Degradation of 2-chlorobenzoate by in vivo constructed hybrid pseudomonads

Abstract 5-Chlorosalicylate degrading bacteria were obtained from the mating between Pseudomonas sp. strain WR401 and Pseudomonas sp. strain B13. Further selection of the hybrid organisms for growth on 2-chlorobenzoate allowed the isolation of strains such as JH230. During growth on 2-chlorobenzoate stoichiometric amounts of chloride were released. Steps in the pathway for 2-chlorobenzoate degradation were determined by simultaneous adaptation studies, assays of enzymes in cell extracts and cooxidation of the analogous substrate 2-methylbenzoate. Results indicate that 2-chlorobenzoate was degraded to 3-chlorocatechol. Ring cleavage of 3-chlorocatechol was by a catechol 1,2-dioxygenase to from 2-chloro- cis, cis - muconate. Further degradation runs via 4-carboxymethylenebut-2-en-4-olide.     

Degradation of 3-chlorobiphenyl by in vivo constructed hybrid pseudomonads

Abstract 3-Chlorobiphenyl-degrading bacteria were obtained from the mating between Pseudomonas putida strain BN10 and Pseudomonas sp. strain B13. Strains such as BN210 resulted from the transfer of the genes coding the enzyme sequence for the degradation of chlorocatechols from B13 into BN10, whereas B13 derivatives such as B131 have acquired the biphenyl degradation sequence from BN10. During growth of the hybrid strains on 3-chlorobiphenyl 90% chloride was released. Activities of phenylcatechol 2,3-dioxygenase, benzoate dioxygenase, catechol 1,2-dioxygenase, chloromuconate cyloisomerase and 4-carboxymethyl-enebut-2-en-4-olide hydrolase were found in 3-chlorobiphenyl-grown cells. The hybrid strains were found to convert some congeners of the Aroclor 1221 mixture such as mono- and dichloro-substituted biphenyls.     

The suppressive effect of bifidobacteria on Bacteroides vulgatus,a putative pathogenic microbe in inflammatory bowel disease

Bacteroides, a predominant commensal bacteria in the gut, are thought to be responsible for the development of inflammatory bowel disease (IBD). In the present study, we examined whether or not bifidobacteria suppress B. vulgatus, a representative pathogenic Bacteroides species, in both the coculture system and the gnotobiotic murine model. As a result, Bifidobacterium infantis 1222 highly inhibited the growth of B. vulgatus in the coculture and also significantly suppressed the systemic antibody response raised by B. vulgatus colonizing the gut in gnotobiotic mice. Colonization of the mice by B. vulgatus increased the number of Peyer's patch (PP) cells bearing PNA (peanut agglutinin)+/anti-kappa+ phenotype, which represents plasma cell-like B cells. Moreover, treatment of those B. vulgatus-implanted mice with B. infantis 1222 abrogated such increase in the number of PNA+/anti-kappa+ cells. These results thus suggested that B. infantis 1222 protected the gut epithelial layer including the PP from being invaded by Bacteroides, thereby suppressing the systemic antibody response raised by Bacteroides.     

Inhibition of saliva-induced oral streptococcal aggregation by blood group glycoproteins

Abstract The inhibition of saliva-induced oral streptococcal aggregation with anti-sera (anti-A, anti-B, anti-AB and anti-B treated with galactose), normal human serum (NHS), blood group-specific lectins (UEA-I, HBA, GPA, BSI-B₄, GS-I), non-specific blood group lectins (MPA, SBA) and carbohydrates (galactose, N -acetylgalactosamine, l -fucose) was studied. Streptococcal species and strains included S. mutans 318, S. mutans 10449, S. mutans NG-8, S. salivarius and S. cricetus HS-6. The saliva was obtained from three subjects with secretor status (2 blood group B persons, 1 blood group A person). The data obtained from experiments performed with S. mutans 10449 and S. mutans NG-8 suggest the involvement of the H-antigenic determinant in the aggregation mechanism of the first strain and of the group B determinant for the second strain. The aggregation of S. salivarius only by B saliva might be related to a galactose-specific lectin on this strain and to some properties of its cell surface (hydrophobicity and the fibrillar surface layer). S. cricetus HS-6 aggregation was inhibited in different degrees by all the inhibitors used. The results demonstrate that interactions between oral streptococci and salivary components depend on the strain and species and on the individual saliva samples.     

Isolation of Helicobacter pylori from the intestinal mucosa of patients with Crohn's disease

BACKGROUND: Helicobacter species are associated with inflammatory bowel disease in rodents and in nonhuman primates. Therefore, we prospectively investigated the presence of Helicobacter species in the intestinal mucosa of patients with and without Crohn's disease by culture and polymerase chain reaction (PCR) assays. MATERIALS AND METHODS: Mucosal fragments were obtained from the ileum, different colon regions, and rectum of 43 patients with Crohn's disease and of 74 patients without inflammatory bowel disease. RESULTS: Helicobacter pylori strains, identified by 16S rRNA gene sequencing, were more frequently isolated and PCR-detected in the intestinal mucosa of patients with ulcerative colitis-like Crohn's disease than in intestinal mucosa of the control group. Otherwise, anti-H. pylori immunoglobulin G levels were significantly lower in fibrostenosing and fistulating Crohn's disease subgroups. No other Helicobacter species were found in the intestinal mucosa of the patients. CONCLUSIONS: Although our results suggest an association between the presence of H. pylori in the intestine and ulcerative colitis-like phenotype of Crohn's disease, H. pylori infection in the actual causality of Crohn's disease is still to be determined.     

益生菌对克罗恩病患者肠道屏障功能和免疫调节的影响

研究益生菌对克罗恩病患者肠道屏障功能和免疫调节的影响,为该类患者的治疗提供参考。

选择2018年1月至2020年6月我院收治的94例克罗恩病患者进行前瞻性随机对照研究,根据住院尾号单双数随机分为单号的对照组（n = 45）和双号的益生菌组（n = 49）。对照组患者给予柳氮磺胺吡啶联合安慰剂治疗,益生菌组患者给予柳氮磺胺吡啶联合双歧杆菌三联活菌片治疗。治疗前及治疗后2个月时,分别检测患者粪便中肠道菌群数量,简化克罗恩病活动指数（CDAI）,血沉（ERS）,血清中超敏C反应蛋白（hs-CRP）、二胺氧化酶、D-乳酸、干扰素-γ（IFN-γ）、白介素（IL）-4、IL-10、IL-17的水平以及外周血中Th1、Th2、Th17、Treg细胞的数目。随访2组患者预后不良终点事件的累积发生率。

治疗后2个月,益生菌组患者粪便中拟杆菌、肠杆菌、肠球菌的数量,简化CDAI、ERS水平,血清中hs-CRP、二胺氧化酶、D-乳酸、IFN-γ、IL-17水平,外周血中Th1、Th17的数量均低于对照组;而粪便中双歧杆菌、乳杆菌数量,血清中IL-4、IL-10水平,外周血中Th2、Treg的数量均高于对照组（均P＜0.05）。随访过程中,益生菌组患者预后不良终点事件的累积发生率低于对照组（P＜0.05）。

益生菌能够改善克罗恩病患者病情及肠道屏障功能,调节免疫应答,同时也对预后具有改善作用。

     

Degradation of vasoactive intestinal polypeptide by tissue homogenates

Extracts of liver, kidney and brain contain an enzyme that is highly specific for degradation of vasoactive intestinal polypeptide (VIP). The Michaelis constants (K_m's) appear to be nearly identical in all three tissues, averaging about 10^?5 mol/liter. The V_max for kidney and liver are about the same but that for cerebral cortex is about two-fold lower. Since the relative V_max in the three organs differ for insulin and VIP, it is concluded that it is unlikely that the same enzyme is responsible for the degradation of both peptides.     

新生溶血病患儿红细胞上致敏抗体对Rh 血型检测的影响

目的:探讨新生溶血病患儿红细胞致敏抗体对其Rh血型鉴定的影响。方法:采用抗球蛋白法、盐水法、微柱凝胶法(Rh血型测定型)、凝聚胺法和抗血清微柱凝胶法(Ig G型)五种方法对近三年来我院收集的163例新生溶血病患儿红细胞进行Rh血型检测,对五种检测结果不一致的患儿红细胞进行0.2 M 2-巯基乙醇抗体放散,比较放散后五种方法检测结果并验证其准确性。结果:29例直接抗体试验阳性患儿的五种Rh血型检测结果不一致,经0.2 M 2-巯基乙醇抗体放散后检测结果均一致。Rh血型准确性验证表明,红细胞放散测定的Rh血型完全符合临床现象。结论:患儿红细胞的致敏抗体达一定数量后,会影响抗球蛋白法、盐水法、微柱凝胶法(Rh血型测定型)、凝聚胺法和抗血清微柱凝胶法(Ig G型)对Rh血型鉴定,0.2M 2-巯基乙醇抗体放散法是一种正确鉴定新生儿Rh血型的简单可行的方法。     

Recovery of protein and chlorophyll from alfalfa by simultaneous lactic acid fermentation and enzyme hydrolysis (ENLAC)

Simultaneous lactic acid fermentation and enzyme hydrolysis by cell wall degrading enzymes (ENLAC for short) was tested for improving the recovery of cell content, such as protein and chlorophyll, from alfalfa. Alfalfa was ensiled with the addition of 1.0% (wet weight) of a variety of commercial enzymes or enzyme cocktails. The best result was achieved by the addition of a 1:1 mixture of Novo Viscozyme (containing mainly hemicellulases, cellulases, and pectinases) and Novo Celluclast 1.5 L or Genencore Cellulase 150 L (containing T. reesei cellulases). ENLAC improved the recovery of protein by 160%, chlorophyll by 240%. The same enzyme treatment in a 24-h reaction without ensiling resulted in only a 14% increase in protein recovery. ENLAC provided optimal acidic conditions for enzyme action, and due to the preservation of the plant material during ensiling, it made possible the efficient use of a low concentration of enzymes during a longer reaction time, compared to conventional 24-h enzyme treatments.     

免疫磁珠技术分离唾液A/B血型抗原并用于吸附血清A/B抗体

目的:应用免疫磁珠分离技术获得具有良好抗原性的A/B血型抗原,并探究其作为ABO血型抗体吸附剂去除A/B抗体的可行性。方法:将含有血型物质的唾液进行预处理,再与包被了抗体的磁珠混合,分离出纯度较高的A/B抗原,运用酶联免疫及凝集抑制试验验证所得抗原的抗原性及是否存在交叉反应。用未纯化A/B抗原和纯化A/B抗原包被磁珠,对含有抗A/B IgM、IgG的血清进行抗体吸附,用纯化A/B抗原对100份来自O型血孕妇的临床血清样本进行抗体吸附,分别评价其吸附效果。结果:纯化抗原与对应抗体反应后,其吸光度显著高于对照组(A抗原与A抗体0.85±0.12 vs.0.27±0.03,P0.01;B抗原与B抗体0.86±0.09 vs.0.24±0.06,P0.01),与其它类型抗体反应后的吸光度值与对照组比较差异无统计学意义(P0.05)。进行红细胞凝集抑制试验时,纯化抗原可显著抑制相应抗体与红细胞的凝集反应,对其它类型抗体与红细胞的凝集没有抑制作用。血清抗体吸附实验表明纯化抗原的吸附效率比未纯化抗原的高(97.00%vs.88.00%,P0.001)。临床样本抗体吸附实验显示,纯化A抗原对抗A IgM/IgG的吸附效率分别为96.88%、98.44%;纯化B抗原对抗B IgM/IgG的吸附效率分别为96.88%、98.44%。结论:磁珠纯化抗原能特异性地与对应抗体结合,有效吸附血清中的血型抗体,有望作为合成A/B抗原的替代品。     

Characterization of Quinolinic Acid Phosphoribosyltransferase in Human Blood and Observations in Huntington''s Disease

Quinolinic acid (QUIN), an excitotoxic compound present in the mammalian CNS and periphery, has been hypothetically linked to human neurodegenerative disorders such as Huntington's disease and epilepsy. Quinolinic acid phosphoribosyltransferase (QPRT), the catabolic enzyme of QUIN, is found in the CNS and peripheral organs where it may be a major influence on the tissue levels of QUIN. We have measured QPRT activity in human blood as a means of assessing one aspect of QUIN metabolism in humans. The enzyme was present in blood cells, platelets having a sixfold greater activity than erythrocytes, but was essentially absent from the plasma. In a blood cell fraction, enzyme activity was potently inhibited by phthalic acid (IC50 = 6.1 microM). Kinetic analyses conducted over a range of QUIN concentrations yielded Km values of 1.89-3.75 microM and Vmax values of 33.4-72.5 fmol nicotinic acid mononucleotide/h/mg protein. Enzyme activity varied 2.2-fold between normal individuals, was reasonably constant over a series of sampling intervals, and showed some diminution when blood was stored for 1 month at -20 degrees C. No differences of enzyme activity in erythrocytes or platelets were apparent between three Huntington's disease patients and their unaffected spouses. These data indicate that measurements of QPRT activities in blood are a convenient means to monitor QUIN metabolism in human subjects and that a deficiency of the enzyme is not apparent in Huntington's disease.     

Microbial metabolism of chloroanilines: enhanced evolution by natural genetic exchange

2-, 3-, and 4-chloroaniline degrading bacteria were obtained by natural genetic exchange between an aniline or toluidine degrading Pseudomonas strain and the chlorocatechol assimilating Pseudomonas sp. B13. Hybrid organisms were isolated through cocultivation of the parent strains in the chemostat as well as through conjugation on solid media in presence of chloroanilines as the selective substrates. Biochemical analysis of the gene products in the hybrid strains clearly showed that the genes coding for the aniline dioxygenase or the genes for the chlorocatechol assimilatory sequence had been transferred.     

Enhanced Hydrocarbons Degradation in the Rhizosphere of Mangrove Plants by a Halophilic Bacillus Subtilis Subtilis Strain

Sulaibikhat Embayment is a severely contaminated coastline in the State of Kuwait. The contaminating pollutants include hydrocarbons, heavy metals, and suspended particles. The objective of this study is to assess the ability of mangroves planted in the Sulaibikhat Embayment to enhance hydrocarbons degradation by the activities of rhizospheric hydrocarbon degrading bacteria (HDB). Accordingly, samples were collected from the rhizosphere of selected mangrove plants and from sediments in the same location but away from mangrove marshes. The samples were analyzed chemically and microbiologically before being enriched with a mixture of hydrocarbon compounds (HC) to isolate HDB.

A number of halophilic HDB were isolated from mangroves rhizosphere and the surrounding sediments such as Pseudomonas balearica, Microbacterium barkeri and Gordonia soli. On the other hand, Bacillus velezensis and Bacillus subtilis subtilis were both isolated only from mangroves rhizosphere. Among the isolated HDB, Bacillus subtilis subtilis was distinguished with its high degradation rates of the tested HC including poly aromatic hydrocarbons. According to our knowledge, this is the first Bacillus subtilis HC-degrading strain that was isolated from Kuwait Bay and from mangroves rhizosphere.     

Degradation of low rank coal by Trichoderma atroviride ES11

A new isolate of Trichoderma atroviride has been shown to grow on low rank coal as the sole carbon source. T. atroviride ES11 degrades approximately 82% of particulate coal (10 g l(-1)) over a period of 21 days with 50% reduction in 6 days. Glucose (5 g l(-1)) as a supplemented carbon source enhanced the coal solubilisation efficiency of T. atroviride ES11, while 10 and 20 g l(-1) glucose decrease coal solubilisation efficiency. Addition of nitrogen [1 g l(-1) (NH(4))(2)SO(4)] to the medium also increased the coal solubilisation efficiency of T. atroviride ES11. Assay results from coal-free and coal-supplemented cultures suggested that several intracellular enzymes are possibly involved in coal depolymerisation processes some of which are constitutive (phenol hydroxylase) and others that were activated or induced in the presence of coal (2,3-dihydrobiphenyl-2,3-diol dehydrogenase, 3,4-dihydro phenanthrene-3,4-diol dehydrogenase, 1,2-dihydro-1,2-dihydroxynaphthalene dehydrogenase, 1,2-dihydro-1,2-dihydroxyanthracene dehydrogenase). GC-MS analysis of chloroform extracts obtained from coal degrading T. atroviride ES11 cultures showed the formation of only a limited number of specific compounds (4-hydroxyphenylethanol, 1,2-benzenediol, 2-octenoic acid), strongly suggesting that the intimate association between coal particles and fungal mycelia results in rapid and near-quantitative transfer of coal depolymerisation products into the cell.     

Rapid identification of the species of the Bacteroides fragilis group by multiplex PCR assays using group- and species-specific primers

We report a rapid and reliable two-step multiplex polymerase chain reaction (PCR) assay to identify the 10 Bacteroides fragilis group species - Bacteroides caccae, B. distasonis, B. eggerthii, B. fragilis, B. merdae, B. ovatus, B. stercoris, B. thetaiotaomicron, B. uniformis and B. vulgatus. These 10 species were first divided into three subgroups by multiplex PCR-G, followed by three multiplex PCR assays with three species-specific primer mixtures for identification to the species level. The primers were designed from nucleotide sequences of the 16S rRNA, the 16S-23S rRNA intergenic spacer region and part of the 23S rRNA gene. The established two-step multiplex PCR identification scheme was applied to the identification of 155 clinical isolates of the B. fragilis group that were previously identified to the species level by phenotypic tests. The new scheme was more accurate than phenotypic identification, which was accurate only 84.5% of the time. The multiplex PCR scheme established in this study is a simple, rapid and reliable method for the identification of the B. fragilis group species. This will permit more accurate assessment of the role of various B. fragilis group members in infections and of the degree of antimicrobial resistance in each of the group members.     

Degradation of Sulfhydryl Groups in Soymilk by Lipoxygenases during Soybean Grinding

The process of degradation of sulfhydryl (SH) groups in soymilk was investigated by using Glycine max var. Suzuyutaka (wild type) and the following seven mutant lines lacking lipoxygenase(s): L-1-, L-2-, L-3-, L-1,-2-, L-2,-3-, L-1,-3-, and L-1,-2,-3-null. The soymilk prepared from the L-1,-2,-3-null line of all the mutants had the highest SH content. The content of SH groups was the lowest with the L-1,-3-null line of the three double-null lines and the highest with the L-2-null line of the three single-null lines. These results show that lipoxygenases strongly participated in the degradation of SH groups in soymilk and that the L-2 isozyme had the greatest SH-degrading capability. When these soybean samples were ground under low-temperature conditions and in a nitrogen (N₂) atmosphere to inhibit the degradation of SH groups caused by lipoxygenases, SH degradation of the L-2,-3- and L-1,-3-null lines was strongly inhibited at low temperature, while that of the L-1,-2-null line was strongly inhibited in the N₂ atmosphere. In view of the strong inhibition of SH degradation in an N₂ atmosphere with Suzuyutaka (wild type), which has three L-1,-2,-3 isozymes, these results suggest that not only the L-2 isozyme but also that the L-3 isozyme of the three in Suzuyutaka played an important role in SH degradation during soybean grinding.     

Relationship between the secretor status and the expression of ABH blood group antigenic determinants in human intestinal brush-border membrane hydrolases

At least six hydrolases of the human intestinal brush-border membrane bear ABH blood group antigenic determinants related to the erythrocyte phenotype: the intestinal glycoproteins of blood group A and B subjects express A or B determinants, respectively, while blood group O subjects express the H determinant identified with Ulex europaeus lectin I. These expressions are under the control of the secretor gene: ABH antigens were not detected in the hydrolases of non-secretor subjects.     

Degradation of polychlorinated biphenyls by extracellular enzymes of Phanerochaete chrysosporium produced in a perforated plate bioreactor

The white rot fungus Phanerochaete chrysosporium was cultivated in a perforated plate bioreactor and the expression of activities of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) was measured. Peak activities of the two enzymes were reached close to day 11 and therefore the cultivation was terminated on that day. Extracellular proteins were concentrated and both peroxidases separated by isoelectric focusing. Degradation of technical PCB mixtures containing low and highly chlorinated congeners (Delor 103 and Delor 106 as equivalents of Aroclor 1242 and Aroclor 1260, respectively) was performed using intact mycelium, crude extracellular liquid and enriched MnP and LiP. A decrease in PCB concentration caused by a 44-h treatment with mycelium (74% w/w for Delor 103 and 73% for Delor 106) or crude extracellular liquid (62% for Delor 103 and 58% for Delor 106) was observed. The degradation was not substrate-specific, because no significant differences between the respective degradation rates were observed with di-, tri-, tetra-, penta-, hexa-, hepta-, and octachlorinated congeners. In contrast, MnP and LiP isolated from the above-mentioned extracellular liquid did not catalyse any degradation.     

探讨不同浓度的清郁和降汤对反流性食管炎大鼠肠道菌群的影响

【目的】观察不同浓度的清郁和降汤对反流性食管炎（reflux esophagitis,RE）的治疗效果,并探讨其对肠道菌群的影响。【方法】将36只健康雄性SD大鼠随机分为6组,其中1组为假手术组,剩余5组大鼠采用“前胃结扎+外置幽门部分结扎术”手术造模方法建立反流性食管炎模型。造模2周后将术后全部存活的30只大鼠随机分成对照组、中药高剂量组（予高剂量清郁和降汤）、中药中剂量组（予中剂量清郁和降汤）、中药低剂量组（予低剂量清郁和降汤）、西药组（予泮托拉唑钠肠溶胶囊+枸橼酸莫沙比利分散片+复方嗜酸乳杆菌片）,每组6只。于术后第15天开始灌胃,其中假手术组及对照组予蒸馏水灌胃,其他组分别给予相应的药物灌胃,持续灌胃14 d后将所有大鼠处死后进行取材。以苏木精-伊红（hematoxylin-eosin,HE）染色观察大鼠食管组织的病理学改变;采用16S rRNA基因高通量测序检测其肠道黏膜的菌群构成。【结果】反流性食管炎大鼠存在着较为明显的肠道菌群结构变化及肠道菌群多样性较低的情况,B组（对照组）中厚壁菌门和拟杆菌门占比减少,变形菌门占比增多,且假单胞菌属、青枯菌属等细菌增多。低、中、高3种浓度的清郁和降汤均能够提升RE大鼠的肠道菌群多样性,增加拟杆菌门及厚壁菌门,降低变形菌门的占比,从属水平上看,清郁和降汤能够提升大鼠肠道中拟杆菌属、乳杆菌属、瘤胃球菌属、颤螺旋菌属、双歧杆菌属和狄氏副拟杆菌属等益生菌占比。以D组（中剂量组）对大鼠肠道菌群多样性提升最为明显,其效果最接近假手术组。特征微生物方面,B组以变形菌门为特征性微生物,D、E两组出现了放线菌门及拟杆菌门下属细菌为特征微生物的情况,在门水平与F组相同。【结论】清郁和降汤能够有效地治疗反流性食管炎,其机制可能与改变RE大鼠的肠道菌群结构、减少有害菌、提升益生菌的占比和改善肠道菌群多样性有关。