Branching of o-nitrobenzoate degradation pathway in Arthrobacter protophormiae RKJ100: identification of new intermediates

We have earlier reported a novel reductive pathway for o-nitrobenzoate (ONB) degradation (at 0.5 mM) in Arthrobacter protophormiae RKJ100, which proceeds via the formation of o-hydroxylaminobenzoate (HABA) and anthranilate (AA). During growth of this organism at 40 times higher concentration (20 mM) of ONB, 3-hydroxyanthranilate (HAA) was identified as an intermediate by thin layer chromatography, gas chromatography and high performance liquid chromatography studies. Crude cell extracts of ONB-grown cells showed HAA 3,4-dioxygenase activity suggesting HAA as a terminal aromatic intermediate of the catabolic energy-yielding pathway as shown before in Pseudomonas fluorescens strain KU-7. HAA is further cleaved to 2-amino-3-carboxymuconic-6-semialdehyde by the action of HAA 3,4-dioxygenase. In this report we propose that ONB degradation occurs via the formation of HABA and the pathway branches at this point to form the two different aromatic intermediates AA and HAA by the action of a reductase and a mutase, respectively.     

Biochemical and phylogenetic analyses of psychrophilic isolates belonging to the Arthrobacter subgroup and description of Arthrobacter psychrolactophilus, sp. nov.

During our work on psychrophilic microorganisms we obtained a large collection of new isolates. In order to identify six of these, we examined their growth properties, cell wall compositions, and their 16S rRNA gene sequences. The results showed that all of the isolates are gram-positive, aerobic, contain lysine in their cell walls, and belong to the high mol% G+C Arthrobacter subgroup. Phylogenetic analysis of the 16S rRNA genes grouped five isolates obtained from a small geographical region into a monophyletic clade. Isolate B7 had a 16S rRNA sequence that was 94.3% similar to that of Arthrobacter polychromogenes and 94.4% similar to that of Arthrobacter oxydans. Primary characteristics that distinguish isolate B7 from the Arthrobacter type strain (Arthrobacter globiformis) and A. polychromogenes include lack of growth at 37 degrees C, growth at 0-5 degrees C, the ability to use lactose as a sole carbon source, and the absence of blue pigments. Because of these differences, isolate B7 was chosen as a type strain representing a new Arthrobacter species, Arthrobacter psychrolactophilus. The sixth isolate, LV7, differed from the other five because it did not have the rod/ coccus morphological cycle and was most closely related to Arthrobacter agilis.     

Production,Purification, and Antibiofilm Activity of a Novel Exopolysaccharide from Arthrobacter sp. B4

A novel exopolysaccharide (EPS), namely, B4-EPS, is produced by Arthrobacter sp. B4. Response surface methodology (RSM) was employed to optimize the fermentation medium for increasing B4-EPS production. Based on Plackett–Burman design (PBD), glucose, yeast extract, and KH₂PO₄ were selected as significant variables, which were further optimized by a central composite design (CCD). According to response surface and canonical analysis, the optimal medium was composed of 16.94 g/L glucose, 2.33 g/L yeast extract, and 5.32 g/L KH₂PO₄. Under this condition, the maximum yield of B4-EPS reached about 8.54 g/L after 72 hr of batch fermentation, which was pretty close to the predicted value (8.52 g/L). Furthermore, B4-EPS was refined by column chromatography. The main homogeneous fraction (B4-EPS1) was collected and applied to assay of antibiofilm activity. B4-EPS1 exhibited a dose-dependent inhibitory effect on biofilm formation of Pseudomonas aeruginosa PAO1 without antibacterial activity. About 86.1% of biofilm formation of P. aeruginosa PAO1 was inhibited in the presence of 50 µg/mL B4-EPS1, which was more effective than the peer published data. Moreover, B4-EPS1 could prevent biofilm formation of other strains. These data suggest B4-EPS may represent a promising strategy to combat bacterial biofilms in the future.     

Adaptation of the psychrotroph Arthrobacter chlorophenolicus A6 to growth temperature and the presence of phenols by changes in the anteiso/iso ratio of branched fatty acids

Arthrobacter chlorophenolicus is a previously described Gram-positive bacterium capable of degrading high concentrations of several phenolic compounds under optimal mesophilic (28 degrees C) as well as psychrophilic (5 degrees C) conditions. However, the exact mechanisms by which this organism is able to tolerate such extremes in temperature and high levels of toxic compounds are currently not known. In this study, we monitored changes in the fatty acid composition of the cell membrane under different extreme growth conditions. Arthrobacter chlorophenolicus adapts to differences in temperature and phenol concentrations by altering the anteiso/iso ratio of fatty acids in the cell membrane to different extents. According to the different physico-chemical properties of those two species of branched fatty acids, the bacteria showed an increased amount of anteiso fatty acids when grown under psychrophilic conditions to decrease the viscosity of their membranes. On the other hand, at higher growth temperatures as well as in the presence of toxic concentrations of phenol, 4-chlorophenol and 4-nitrophenol, the cells adapted their membrane by a dose-dependent decrease in the anteiso/iso ratio, leading to a more rigid membrane and counteracting the fluidity increase caused by the higher temperature and the organic solvents.     

Growth of epithelium from a preneoplastic mammary outgrowth in response to mammary adipose tissue

Summary We investigated the effects of conditioned media derived from mouse mammary fat pads on the proliferation of CL-S1 cells, an epithelial cell line originally isolated from a preneoplastic mammary outgrowth line. Cell proliferation in vitro in serum-free defined medium was compared to that in this medium conditioned using intact mammary fat pad pieces or isolated fat pad adipocytes. Culture medium was conditioned by incubating the conditioning material in defined culture medium for 24 h at 37°C. Conditioned medium induced CL-S1 proliferation as much as 10- to 20-fold above the minimal levels of growth in control cultures after 13 d of culture. The growth-stimulatory factor(s) had an apparent molecular weight of greater than 10 kDa. This growth-stimulatory activity was both heat and trypsin stable. Because the role of adipose tissue is to store and release lipids, we next tested whether lipids are released during medium conditioning. The lipid composition of the fat pad conditioned medium was characterized using both thin layer and gas liquid chromatography. These lipid analyses indicated that the fat pad pieces released significant amounts of fatty acids and phospholipids into the medium during the conditioning period. The free fatty acid composition included both saturated and unsaturated molecules, and about 80% of the total fatty acids consisted of palmitate, stearate, oleate, and linoleate. These same fatty acids were a structural component of the majority of phospholipid found in the medium. The addition of palmitate or stearate to defined medium had no effect or was inhibitory for CL-S1 proliferation, depending on the concentration used. Defined medium supplemented with oleate, arachidonate, or linoleate induced CL-S1 proliferation, and the inhibitory effects of palmitate and stearate were overcome by addition of oleate and linoleate. These data indicate that both unsaturated and saturated fatty acids are released from intact adipose cells of the mouse mammary fat pad and that fatty acids can influence the growth of prenoplastic mouse mammary epithelium. Thus, unsaturated fatty acids, perhaps in conjunction with other substances released simultaneously, are candidate molecules for the substances that mediate the effect of adipose tissue on growth of epithelium. This work was supported in part by a grant from the American Institute for Cancer Research; grant CA 46885 from the National Institutes of Health, Bethesda, MD; and by State of Washington initiative 171.     

Variation amongst survivor populations of white clover collected from sites across Europe: growth attributes and physiological responses to low temperature

Experiments were carried out at IGER, Aberystwyth, UK to investigate traits of direct relevance to the processes of overwintering and spring growth in white clover (Trifolium repens L.). The plant material used was derived from baseline populations of the cultivar AberHerald and survivor populations generated after 2-3 years' growth in Germany (Kiel), Sweden (Uppsala) and Switzerland (Zürich). The aims of the experiments were to measure the level of genetic shift that had occurred in certain traits due to selection in the survivor populations by comparing these with the baseline population. The adaptive significance of traits was assessed by determining the extent to which stabilizing selection had operated to reduce levels of intra-population variation. Significant differences were found in the responses of leaf production to two temperature treatments in the survivor populations from Germany and Sweden compared with the Swiss and baseline material. Plants of the former two populations produced much more leaf than the others at the higher temperature, but leaf production rates at the lower temperature did not differ. As this experiment used cloned genotypes in the two treatments, the result suggests that a higher degree of phenotypic plasticity for this trait had been selected for in the German and Swedish populations. These populations also showed greater rates of regrowth of leaves from terminal buds exposed to sub-zero temperatures, but there were no differences between populations in levels of freezing tolerance, or in stolon carbohydrate content. Genetic shift occurred in the degree of unsaturation of stolon lipids, with all three survivor populations possessing higher proportions of unsaturated fatty acids than the baseline. Stabilizing selection also operated on this trait in the survivor populations, suggesting that it is of adaptive significance in cool climates.     

Investigation into the effect of detergents on disinfectant susceptibility of attached Escherichia coli and Listeria monocytogenes

Aims:  Investigate the effect of detergent treatment on susceptibility of attached Escherichia coli and Listeria monocytogenes to subsequent disinfectant treatment.
Methods and Results:  Plate counts show that E. coli attached to stainless steel surfaces became significantly more susceptible to benzalkonium chloride (BAC) after treatment with sodium alkyl sulfate (SAS) and fatty alcohol ethoxylate (FAE). No change in susceptibility was observed with Sodium dodecyl sulfate (SDS). L. monocytogenes became significantly less susceptible to BAC after treatment with SAS and SDS yet no change in susceptibility was observed with FAE. Flow cytometry using the fluoresceine propidium iodide revealed significant increases in cell membrane permeability of both organisms by SAS and FAE, although the effect was much greater in E. coli . No change was observed with SDS. Hydrophobic interaction chromatography showed that both organisms became less hydrophobic following treatment with SAS and SDS but FAE had no effect.
Conclusions:  In E. coli, detergents that increase susceptibility to BAC increase membrane permeability. In L. monocytogenes, detergents that reduce susceptibility to BAC lower cell surface hydrophobicity.
Significance and Impact of the Study:  Detergents can influence the sensitivity of pathogenic food borne micro-organisms to BAC.     

The phospholipid fatty acids of Porphyridium purpureum cultured in the presence of triton x-100 and sodium desoxycholate

The phospholipid fatty acid composition of Porphyridium purpureum grown on a solid medium was studied in the presence of Triton X-100 (TX) and sodium desoxycholate (SDC). The most common fatty acids in PC and PE were palmitic (16:0), stearic (18:0), linoleic (18:2ω6), arachidonic (20:4ω6) and eicosapentaenoic (20:5ω3) acids, 20:4ω6 being very abundant. In PG the most common acids were 16:0, trans-hexaenoic acid (tr16:1ω3), oleic acid (18:1) and 20:4ω6. Both detergents caused an increase in the saturation of PC and, to a lesser extent, of PE. The relative amounts of short chain fatty acids increased. Both detergents increased the amounts of 16:0 and, correspondingly, decreased the amounts of 20:4ω6. In PG the amounts of both 16:0 and tr 16:1ω3 increased and the amounts of 18:0, 18:2ω6 and 20:4ω6 decreased in the presence of detergents. The changes were always greatest at the concentrations of 5–10 ppm TX or SDC. At 20 ppm the fatty acid compositions, especially with SDC, were very similar to the controls, which suggests a change in the detergent effect between 10–20 ppm. The normal PC/PE ratio was 5.6 and the (PC+ PE)/PG ratio 39.0. Both detergents caused a marked decrease in these ratios. Because the detergent effects are not linear, it seems that even very low detergent concentrations have an important influence on algae in polluted waters.     

Genetic and physiological response to fumonisin and AAL-toxin by intact tissue of a higher plant

The differential phytotoxicity of purified AAL-toxin to lines of tomato isogenic for the Asc gene parallels resistance to Alternaria alternata f.sp. lycopersici. This relationship, as reported earlier, is consistent with the role of AAL-toxin as a host-specific toxin with the role of a primary chemical determinant of Alternaria stem canker. Current results indicate the pathogen and the AAL-toxin also can be recovered from ripe fruit with symptoms of the disease known as black mold. Fumonisins are structurally similar to the AAL-toxins but are secreted by Fusarium moniliforme which is taxonomically distinct from A. alternata. F. moniliforme, is not pathogenic on living tomato tissues but was recovered from ripe tomato fruit with symptoms of black mold. The penetration of ripe fruit and subsequent colonization by both fungi appears to be saprophytic. Fumonisins and AAL-toxins express equivalent genotype-specific activity against the isogenic Asc lines of tomato and produce equivalent necrotic symptoms in tomato leaflet bioassays. Evidence was obtained that the biosynthetic pathway for production of these toxins is present in several species of both Alternaria and Fusarium. Toxin biosynthesis was sensitive to nutritional regulation in both genera. However, pathogenicity on tomato was not altered by the medium used for inoculum production in either genera and remained restricted to A. alternata f.sp. lycopersici in the studies reported here. Differences in the amount of toxin produced were found among isolates of both genera while the magnitude of the differences was defined by the substrate on which the fungi were grown.     

Differential Regulation of Intestinal and Liver Fatty Acid-Binding Proteins in Human Intestinal Cell line (Caco-2): Role of Collagen

Fatty acid-binding proteins (FABP) are small cytosolic proteins which are thought to play a key role in fatty acid metabolism. The intestine contains the intestinal (I-FABP) and the liver (L-FABP) isoforms, but their regulation is still poorly documented. In order to find suitable conditions for studying the regulation of the two FABP isoforms in Caco-2 cells, we investigated the effects of the presence of collagen during cell proliferation or differentiation. When collagen was present only during cell proliferation on culture dishes, I-FABP expression was enhanced, whereas sucrase-isomaltase was unaffected and L-FABP expression was merely accelerated. In contrast, when collagen was present during cell differentiation on filter inserts, both I-FABP and sucrase-isomaltase were strongly reduced, but L-FABP was not affected. Under the former conditions (the more suitable for studying FABP regulation), the peroxysome proliferator-activated receptor (PPAR) activators, clofibrate and α-bromopalmitate, enhanced the two isoforms. This study, which is the first one providing a quantitative protein analysis of I-FABP and L-FABP in Caco-2 cells, demonstrates different time courses of expression of these proteins during cell differentiation. It also shows that I-FABP is specifically regulated by collagen and that, under conditions optimal for their expression, both isoforms are modulated by metabolic factors.     

Growth and pectate-lyase activity of the ruminal bacterium Lachnospira multiparus in the presence of short-chain organic acids

AIMS: Acetic, propionic, butyric and lactic acids are end products of feed fermentation by rumen microbes. The effects of these short chain acids on growth and pectate-lyase (PL) activity of Lachnospira multiparus were studied. METHODS AND RESULTS: The bacterial strain used was L. multiparus D32. Acids were tested between 50 and 300 mmol l(-1). Growth and PL activity were measured by the increase in total protein content and by the increase in absorbance at 235 nm in the reaction medium respectively. With the exception of lactic acid, all acids decreased bacterial growth rates; generally, these effects were more pronounced at higher concentrations and with acids of longer chains. PL activity was inhibited by all the acids except by butyric acid at 50 and 100 mmol l(-1). Enzyme inhibition increased with the concentrations of the acids and lactic acid was the most inhibitory. CONCLUSIONS: High concentrations of short chain acids can differentially inhibit the growth rate and the PL activity of L. multiparus. SIGNIFICANCE AND IMPACT OF THE STUDY: Products of fermentation generated by the ruminal microbiota could modify the degradation of pectic substances by this bacterium.     

血清H-FABP和S-100B 在急性脑梗死患者早期诊断及病情评估中的应用

目的:探索血清心型脂肪酸结合蛋白(H-FABP)和S-100B蛋白(S-100B)在急性脑梗死(ACI)患者早期诊断及病情评估中的应用。方法:选择自2012年9月至2015年3月我院收治的ACI患者91例作为观察组,根据Adama分型法、神经缺损程度和不同的病发时间进行分组,另外选取健康体检者73例作为对照组。采集受试者血液,应用酶联免疫吸附法检测血清中H-FABP和S-100B水平,并进行相关性分析。结果:观察组患者发病后第1、3、12 h血清中H-FABP和S-100B水平明显高于对照组(P0.05)。观察组患者血清中H-FABP水平发病第1 h开始升高,在第3 h达到最高水平,之后开始逐渐下降;S-100B水平变化趋势同H-FABP,发病第12 h达到最高水平,之后开始下降。病灶直径越大,患者血清中H-FABP和S-100B水平越高,且组间差异均具有统计学意义(P0.05);重度神经缺损患者血清中H-FABP和S-100B水平明显高于中度和轻度缺损患者,中度神经缺损患者血清中H-FABP和S-100B水平显著高于轻度缺损患者(P0.05);ACI患者血清中H-FABP和S-100B水平与病灶直径和神经缺损程度呈正相关(P0.05)。结论:ACI患者血清中H-FABP和S-100B水平可以作为疾病的早期诊断标准之一,同时可以准确评估病情发展,对于临床检测和确诊具有重要意义,值得在临床上广泛使用。     

Strain variability in fatty acid composition of Chattonella marina (Raphidophyceae) and its relation to differing ichthyotoxicity toward rainbow trout gill cells

Lipid profiles of three strains (Mexico, Australia, Japan) of Chattonella marina (Subrahmanyan) Hara et Chihara were studied under defined growth (phosphate, light, and growth phase) and harvest (intact and ruptured cells) conditions. Triacylglycerol levels were always <2%, sterols <7%, free fatty acids varied between 2 and 33%, and polar lipids were the most abundant lipid class (>51% of total lipids). The major fatty acids in C. marina were palmitic (16:0), eicosapentaenoic (EPA, 20:5ω3), octadecatetraenoic (18:4ω3), myristic (14:0), and palmitoleic (16:1ω7c) acids. Higher levels of EPA were found in ruptured cells (21.4–29.4%) compared to intact cells (8.5–25.3%). In general, Japanese N‐118 C. marina was the highest producer of EPA (14.3–29.4%), and Mexican CMCV‐1 the lowest producer (7.9–27.1%). Algal cultures, free fatty acids from C. marina, and the two aldehydes 2E,4E‐decadienal and 2E,4E‐heptadienal (suspected fatty acid‐derived products) were tested against the rainbow trout fish gill cell line RTgill‐W1. The configuration of fatty acids plays an important role in ichthyotoxicity. Free fatty acid fractions, obtained by base saponification of total lipids from C. marina showed a potent toxicity toward gill cells (median lethal concentration, LC₅₀ (at 1 h) of 0.44 μg · mL^?1 in light conditions, with a complete loss of viability at >3.2 μg · mL^?1). Live cultures of Mexican C. marina were less toxic than Japanese and Australian strains. This difference could be related to differing EPA content, superoxide anion production, and cell fragility. The aldehydes 2E,4E‐decadienal and 2E,4E‐heptadienal also showed high impact on gill cell viability, with LC₅₀ (at 1 h) of 0.34 and 0.36 μg · mL^?1, respectively. Superoxide anion production was highest in Australian strain CMPL01, followed by Japanese N‐118 and Mexican CMCV‐1 strains. Ruptured cells showed higher production of superoxide anion compared to intact cells (e.g., 19 vs. 9.5 pmol · cell^?1 · hr^?1 for CMPL01, respectively). Our results indicate that C. marina is more ichthyotoxic after cell disruption and when switching from dark to light conditions, possibly associated with a higher production of superoxide anion and EPA, which may be quickly oxidized to produce more toxic derivates, such as aldehydes.     

Fat-1基因真核表达载体的构建及其对人口腔鳞癌细胞脂肪酸含量的影响

目的：构建真核表达载体pcDNA3．1-Fat1,线性化稳定转染人口腔鳞癌细胞株Tca8113,检测其细胞内脂肪酸含量变化。方法：通过重叠延伸PCR方法人工合成利于真核表达的Fat-1基因,用基因重组技术构建真核表达载体pcDNA3．1-Fat—1,用脂质体转染真核细胞的方法转染人1：／腔鳞癌细胞株Tca8113,用气相色谱仪检测脂肪酸的变化情况。结果：测序及酶切鉴定成功合成真核偏好表达的Fat-1基因。与对照组相比,转染Fat—1基N的口腔癌细胞的n-3脂肪酸明显增多,n-6／n-3明显下降。结论：成功构建真核表达载体pcDNA3．1-Fat1,并对口腔鳞癌细胞内脂肪酸含量产生明显影响,为进一步研究Fat-1基因在口腔鳞癌中的生物学功能奠定了基础。     

Impact of the exopolysaccharides Pel and Psl on the initial adhesion of Pseudomonas aeruginosa to sand

In this study, the impact of the exopolysaccharides Pel and Psl on the cell surface electron donor-electron acceptor (acid-base) properties and adhesion to quartz sand was investigated by using Pseudomonas aeruginosa PAO1 and its isogenic EPS-mutant strains Δpel, Δpsl and Δpel/Δpsl. The microbial adhesion to hydrocarbon (MATH) test and titration results showed that both Pel and Psl contribute to the surface hydrophobicity of the cell. The results of contact angle measurement, however, showed no correlation with the cell surface hydrophobicity measured by the MATH test and the titration method. Packed-bed column experiments indicated that the exopolysaccharides Pel and Psl are involved in the initial cell attachment to the sand surface and the extent of their impact is dependent on the ionic strength (IS) of the solution. Overall, the Δpel/Δpsl double mutant had the lowest adhesion coefficient to sand compared with the wild-type PAO1, the Δpel mutant and the Δpsl mutant. It is hypothesized that in addition to bacterial surface hydrophobicity and DLVO forces, other factors, eg steric repulsion caused by extracellular macromolecules, and cell surface appendages (flagella and pili) also contribute significantly to the interaction between the cell surface and a sand grain.     

Regulation of hormone-sensitive lipase expression by saturated fatty acids and hormones in bovine mammary epithelial cells

Hormone-sensitive lipase was firstly identified as an epinephrine-induced lipase in adipocyte. HSL mRNA was detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and bovine lactating mammary gland. Saturated fatty acids (stearate and palmitate), but not unsaturated fatty acids (oleate and linoleate) induced up-regulation of HSL mRNA in a time- and concentration-dependent manner in bMEC. Treatment with insulin (5-10 ng/ml), dexamethasone (50-250 nM) or GH (50 ng/ml) induced down-regulation of HSL. These results suggest that HSL was regulated by fatty acids and some hormones in mammary epithelial cells and thereby play an important role of lipid and energy metabolism.     

Nicotine protects against arachidonic-acid-induced caspase activation, cytochrome c release and apoptosis of cultured spinal cord neurons

Hydrolysis of membrane phospholipids of spinal cord neurons is one of the first events initiated in spinal cord trauma. In this process, free fatty acids, and in particular arachidonic acid, are released. Exposure of spinal cord neurons to free arachidonic acid can compromise cell survival and initiate apoptotic cell death. In order to determine potential mechanisms of apoptosis induced by arachidonic acid, activation of caspases -3, -8, and -9, as well as the release of cytochrome c into the cytoplasm were measured in cultured spinal cord neurons exposed to 10 microM of this fatty acid. In addition, because nicotine can exert a variety of neuroprotective effects, we hypothesized that it can prevent arachidonic acid induced apoptosis of spinal cord neurons. To study this hypothesis, spinal cord neurons were pretreated with nicotine (10 microM for 2 h) before arachidonic acid exposure and caspase activation as well as markers of apoptotic cell death were studied. Treatment of spinal cord neurons with arachidonic acid for up to 24 h significantly increased cytoplasmic levels of cytochrome c, induced caspase activation and induced DNA laddering, a hallmark of apoptotic cell death. Nicotine pretreatment markedly attenuated all these effects. In addition, antagonist studies suggest that the alpha7 nicotinic receptor is primarily responsible for these anti-apoptotic effects of nicotine. These results indicate that nicotine can exert potent neuroprotective effects by inhibiting arachidonic acid induced apoptotic cascades of spinal cord neurons.     

毛乌素沙地旱柳生长和生理特征对遮荫的反应

在一个控制试验中,旱柳经历了全不遮荫、部分遮荫和全部遮荫处理．比较了全不遮荫枝、全部遮荫枝、部分遮荫阳生枝(阳生枝)和部分遮荫阴生枝(阴生枝)的生长和生理特征,结果表明：阳生枝和全部遮荫枝的叶出生率和死亡率分别大于全不遮荫枝和阴生枝;遮荫处理明显影响净光合速率和夜间呼吸速率;阳生枝的分枝生物量、总校长度、枝叶生物量和枝叶重比显著大于全不遮荫枝,而阴生枝的分枝数、分枝生物量、叶面积、叶生物量、基茎、总校长度、枝叶生物量和枝叶重比都显著小于全部遮荫枝．