Superoxide dismutases of virulent and avirulent strains of Brucella abortus

Extracts of Brucella abortus strains 2308,RB51,45/20 and ST 19 had no significant differences in superoxide dismutase (SOD) activity as measured by the epinephrine assay. These B. abortus strains represent smooth, intermediate and rough colony forms. SOD activity was inhibited 60 to 75% by 2 mM KCN and suggests the presence of Cu/Zn SOD. The SOD activities were similar when the strains were grown in trypticase soy broth containing either 0.5% glucose or erythritol. There were two distinct SOD activity bands in native polyacrylamide gel electrophoresis with identical mobilities for each of the strains. When the native gel was stained for SOD activities in the presence of 2 mM KCN, the SOD band that co-migrated with the bovine erythrocyte Cu/Zn SOD activity disappeared. The band of SOD activity that migrated similar to E. coli iron SOD activity was unaffected by KCN. There were no significant differences in either the total SOD or Cu/Zn SOD activities among the strains. As the Brucella strains represent ranges of virulence, it is difficult to associate any primary role for SOD as a virulence factor.     

低温对青海扁茎早熟禾活性氧代谢的影响和相关基因表达分析

为研究青海扁茎早熟禾(BJ)在低温胁迫下活性氧积累、抗氧化酶防御与逆境相关基因表达的机制,以WY-2(草地早熟禾‘午夜2号')为对照,对这两种材料低温适应(10/5 ℃) 3 d和冷冻胁迫(-10 ℃) 24 h,测定了O^-·₂产生速率、H₂O₂含量、丙二醛(MDA)含量、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)活性及其抗氧化酶相关基因、冷胁迫相关转录因子的相对表达量。结果表明,低温胁迫可引起BJ和WY-2叶片内的O^-·₂产生速率、H₂O₂含量和MDA含量的增加,BJ活性氧的积累程度和MDA含量均低于WY-2;低温胁迫可导致BJ的SOD、APX和GR活性的降低,CAT活性呈先升后降的趋势,但只有APX活性表现为BJ高于WY-2,同时,BJ和WY-2叶片内的Cu/ZnSOD、CAT、APX和GR基因的表达量在低温胁迫后被明显的上调,且BJ抗氧化酶相关基因的相对表达量普遍高于WY-2;BJ叶片内的DREB2是下调表达的,ERF、DREB1、CSP和HSP基因在低温胁迫后均表现出明显的上调表达,且ERF、CSP和HSP的相对表达量高于WY-2。从而得出结论,扁茎早熟禾在低温胁迫时有害物质的积累低于午夜二号,抗氧化酶相关基因表达上调幅度,以及一些与冷胁迫相关转录因子的表达调控均大于午夜二号。这些物质合成和基因表达的差异可能共同导致扁茎早熟禾的高耐冷性。     

Molecular forms of selected antioxidant enzymes in dog semen--electrophoretical identification

The aim of the study was the electrophoretical identification of molecular forms of selected antioxidant enzymes in dog semen. Ejaculates to be studied were chosen from five dogs, aged from two to eight years. Polyacrylamide gel electrophoresis was carried out under non-denaturing conditions and then gels were stained for the activity of the following enzymes: superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Sperm homogenates and all fractions (pre-spermatic, spermatic and post-spermatic) of dog ejaculate demonstrated one protein band with SOD activity characterized by low electrophoretic mobility. Based on the confirmed sensitivity to H2O2, it can be assumed that the detected SOD is an enzyme containing ions of Zn2+ and Cu2+ (Cu,Zn SOD). In sperm homogenates one protein band with GPx activity was characterized by high electrophoretic mobility, whereas in the spermatic and post-spermatic fractions of dog ejaculate three protein bands with different (low, medium and high) electrophoretic mobility were identified. CAT molecular forms were not found in either sperm homogenates or in the analyzed fractions of ejaculate.     

利用柞蚕蛹粉提取超氧化物歧化酶

以柞蚕蛹冻干粉为材料 ,经提取、盐析、浓缩、透析和DEAE 纤维素离子交换柱层析等纯化步骤 ,获得了一种超氧化物歧化酶 (SOD)。该酶蛋白结晶呈菱形 ,PAGE法显示为均一的蛋白谱带。该酶对H2 O2 和KCN敏感 ,对氯仿 乙醇不敏感 ,是一种Cu·Zn SOD。研究结果为SOD的提取提供了新酶源     

Cloning and expression of the superoxide dismutase gene of the feline strain of Porphyromonas gingivalis: immunological recognition of the protein by cats with periodontal disease

Recent evidence suggests that feline members of the genus Porphyromonas are of consequence in periodontal disease in cats. Several possible virulence factors from feline strains of Porphyromonas gingivalis have been described that have similarities to those of human P. gingivalis. Both human and feline strains of P. gingivalis produce superoxide dismutase (SOD) which has been proposed as modulator of the inflammatory response during infection. The objective of this study was to clone the superoxide dismutase gene of feline P. gingivalis, to compare the characteristics of its product with that of the native enzyme and to determine its immunoreactivity in cats with periodontal disease. The sod gene of the feline strain Veterinary Pathology and Bacteriology (VPB) 3457 of P. gingivalis was amplified by PCR and cloned in frame with the alpha-peptide of the LacZ gene of E. coli in plasmid pUC19. This construct expressed SOD activity in E. coli with characteristics similar to those of the native SOD enzyme of P. gingivalis human strain 381 and the parent feline strain VPB 3457. The recombinant SOD had an apparent molecular weight of 54,700+/-1300 (S.E.M.) and was inactivated by 5mM hydrogen peroxide but not by 2mM KCN. There was a significant association (P=0.005) between the immunoreactivity of cats to P. gingivalis VPB 3457 soluble whole cell proteins on immunoblots and their responsiveness to the SOD protein. This suggests that cats showing a marked serum responsiveness to P. gingivalis itself, react to the SOD enzyme and further supports the role of feline P. gingivalis in periodontal disease.     

家蚕血淋巴超氧化物歧化酶初步研究

采用聚丙烯酰胺凝胶垂直平板电泳，浓缩胶３．３％，分离胶６．５％，分析家蚕血淋巴超氧化物歧化酶（ＳＯＤ）。结果表明，家蚕血淋巴ＳＯＤ在大部分供试品种中能明显看到两茶酶带，少数品种只能见到两条弱带或仅存在一条活性带。品种间酶带的宽度及染色程度亦存在明显的差异。家蚕血淋巴ＳＯＤ以Ｃｕ／Ｚｎ－ＳＯＤ型为主，少数品种中亦可能存在Ｍｎ－ＳＯＤ型与Ｆｅ－ＳＯＤ型。家蚕肠液ＳＯＤ同工酶也有两条弱酶带，其ＳＯＤ酶活力较低。     

Hemagglutination and hemagglutination inhibition of turkey red blood cells with Mycoplasma hyopneumoniae

The ability of Mycoplasma hyopneumoniae to agglutinate RBC was evaluated to develop an in vitro cytadsorption assay. Using swine RBC in a microtitration hemagglutination test, no agglutination or partial agglutination was detected. Comparison of RBC from various other species indicated that improved hemagglutination was obtained with RBC from turkeys. This hemagglutination was detected only when mycoplasma cells used in the assay had been frozen and thawed, heated at 50 C for 30 minutes, or treated with trypsin. Treatment of RBC with trypsin or neuraminidase enhanced hemagglutination. Possible surface lectin activity in M hyopneumoniae was evaluated by use of carbohydrates in a blocking assay; hemagglutination was not inhibited by any of 13 carbohydrates evaluated. Mycoplasma hyopneumoniae convalescent porcine serum and monoclonal antibodies against 2 M hyopneumoniae immunogens of molecular weights of 64,000 and 41,000 inhibited hemagglutination.     

1,25‐Dihydroxyvitamin D3 and its analogues increase catalase at the mRNA,protein and activity level in a canine transitional carcinoma cell line

Antioxidant enzymes, such as catalase, superoxide dismutases (SOD), MnSOD and Cu/ZnSOD, protect cells by scavenging reactive oxygen species (ROS). Numerous studies have reported the anti‐cancer effects of 1,25‐dihydroxyvitamin D₃ (calcitriol) and its related analogues, seocalcitol and analogue V. In this study, canine bladder transitional cell carcinoma (cbTCC) cells were used to determine effects of calcitriol and its related analogues on antioxidant enzyme gene expression, protein expression and activity. Catalase mRNA was increased in response to calcitriol (10^?7 M), and seocalcitol (10^?7 and 10^?9 M). MnSOD mRNA was decreased in response to calcitriol at 10^?7 M. Catalase was significantly increased in response to calcitriol (10^?7 and 10^?9 M), and seocalcitol (10^?9 M). Catalase enzymatic activity increased in response to calcitriol, seocalcitol and analogue V (10^?9 M). In addition, global gene expression analysis identified the involvement of mitogen‐activated protein kinase (MAPK) signalling in cbTCC's response to calcitriol and seocalcitol treatment.     

Cell-mediated and humoral immune response to Mycoplasma hyopneumoniae in pigs enhanced by dextran sulfate

The effect of dextran sulfate (DS), known to be cytotoxic to macrophages, on the cell-mediated and humoral immune response to nonviable Mycoplasma hyopneumoniae in pigs was investigated. The cell-mediated immune response was determined by means of lymphocyte transformation a test, using uptake of [3H]thymidine in a microculture system and the humoral immune response by means of a microplate complement-fixation test. Peripheral blood lymphocytes from pigs vaccinated with nonviable M hyopneumoniae and DS incorporated substantially more [3H]thymidine than did those from pigs given Mycoplasma or DS alone. The transformation of lymphocytes from M hyopneumoniae-DS vaccinated pigs was enhanced when M hyopneumoniae cells used in the assay system were heated at 60 C for 30 minutes. Similarly prepared M flocculare and M hyorhinis cells also stimulated lymphocytes from M hyopneumoniae-DS vaccinated pigs, but not nearly as great as when M hyopneumoniae cells were used. The humoral antibody response and the cell-mediated immune response to nonviable M hyopneumoniae was markedly enhanced by DS. Pigs were vaccinated with nonviable M hyopneumoniae and/or DS 4 times and challenge exposed intratracheally with viable M hyopneumoniae. Pigs vaccinated with M hyopneumoniae and DS had less severe pneumonia than did nonvaccinated pigs.     

Study of the acetylcholinesterase activity of Ascaridia galli: kinetic properties and the effect of anthelmintics.

Acetylcholinesterase (EC 3.1.1.7) activity was demonstrated in whole worm homogenates of adult Ascaridia galli with acetylthiocholine as substrate. The pH optimum was not measurable because of an autohydrolysis of the substrate. The Michaelis constant (Km) was 4 mM with saturation by excess substrate. Optimum enzyme activity was noted at a protein concentration of 200 mg/ml assay medium and at a temperature of 37 degrees C. Arrhenius plot of temperature dependence of the enzyme activity showed an energy of activation (delta Ea) of 28.962 K joule/mole above, and 25.448 K joule/mole below, the transition temperature (37 degrees C). Complete inhibition by eserine (physostigmine), a specific and classical acetylcholinesterase inhibitor, established the identity of the enzyme. A marginally higher enzyme activity was observed in females than in males as well as in homogenates from worms of mixed sexes. The enzyme was markedly activated by divalent metal cations such as Fe2+, Mg2+, Cd2+, Cu2+, Zn2+ and Ca2+, while Co2+ and Mn2+ inhibited the activity. Piperazine adipate at a concentration of 10(-3) M caused 45.5% and albendazole, a benzimidazole anthelmintic, 37.5% inhibition in the enzyme activity, while levamisole and mebendazole proved to be practically ineffective, causing an inhibition of 12 and 9%, respectively.     

Temperature and metal ions-dependent activity of the family I inorganic pyrophosphatase from the swine roundworm Ascaris suum

Temperature dependence, heat stability and metal ions-dependent activity were examined on the Family I inorganic pyrophosphatase (PPase) recently identified from Ascaris suum. Recombinant A. suum PPase (rAsPPase) showed an optimal activity at 55 degrees C. The rAsPPase was heat stable at 40 degrees C in the absence of added Mg(2+) and at 50 degrees C in its presence. The enzyme required divalent metal ions for its activity. The preferences for the metal ions (5 mM concentration) were in the order: Mg(2+)> Co(2+)> Cu(2+)> Fe(2+)> Zn(2+)> Mn(2+). On the contrary, enzyme activity was inhibited by Ca(2+). These findings suggest that catalytic features of AsPPase are consistent with the Family I PPases reported from a wide range of organisms.     

紫花苜蓿种子萌发对钴胁迫的生理生化响应

以“甘农3号”紫花苜蓿为试验材料,研究不同浓度Co²⁺(0,0.25,0.50,1.00,2.50,5.00 mmol/L CoCl₂)胁迫对紫花苜蓿种子萌发及幼苗生理生化特性的影响。结果表明:Co胁迫对紫花苜蓿种子的萌发及幼苗的生长有明显的抑制作用,随着Co胁迫浓度的增大,种子发芽势、发芽率、发芽指数、活力指数及幼苗的胚芽长、胚根长、根系活力和干重均显著降低,而且Co胁迫对发芽势的抑制作用大于发芽率,对胚根生长的抑制作用大于胚芽;低浓度Co胁迫(0.25和0.50 mmol/L)下,苜蓿幼苗叶片中可溶性蛋白、可溶性糖含量和蛋白水解酶、超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、愈创木酚过氧化物酶(GPX)、过氧化氢酶(CAT)活性与CK均无显著差异,而游离脯氨酸含量显著升高;随着Co胁迫浓度的升高,可溶性蛋白、可溶性糖、游离脯氨酸含量及蛋白水解酶、SOD、APX、GPX、CAT活性均显著下降,超氧阴离子自由基(O2·-)产生速率、羟自由基(OH^·)浓度、H₂O₂及丙二醛(MDA)含量均显著增加。说明高浓度Co胁迫使得苜蓿幼苗抗氧化系统活性下降,活性氧清除能力降低,膜脂过氧化程度加剧,从而抑制了紫花苜蓿种子的萌发及幼苗的生长。     

Suppressive effect of nonviable Mycoplasma hyopneumoniae on phytohemagglutinin-induced transformation of swine lymphocytes

The effect of nonviable Mycoplasma hyopneumoniae on transformation of swine peripheral blood lymphocytes by mitogen was investigated. Lymphocyte transformation was evaluated as incorporation of [3H]-thymidine, using a microculture system. Mycoplasma hyopneumoniae was grown in Friis medium, inactivated with sodium azide, and washed with phosphate-buffered saline solution. Four strains of M hyopneumoniae, strain J, strain 11, and 2 low-passage isolates (1361A, 1375C), were found to suppress phytohemagglutinin-induced lymphocyte transformation. Mycoplasma hyopneumoniae strains J, 11, and 1361A reduced lymphocyte transformation by about 50%, whereas strain 1375C reduced lymphocyte transformation by 98.7%. The suppressive effect was abrogated by heating M hyopneumoniae at 60 C or at higher temperatures for 30 minutes. Sonication of the heated M hyopneumoniae cells partially restored the suppressive effect.     

Cholesterol oxidase and resistance of Rhodococcus equi to peroxidative stress in vitro in the presence of cholesterol

Rhodococcus equi is a well-characterized bacterial pathogen which lyses cell membranes with the help of cholesterol oxidase (CO). Survival in macrophages is warranted by its ability to resist reactive radicals via catalase and superoxide dismutase (SOD). Therefore, CO production in the absence or presence of 0.1 % cholesterol and sensitivity to exogenous hydrogen peroxide (H2O2) and superoxide anion (SOA) were tested in seven strains of R. equi in vitro. When R. equi strains were grown on agar plates with cholesterol, the bacterial growth [colony-forming units (cfu)/plate] did not increase significantly in comparison with the growth on plates without cholesterol. The activity of CO increased, significantly for extracellular CO. In subsequent experiments, R. equi strains grown on cholesterol were stressed with H2O2 or SOA so that approximately 10 % of cfu/plate survived. During stress induced by SOA, membrane CO and SOD activity increased significantly. Catalase activity increased 2-fold with H2O2 and 3-fold with SOA exposure. These data suggest that the presence of cholesterol induces CO in bacteria grown on agar plates. Catalase, SOD and even membrane-bound CO respond to reactive oxygen species.     

A metallo phosphatase activity present on the surface of Trypanosoma brucei procyclic forms

In this work, we describe how living cells of Trypanosoma brucei procyclic forms were able to hydrolyze extracellular p-nitrophenylphosphate (pNPP). These intact parasites, which had their viability determined by motility and the Trypan blue method, presented a low level of pNPP hydrolysis in the absence of any divalent metal (0.72+/-0.07 nmol pNP/mg min). Interestingly, in the presence of 5mM MgCl(2), ectophosphatase activity of 1.91+/-0.21 nmol pNP/mg min was observed. The ectophosphatase activity was also stimulated by MnCl(2), CoCl(2) and CuCl(2) but not by CaCl(2) and CdCl(2) and was inhibited by ZnCl(2). The addition of Mg(2+), Mn(2+), Co(2+) and Cu(2+) to extracellular medium increased the ectophosphatase activity in a dose-dependent manner. At 5mM pNPP, half-maximal stimulation of pNPP hydrolysis was obtained with 0.39+/-0.05 mM MgCl(2), 0.33+/-0.03 mM MnCl(2), 1.63+/-0.12 mM CoCl(2) and 2.04+/-0.33 mM CuCl(2). In the absence of any divalent metal (basal activity) the apparent K(m) for pNPP was 0.66+/-0.09 mM, while at saturating MgCl(2) concentrations the corresponding apparent K(m) for pNPP for Mg(2+)-stimulated phosphatase activity (difference between total minus basal phosphatase activity) was 0.27+/-0.03 mM. The Mg(2+)-stimulated pNPP hydrolysis was strongly inhibited by ZnCl(2) and vanadate, while the metal-independent basal phosphatase activity was less inhibited by these phosphotyrosyl phosphatase inhibitors.     

In vitro stimulation of antibody production to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells.

A method to stimulate and detect the in vitro production of antibodies to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells (PBMC) was established. PBMC were cultured in microtiter plates coated with a sonicated M. hyopneumoniae whole cell antigen and the amount of antibody bound to the coating antigen was determined by an enzyme linked immunosorbent assay (ELISA). In addition, the amount of non-bound antibody was determined by testing the culture supernatants in the ELISA which detects porcine antibodies to M. hyopneumoniae. The production of antibodies, in terms of total absorbance values, was enhanced by including 2.5 ng pokeweed mitogen (PWM) per ml growth medium without altering the specificity of the assay. In a pilot experiment, the applicability of the method to follow the development of antigen-reactive cells during primary and secondary immunizations with M. hyopneumoniae was evaluated. Antigen-reactive cells, identified by their ability to produce antibodies to M. hyopneumoniae in vitro, were detected seven days after the primary immunization and reached their highest antigen reactivity one week later. In comparison, antigen-reactive cells could be detected three days after the booster immunization and remained in the circulation for 2 weeks.     

Beta-adrenergic receptor binding in crude porcine adipose tissue plasma membranes.

Methods have been detailed to prepare a crude membrane fraction from isolated porcine adipose tissue cells. Adipocytes were obtained after incubation of 5 g of adipose tissue slices with 4,500 units of a selected lot of collagenase in a total volume of 15 mL at 37 degrees C for 90 min. There was no bovine serum albumin present during cell isolation because albumin did not enhance cell yield or yield of lipolytic activity. Isolated cells were lysed by exposure to hypotonic conditions in the presence of 7.5 mM ethylene glycol tetraacetic acid (EGTA) and .8 mM phenylmethylsulfonyl fluoride (PMSF). A 30,000 x g centrifugal pellet was used as the crude membrane preparation. Binding of tritiated dihydroalprenolol (DHA), a beta-adrenergic antagonist, was measured in the presence of 7.5 mM EGTA and .2 mM PMSF, because these protease inhibitors improved specific binding by approximately 50% to greater than 150 fmol/mg of protein and decreased non-specific binding to less than 10% at 2.5 nM DHA.     

Experimental dual infection of pigs with an H1N1 swine influenza virus (A/Sw/Hok/2/81) and Mycoplasma hyopneumoniae

Dual infection of pigs with swine influenza virus (SIV) and Mycoplasma hyopneumoniae was carried out to compare the clinical and pathological effects of dual infection in caesarian derived and colostrums deprived (CDCD) pigs, with that of a single infection with M. hyopneumoniae. In Experiment 1, 40-day-old CDCD pigs were inoculated only with SIV (A/Sw/Hok/2/81, H1N1). The virus was isolated from nasal swabs for 5-6 days. None of these pigs showed clinical signs of infection throughout the experimental period. These results suggested that this strain can infect pigs but is only slightly pathogenic when it is inoculated singly to a CDCD pig. In Experiment 2, 60-day-old CDCD pigs were inoculated with M. hyopneumoniae and then were inoculated with SIV (A/Sw/Hok/2/81) at 1 week (MHYO-7d-SIV-7d group) or 3 weeks (MHYO-21d-SIV-7d group) after M. hyopneumoniae inoculation. Macroscopically, dark red-to-purple lung lesions were observed in all of pigs at 14 or 28 days post-inoculation. Percentages of dark red-to-purple lung lesions in dual infection groups (MHYO-7d-SIV-7d group: 18.7 +/- 4.2%, MHYO-21d-SIV-7d group: 23.0 +/- 8.0%) were significantly (P < 0.05) increased compared to those of each control group in which pigs were inoculated only with M. hyopneumoniae (MHYO-14d group: 4.7 +/- 2.9%, MHYO-28 group: 3.3 +/- 2.4%). Microscopically, bronchial epithelial lesions (epithelial disruption, degeneration, hyperplasia and formation of microabscess) were frequently observed in dark red-to-purple lung lesions of only the dual infection groups. These results demonstrate that the lung lesion of pigs inoculated with M. hyopneumoniae and SIV is more severe than that of pigs inoculated only with M. hyopneumoniae.     

In vitro activity of tiamulin against porcine mycoplasmas

The activity of tiamulin against Mycoplasma hyopneumoniae, M flocculare, M hyorhinis and M hyosynoviae grown in liquid medium was assessed in vitro. With the first three of these mycoplasmas, the activity of tylosin and oxytetracycline was observed in parallel. Tiamulin was more active against M hyopneumoniae and M flocculare, but there was less disparity between the three antibiotics with the strain of M hyorhinis tested. Tiamulin was notably more active against M hyosynoviae than against M hyopneumoniae. It was more difficult to suppress M hyopneumoniae than the other mycoplasmas with tiamulin. This persistence of M hyopneumoniae was more striking when M hyopneumoniae and M hyosynoviae were tested in parallel.     

Epigallocatechin-3-gallate from green tea negatively affects swine granulosa cell function

The use of herbs as additives in livestock nutrition as an alternative to antibiotics is becoming a new goal in animal production. It is known that green tea exerts antimicrobial activity owing to specific flavonoid compounds named catechins, primarily represented by epigallocatechin-3-gallate (EGCG). Remarkably, despite many potential benefits of green tea and EGCG consumption, it is also important to get an insight on the possible reproductive-related consequences of feeding supplementation. To this purpose, granulosa cells were harvested from follicles > 5mm and treated with 5 and 50 microg/ml of EGCG in order to evaluate the effects on the main parameters of granulosa cell function: steroidogenesis, by measuring progesterone and estradiol-17beta production, and proliferation, one of the major feature of ovarian follicular growth. Moreover, as the genesis of new vessels has been demonstrated to be fundamental for follicle development, we evaluated the effect of EGCG on the production of the main angiogenetic factor, VEGF, by swine granulosa cells. Finally, since reactive oxygen species (ROS) might be involved in the control of female reproductive activity, we studied the effect of EGCG on superoxide anion (O2-) and hydrogen peroxide (H2O2) production by swine granulosa cells and on the activity of the scavenging enzyme superoxide dismutase (SOD). EGCG significantly (p < 0.05) inhibited proliferation, steroidogenesis, VEGF and O2- production by swine granulosa cells; on the contrary, H2O2 levels and SOD activity were stimulated (p < 0.05) by the catechin. Therefore, since our data demonstrate that EGCG has a negative effect on reproductive performances in swine, feeding supplementation should be carefully considered.