chains with particular reference to autotrophs and the handling of C1 compounds

Abstract The behaviour, functions and biosynthesis of c -type cytochromes of the bacterial periplasm are reviewed. Particular emphasis is placed on cytochromes involved in (i) the metabolism of C₁ compounds and (ii) the electron transport reactions of Paracoccus denitrificans .     

Interaction of Xenorhabdus nematophilus subsp. nematophilus with the haemolymph of Galleria mellonella

The lethal dose (LD)₅₀ values and probit-mortality regression slopes of the primary and secondary forms of Xenorhabdus nematophilus subsp. nematophilus for Galleria mellonella were equal. The two bacterial forms grew at equal rates in larval serum-supplemented media. The secondary form grew less well in larval serum-supplemented media than in synthetic larval serum.The secondary bacteria adhered to the haemocytes to a greater extent than did the primary bacteria. Both types of bacteria did not produce metabolites suppressing the ability of the haemocytes to respond to Bacillus cerues.Differences were observed in the rate of clearance of the primary and secondary bacteria from and their subsequent re-entrance into the haemolymph in vivo. This appeared to be independent of bacterial metabolism. Evidence is presented showing multiplication of the primary bacteria during their association with the haemocytes.The total haemocyte counts increased during bacterial infection. All the haemocytes were killed. The rate and pattern of change of the total haemocyte counts was influenced by the form of bacteria and independent of bacterial metabolism.     

Axenic incorporation of [U-14C]palmitic acid into the phenolic glycolipid-I of Mycobacterium leprae

Abstract Incorporation of [U-¹⁴C]palmitic acid ([¹⁴C]PA) into the specific phenolic glycolipid-I (PGL-I) of freshly harvested, nude mouse-derived Mycobacterium leprae was investigated in an axenic modified Dubos medium. Incorporation was approximately linear for 10–14 days at pH 7.2, 33°C. No incorporation of radiolabeled phenol, acetate, tyrosine, phenylalanine, bicarbonate, proprionate or UDP-glucose was detected. Procedures known to remove residual host tissue did not diminish the rate of [¹⁴C]PA incorporation, indicating that bacterial metabolism was being measured. The antileprosy compounds, rifampicin and dapsone, significantly reduced incorporation of the label. The ability to quantitate PGL-I synthesis in the extracellular bacillus should facilitate a better understanding of the optimum conditions for metabolism in M. leprae .     

ASPP2 suppresses tumour growth and stemness characteristics in HCC by inhibiting Warburg effect via WNT/β-catenin/HK2 axis

Abnormal energy metabolism is one of the characteristics of tumours. In the last few years, more and more attention is being paid to the role and regulation of tumour aerobic glycolysis. Cancer cells display enhanced aerobic glycolysis, also known as the Warburg effect, whereby tumour cells absorb glucose to produce a large amount of lactic acid and energy under aerobic conditions to favour tumour proliferation and metastasis. In this study, we report that the haploinsufficient tumour suppressor ASPP2, can inhibit HCC growth and stemness characteristics by regulating the Warburg effect through the WNT/β-catenin pathway. we performed glucose uptake, lactate production, pyruvate production, ECAR and OCR assays to verify ASPP2 can inhibit glycolysis in HCC cells. The expression of ASPP2 and HK2 was significantly inversely correlated in 80 HCC tissues. Our study reveals downregulation of ASPP2 can promote the aerobic glycolysis metabolism pathway, increasing HCC proliferation, glycolysis metabolism, stemness and drug resistance. This ASPP2-induced inhibition of glycolysis metabolism depends on the WNT/β-catenin pathway. ASPP2-regulated Warburg effect is associated with tumour progression and provides prognostic value. and suggest that may be promising as a new therapeutic strategy in HCC.     

细菌溶血磷脂的生物合成及生物学功能

溶血磷脂(lysophospholipids, LPLs)是细胞膜中的一类脂质代谢中间产物，主要由磷脂分子被水解后生成。LPL的生物学功能与其前体磷脂有明显的区别。在真核细胞中，LPL是一种参与多种胞内生物信号调控的重要活性分子，但在细菌中，LPL的生物学功能还未被充分揭示。LPL通常是细菌细胞膜中的次要组分，在环境压力条件下其含量可显著升高。除了参与细胞膜磷脂代谢，LPL被认为在细菌环境适应性及致病性中发挥重要作用。其在细胞膜中的累积可以显著提高细菌在环境压力下的存活及增殖效率，同时还是细菌感染过程中重要的信号分子。近期有研究表明，LPL可能是细菌新发现的潜在毒力因子。本文因此将结合最新研究数据，对不同种类LPL的从头合成通路以及LPL在细菌抵御环境压力和细菌-宿主互作等方面所发挥的生物学功能进行综述，为对细菌致病机制和防治细菌感染的相关研究提供新的思路和参考借鉴。     

Dehalogenation of dichloromethane by dichloromethane dehalogenase/glutathione S-transferase leads to formation of DNA adducts

Formation of DNA adducts following conversion of dichloromethane by bacterial dichloromethane dehalogenase/glutathione S-transferase was demonstrated. Adducts included dichloromethane carbon and glutathione sulfur atoms. A reaction with DNA occurred preferentially at guanine bases. Increased DNA degradation in a polA mutant of Methylobacterium dichloromethanicum DM4 grown with dichloromethane confirmed the genotoxicity associated with dichloromethane degradation, suggesting an important role of DNA repair in the metabolism of halogenated, DNA-alkylating compounds by bacteria.     

Bacterial [methyl-3H]thymidine incorporation in substrate-amended estuarine sediment slurries

Abstract Five different bacterial communities were enriched in substrate-amended slurries of sediment from the Tay Estuary, Scotland. During incubation of the slurries, concentrations of volatile fatty acids, sulphate, sulphide and methane were monitored to clearly define the activity of the stimulated populations. An aerobic population, a ‘microaerophilic’ population and three anaerobic populations (fermentative heterotrophs, sulphate-reducing bacteria and methanogens plus acetogens) were established to reflect community growth and metabolism both in surface oxic and deeper anoxic layers. Similar numbers of cells involved in division were observed in all five slurries, demonstrating the potential for bacterial production. Thymidine incorporation rates in glucose-stimulated slurries under both aerobic and fully anaerobic conditions were similar, confirming the ability of fermentative anaerobic heterotrophs to incorporate [ methyl -³H]thymidine into DNA during growth. Although anaerobic communities of sulphate-reducing, acetogenic plus methanogenic bacteria were stimulated and actively growing, they did not incorporate [ methyl -³H]thymidine into DNA. Since the thymidine technique does not measure the growth of these important groups, calculated productivity values based upon thymidine incorporation within anoxic sediment systems will be substantially underestimated, even if growth substrates are not limiting.     

The relationship between photosynthetic electron transport and photorespiratory 14CO2 release after DCMU treatment in the duckweed, Lemna gibba

The photosynthetic rate of Lemna gibba was measured as ¹⁴CO₂ uptake at the beginning of and after 1 h DCMU treatment during the separate excitation of PS I (703 nm), mainly PS II (662 nm) and the combined excitation of both photosystems (662 + 703 nm) in 2 and 21% oxygen. The results show the Warburg effect. Photosynthesis was significantly reduced by DCMU whenever PS II was excited, at 662 nm and 662 + 703 nm. Photosynthetic enhancement was greater in 21 than in 2% oxygen in both the treated and untreated plants.
Photorespiratory ¹⁴CO₂ release was only affected by DCMU treatment at 662 + 703 nm. It was significantly decreased in 21% O₂ and significantly increased in 2% O₂ as compared to the controls without DCMU. The ¹⁴C-glycolate remaining in the plant after photosynthesis/photorespiration measurements was reduced whenever the electron supply to PS I was low.
These data support the hypothesis that a relationship exists between glycolate metabolism and photosynthesis via the electron transport chain where electrons from the oxidation of glycolate are donated to PS I when the electron supply from water is low.     

嗜水气单胞菌感染青鱼肝脏的蛋白质组学分析

采用同重同位素标记相对与绝对定量(iTRAQ)结合液相色谱串联质谱(LC-MS/MS) 技术, 分析嗜水气单胞菌(Aeromonas hydrophila)感染青鱼(Mylopharyngodon piceus)后青鱼肝脏组织的差异表达蛋白。以致病性嗜水气单胞菌菌株BCK0712注射感染健康青鱼, 24h后采集感染组和对照组青鱼肝脏, 开展蛋白质组学分析。通过数据库检索, 共鉴定到4475个肝脏组织蛋白, 从中筛选到188个差异表达蛋白, 其中表达上调的蛋白70个, 表达下调的蛋白118个。经生物信息学分析, 表明这些差异表达蛋白主要参与了补体和凝血级联反应、剪接体、细胞内吞作用、氧化磷酸化、碳代谢、精氨酸和脯氨酸代谢、色氨酸代谢等通路。组织病理分析表明, 青鱼感染嗜水气单胞菌后肝脏出现了明显的病理变化, 主要表现为肝细胞边界不分明、细胞核固缩、肝板排列紊乱、有出血现象等。研究结果为进一步深入探究嗜水气单胞菌的致病机制提供了理论基础。     

The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis

Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is well established as a key regulatory posttranslational modification in eukaryotes, but little is known about its extent and function in prokaryotes. Although protein kinases and phosphatases have been predicted and identified in a variety of bacterial species, classical biochemical approaches have so far revealed only a few substrate proteins and even fewer phosphorylation sites. Bacillus subtilis is a model Gram-positive bacterium in which two-dimensional electrophoresis-based studies suggest that the Ser/Thr/Tyr phosphorylation should be present on more than a hundred proteins. However, so far only 16 phosphorylation sites on eight of its proteins have been determined, mostly in in vitro studies. Here we performed a global, gel-free, and site-specific analysis of the B. subtilis phosphoproteome using high accuracy mass spectrometry in combination with biochemical enrichment of phosphopeptides from digested cell lysates. We identified 103 unique phosphopeptides from 78 B. subtilis proteins and determined 78 phosphorylation sites: 54 on serine, 16 on threonine, and eight on tyrosine. Detected phosphoproteins are involved in a wide variety of metabolic processes but are enriched in carbohydrate metabolism. We report phosphorylation sites on almost all glycolytic and tricarboxylic acid cycle enzymes, several kinases, and members of the phosphoenolpyruvate-dependent phosphotransferase system. This significantly enlarged number of bacterial proteins known to be phosphorylated on Ser/Thr/Tyr residues strongly supports the emerging view that protein phosphorylation is a general and fundamental regulatory process, not restricted only to eukaryotes, and opens the way for its detailed functional analysis in bacteria.     

Footprinting analysis of interactions between the largest eukaryotic RNase P/MRP protein Pop1 and RNase P/MRP RNA components

Ribonuclease (RNase) P and RNase MRP are closely related catalytic ribonucleoproteins involved in the metabolism of a wide range of RNA molecules, including tRNA, rRNA, and some mRNAs. The catalytic RNA component of eukaryotic RNase P retains the core elements of the bacterial RNase P ribozyme; however, the peripheral RNA elements responsible for the stabilization of the global architecture are largely absent in the eukaryotic enzyme. At the same time, the protein makeup of eukaryotic RNase P is considerably more complex than that of the bacterial RNase P. RNase MRP, an essential and ubiquitous eukaryotic enzyme, has a structural organization resembling that of eukaryotic RNase P, and the two enzymes share most of their protein components. Here, we present the results of the analysis of interactions between the largest protein component of yeast RNases P/MRP, Pop1, and the RNA moieties of the enzymes, discuss structural implications of the results, and suggest that Pop1 plays the role of a scaffold for the stabilization of the global architecture of eukaryotic RNase P RNA, substituting for the network of RNA–RNA tertiary interactions that maintain the global RNA structure in bacterial RNase P.     

The influence of temperature on respiration of diapausing pupae of Pieris brassicae (Lepidoptera)

The respiration of diapausing Pieris pupae has been measured at different temperatures between 5 and 35°C in animals maintained at 20°C, either 14 or 74 days after larvo-pupal ecdysis or at 5°C for 30 or 60 days.

The sudden transfer of animals from 5 to 15, 20, 25, 30, 35°C or from 20 to 30, 35°C results in a respiratory overshoot whose characteristics (duration, height, extra-respiration) depend on experimental conditions.

After a certain period of acclimation, overshoots are eliminated. The respiratory rate except for animals maintained during 74 days at 20°C can then be represented as an exponential function of temperature.

The Q₁₀ values change according to the treatment given to pupae.

The respiratory rate of male pupae is higher than that of female ones.

The following points are discussed:

1. 1.|The meaning of overshoots is analysed according to economy and metabolic homeostasis, showing the existence of acclimation.

2. 2.|Exponential curves which are not relevant to non-diapausing pupae or to the diapausing ones taken at larvo-pupal molting are characteristic of steady metabolism. These curves can be interpreted as the result of the temperature effect on a master respiratory reaction which would then be rate limiting.

3. 3.|Wintering leads to gradual and slow adaptation to cold temperatures which brings both a respiratory increase, a decrease of the Q₁₀ and of the activation energy of the master reaction.