Expression and immunological evaluation of elongation factor Tu of Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2) is considered as a major pathogen that causes sepsis and meningitis in piglets and humans, but knowledge of its antigenic proteins remains limited so far. The surface-related proteins of pathogens often play significant roles in bacterium-host interactions and infection. Here, we obtained the elongation factor Tu (EF-Tu) gene of Streptococcus suis and constructed the recombinant expression plasmid successfully. The target recombinant plasmid was then expressed in Escherichia coli and the immuno-protection of the recombinant protein was subsequently evaluated as well. The EF-Tu gene of Streptococcus suis is 1197 bp in length, encodes 398 amino acids. The target recombinant EF-Tu (rEF-Tu) protein can recognize the antiserum of Streptococcus suis and can provoke obvious humoral immune responses in rabbits and conferred protection to rabbits against Streptococcus suis ear-vein challenge, implying that the EF-Tu may be used as an attractive candidate antigen for a component of subunit vaccine.

A genome-wide association study reveals candidate genes for the supernumerary nipple phenotype in sheep (Ovis aries).

Genome-wide association studies (GWASs) have been widely applied in livestock to identify genes associated with traits of economic interest. Here, we conducted the first GWAS of the supernumerary nipple phenotype in Wadi sheep, a native Chinese sheep breed, based on Ovine Infinium HD SNP BeadChip genotypes in a total of 144 ewes (75 cases with four teats, including two normal and two supernumerary teats, and 69 control cases with two teats). We detected 63 significant SNPs at the chromosome-wise threshold. Additionally, one candidate region (chr1: 170.723-170.734 Mb) was identified by haplotype-based association tests, with one SNP (rs413490006) surrounding functional genes BBX and CD47 on chromosome 1 being commonly identified as significant by the two mentioned analyses. Moreover, Gene Ontology enrichment for the significant SNPs identified by the GWAS analysis was functionally clustered into the categories of receptor activity and synaptic membrane. In addition, pathway mapping revealed four promising pathways (Wnt, oxytocin, MAPK and axon guidance) involved in the development of the supernumerary nipple phenotype. Our results provide novel and important insights into the genetic mechanisms underlying the phenotype of supernumerary nipples in mammals, including humans. These findings may be useful for future breeding and genetics in sheep and other livestock.

Genome-wide association analysis identifies the genetic basis of fat deposition in the tails of sheep (Ovis aries).

Fat-tailed sheep (Ovis aries) can survive in harsh environments and satisfy human's intake of dietary fat. However, the animals require more feed, which increases the cost of farming. Thus, most farmers currently prefer thin-tailed, short-tailed or docked sheep. To date, the molecular mechanism of the formation of fat tails in sheep has not been completely elucidated. Here, we conducted a genome-wide association study using phenotypes and genotypes (the Ovine Infinium HD SNP BeadChip genotype data) of two breeds of contrasting tail types (78 Small-tailed and 78 Large-tailed Han sheep breeds) to identify functional genes and variants associated with fat deposition. We identified four significantly (rs416433540, rs409848439, rs408118325 and rs402128848) and three approximately associated autosomal SNPs (rs401248376, rs402445895 and rs416201901). Gene annotation indicated that the surrounding genes (CREB1, STEAP4, CTBP1 and RIP140, also known as NRIP1) function in lipid storage or fat cell regulation. Furthermore, through an X-chromosome-wide association analysis, we detected significantly associated SNPs in the OARX: 88-89 Mb region, which could be a strong candidate genomic region for fat deposition in tails of sheep. Our results represent a new genomic resource for sheep genetics and breeding. In addition, the findings provide novel insights into genetic mechanisms of fat deposition in the tail of sheep and other mammals.

Development of an indirect ELISA for bovine mastitis using Sip protein of Streptococcus agalactiae.

The sip gene encoding for a conserved highly immunogenic surface protein of Streptococcus agalactiae was amplified using polymerase chain reaction (PCR) and subcloned into prokaryotic expression vector pET32a (+) and expressed as a recombinant protein in E. coli BL21 (DE3). An indirect enzyme linked immunosorbent assay (ELISA) was developed using the purified Sip protein as a coating antigen, which could identify S. agalactiae specific antibody in sera. The coating antigen at a concentration of 3.125 µg/ml, serum diluted to 1:160, and HRP-conjugated secondary antibody concentration at 1:4000 was found to be most effective in exhibiting positive result. The ELISA was found to be highly specific for S. agalactiae that may be used for the detection of the pathogen in mastitis cases, for epidemiological studies and for surveillance.

Resumption of menstruation and pituitary response to gonadotropin-releasing hormone in functional hypothalamic amenorrhea subjects undertaking estrogen replacement therapy.

BACKGROUND: Functional hypothalamic amenorrhea (FHA) refers to a functional menstrual disorder with various causes and presentations. Recovery of menstrual cyclicity is common in long-term follow-up but the affecting factors remain unknown. AIM: To explore factors affecting the menstrual resumption and to evaluate the pituitary response to gonadotropin-releasing hormone (GnRH) in FHA. MATERIALS AND METHODS: Thirty cases with FHA were recruited. All subjects were put on continuous 1 mg/day estradiol valerate orally and followed up monthly. Recovery was defined as the occurrence of at least three consecutive regular cycles. Responder referred to those who recovered within two years of therapy. Gonadotropin response to the 50 µg GnRH challenge was tested every three months. RESULTS: Nineteen (63.3%) subjects recovered with a mean time to recovery of 26.8 months. Time to recovery was negatively correlated with body mass index (BMI) before and by amenorrhea. Twentyone cases had undertaken therapy for more than two years and 10 of them recovered. BMI before and by amenorrhea were negatively correlated with the recovery. Significant increase of serum luteinizing hormone (LH) and LH response to GnRH were noted after recovery. CONCLUSIONS: Menstrual resumption was common in FHA undertaking estrogen replacement therapy (ERT). The likelihood of recovery was affected by their BMI before and by amenorrhea but not by the weight gain during therapy. Low serum LH and attenuated LH response to GnRH were the main features of pituitary deficiency in FHA. The menstrual resumption in FHA was accompanied by the recovery of serum LH and the LH response to GnRH.

Design of fuzzy cognitive maps using neural networks for predicting chaotic time series.

As a powerful paradigm for knowledge representation and a simulation mechanism applicable to numerous research and application fields, Fuzzy Cognitive Maps (FCMs) have attracted a great deal of attention from various research communities. However, the traditional FCMs do not provide efficient methods to determine the states of the investigated system and to quantify causalities which are the very foundation of the FCM theory. Therefore in many cases, constructing FCMs for complex causal systems greatly depends on expert knowledge. The manually developed models have a substantial shortcoming due to model subjectivity and difficulties with accessing its reliability. In this paper, we propose a fuzzy neural network to enhance the learning ability of FCMs so that the automatic determination of membership functions and quantification of causalities can be incorporated with the inference mechanism of conventional FCMs. In this manner, FCM models of the investigated systems can be automatically constructed from data, and therefore are independent of the experts. Furthermore, we employ mutual subsethood to define and describe the causalities in FCMs. It provides more explicit interpretation for causalities in FCMs and makes the inference process easier to understand. To validate the performance, the proposed approach is tested in predicting chaotic time series. The simulation studies show the effectiveness of the proposed approach.

Down-regulation of cyclooxygenase-2 is involved in ischemic postconditioning protection against renal ischemia reperfusion injury in rats.

Ischemic postconditioning (IPostC) is a phenomenon whereby rapid intermittent interruptions of blood flow in the early phase of reperfusion protect an organ from ischemia-reperfusion injury. In the present study, we investigated whether the protective effect of IPostC was associated with the cyclooxygenase-2 (COX-2) pathway by evaluating its expression following renal ischemia-reperfusion in rats. Animals underwent 45 minutes of renal pedicle occlusion followed by reperfusion for 1.5, 3, 6, 12, or 24 hours. IPostC was performed by six 10-second cycles of reperfusion and 10 seconds of renal pedicle occlusion at the end of ischemia. Blood and kidney samples were collected at each reperfusion time point. The protein expression of COX-1 and COX-2 were evaluated by Western blotting. Our data showed that IPostC attenuated the renal dysfunction and decreased COX-2 expression induced by ischemia-reperfusion insults. The results indicated that the protective effect of IPostC was related to down-regulation of COX-2 expression.

Ischemic postconditioning modified renal oxidative stress and lipid peroxidation caused by ischemic reperfusion injury in rats.

Several recent studies have shown that ischemic postconditioning (IPostC) protects hears from ischemic reperfusion insults in various animal models. However, the mechanism of IPostC remains unclear. In the present study, we investigated the hypothesis that PostC protected kidneys against ischemic reperfusion injury by modifying renal oxidative stress and lipid peroxidation. Rats underwent 45 minutes of renal pedicle ligature followed by reperfusion for 1, 3, 6, 12, or 24 hours. IPostC was performed using 6, 10 second cycles of reperfusion and 10 seconds of renal pedicle occlusion at the end of the ischemia. Our data showed that IPostC attenuated renal dysfunction, significantly increasing the activity of antioxidases, including superoxide dismutase (SOD), catalase (CAT), and glutathione perokidase (GSH-Px) in renal homogenates, and concentrations of GSH and SOD expression. The level of malondialdehyde (MDA) and the activity of myeloperoxidase (MPO) were significantly decreased in IPostC rats. These results indicated that the protective effects of IPosC may be related to modification of renal oxidative stress and lipid peroxidation caused by ischemic reperfusion injury in rats.

A novel flocculant of Al(OH)3-polyacrylamide ionic hybrid.

A novel flocculant based on hybrid Al(OH)(3)-polyacrylamide (HAPAM) has been synthesized using a redox initiation system ((NH(4))(2)S(2)O(8)-NaHSO(3)) at 40 degrees C in aqueous medium. The HAPAM was characterized by viscometry, IR spectroscopy, TEM, conductivity, and TGA. The flocculation behavior for 0.25 wt% kaolin suspension was evaluated by spectrophotometry and phase contrast microscopy. It was found that an ionic bond exists between Al(OH)(3) colloid and polyacrylamide (PAM) chains in the HAPAM and the flocculation efficiency of HAPAM is much better than that of commercial polyacrylamide (PAM) and PAM/AlCl(3) blend.

Effect of copper aspirinate on contraction of isolated rabbit aortic strips.

AIM: To investigate the effect of copper aspirinate on contraction of vascular smooth muscle. METHODS: Isolated rabbit aortic strips, including intact endothelium strips and endothelial cell-denuded aortic strips, were suspended in modified Krebs'solution to determine effects of copper aspirinate on the contraction induced by norepinephrine (NE), KCl, and CaCl2, while CuSO4, aspirin, and vehicle were used as controls. RESULTS: Copper aspirinate possessed antagonistic effect on contraction of rabbit aortic strips induced by NE with an IC50 value of 31 nmol/L, while CuSO4 had much l ess antagonistic effect with an IC50 value of 0.29 micromol/L, and aspirin did not work in the same preparation. No effect of copper aspirinate were found on the contraction induced by KCl and CaCl2. Effects on endothelial cell denuded aortic strips were similar to those in the normal aortic strips. CONCLUSION: Copper aspirinate possessed different effects from aspirin and CuSO4 on vascular smooth muscles. It inhibited contraction induced by NE with an activity stronger than CuSO4 at the same Cu2+ concentration, this action might be due to blockade of the receptor-operated Ca2+ channels, and it might not be linked to the endothelium.

Paeonilide, a novel anti-PAF-active monoterpenoid-derived metabolite from Paeonia delavayi.

A novel monoterpenoid-derived metabolite, paeonilide, was isolated from the roots of Paeonia delavayi. Its structure was established by a combination of spectroscopic and X-ray crystallographic analyses. It showed an anti-PAF effect with an IC50 value of ca. 8 microg/ml.

Effect of protopine on cytosolic Ca2+ in rabbit platelets.

AIM: To study the influence of protopine (Pro) on the cytoplasmic free Ca2+ concentration ([Ca2+]i) in rabbit platelets. METHODS: Measurement of [Ca2+]i of platelets in vitro by Fura 2-AM fluorescence technique. RESULTS: In the presence of CaCl2 1 mmol.L-1, Pro 10, 20, and 40 mumol.L-1 attenuated the rise in [Ca2+]i evoked by ADP from (420 +/- 57) to (320 +/- 26), (264 +/- 21), and (180 +/- 14) nmol.L-1, respectively, by arachidonic acid (AA) from (280 +/- 36) to (210 +/- 17), (184 +/- 21), and (143 +/- 16) nmol.L-1, respectively, and by platelet-activating factor (PAF) from (350 +/- 42) to (282 +/- 31), (223 +/- 30), and (165 +/- 15) nmol.L-1, respectively. In the presence of egtazic acid 1 mmol.L-1, Pro 10, 20, and 40 mumol.L-1 reduced the Ca2+ release induced by ADP, AA, and PAF, respectively. Pro 10, 20, and 40 mumol.L-1 also decreased ADP-, AA-, and PAF-induced Ca2+ influx. CONCLUSION: Pro inhibited not only Ca2+ release but also the influx of Ca2+.

Effects of copper-aspirin complex on plasma 6-keto-prostaglandin F(1alpha) level and platelet cytosolic calcium in rabbits.

Effects of copper-aspirin complex on washed platelet aggregation, thromboxane B(2) formation and 6-ketoprostaglandin F(1alpha) level were monitored by Born's and Terashita's methods, respectively. The influence of copper-aspirin complex on cytosolic free calcium was examined using the fluorescent indicator, Fura 2-AM. Copper-aspirin complex significantly inhibited arachidonic acid-induced aggregation in washed platelets. The IC(50) value was 9.6 micromol L(-1). Copper-aspirin complex significantly decreased arachidonic acid-induced thromboxane B(2) formation by 87.1% in washed platelets. Ten mg kg(-1) of copper-aspirin complex given intragastrically markedly increased the plasma level of 6-keto-prostaglandin F(1alpha). Aspirin, however, reduced both thromboxane B(2) formation and 6-keto-prostaglandin F(1alpha) level. In the presence of CaCl2 1 mmol L(-1), copper-aspirin complex (20, 40 and 80 micromol L(-1)) markedly lowered arachidonic acid-induced increase in platelet calcium from the resting level (270+/-36 nmol L(-1)) to 213+/-14, 170+/-20 and 135+/-17 nmol L(-1), respectively. In the presence of ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid 1 mmol L(-1), copper-aspirin complex (20, 40 and 80 micromol l L(-1)) significantly suppressed the release of intracellular calcium induced by arachidonic acid from 127+/-23 nmol L-1 to 108+/-17, 93+/-12 and 70+/-13 nmol L(-1).

Effects of copper-aspirin complex on platelet aggregation and thrombosis in rabbits and mice.

The effects of intragastric and intraduodenal copper-aspirin complex on rabbit platelet aggregation were observed by Born's method. Myers's method was used to evaluate the antithrombotic effect of copper-aspirin complex in mice. In-vitro copper-aspirin complex selectively inhibited arachidonic acid-induced platelet aggregation with an IC50 value (concentration resulting in 50% inhibition) of 13.2 microM (95% confidence limits 9.1-16.8 microM). Copper-aspirin complex (10 mg kg(-1) given intragastrically or intraduodenally) was more potent than aspirin in inhibiting arachidonic acid-induced platelet aggregation. Copper-aspirin complex (10 mg kg(-1)) had a stronger inhibitory effect and a longer duration of action when given intragastrically than when given intraduodenally. It was shown by radioimmunoassay that copper-aspirin complex significantly reduced the level of thromboxane B2 in plasma while markedly increasing that of 6-ketoprostaglandin F1alpha (6keto-PGF1alpha). Copper-aspirin complex (10 mg kg(-1) given intragastrically for 7 days) significantly reduced mouse mortality caused by intravenous injection of arachidonic acid. The results suggest that both in-vitro and in-vivo copper-aspirin complex is more potent in selectively inhibiting arachidonic acid-induced platelet aggregation than aspirin. When given intragastrically the complex has a more potent antiplatelet effect and a longer duration of action than when given intraduodenally. The antithrombotic effect of the complex was more potent than that of aspirin.

Inhibitory effects of copper-aspirin complex on platelet aggregation.

AIM: To study the inhibitory effects of copper-aspirin complex (CuAsp) on platelet aggregation. METHODS: With adenosine diphosphate the effects of CuAsp on platelet aggregation in vitro or in vivo were investigated. Radioimmunoassay and fluorophotometry were used to measure thromboxane B2 (TXB2) generation from platelets, the levels of TXB2 and of 6-keto-PGF1 alpha in plasma and the platelet serotonin release reaction. RESULTS: In vitro, CuAsp inhibited arachidonic acid (AA)-induced aggregation (IC50 = 17 mumol.L-1, 95% confidence limits: 9-33 mumol.L-1), the release of 5-HT (IC50 = 19 mumol.L-1, 95% confidence limits: 10-30 mumol.L-1), and TXB2 generation from platelets (P < 0.05). CuAsp 10 mg.kg-1 i.g. selectively inhibited AA-induced aggregation, and increased the 6-keto-PGF1 alpha concentration in plasma while decreased that of TXB2. CONCLUSION: CuAsp, in vitro or in vivo, shows more potent inhibitory effects on AA-induced aggregation than aspirin (Asp), related to the inhibition of platelet cyclooxygenase and the release of active substances from platelets.

Coordination of copper with aspirin enhances its anti-platelet aggregation activity.

Anti-platelet aggregation of copper aspirinate, a copper complex of aspirin, has been studied in vitro and in vivo. The result shows that copper aspirinate is much more effective than aspirin against AA-, ADP- and PAF-induced platelet aggregation. Its mechanism is related to the inhibition of platelet cyclooxygenase and release of active substances from platelets, and to the promotion of PGI(2) level in plasma.

Application of biotin-avidin system, determination of circulating immune complexes, and evaluation of antibody response in different hydatidosis patients.

The avidin-biotin-peroxidase complex enzyme-linked immunosorbent assay (ABC-ELISA) and standard ELISA were used for the detection of Echinococcus granulosus antibody in sera of 101 patients operated on for hydatid disease, 40 patients with miscellaneous nonhydatid diseases, and 61 normal subjects. Sensitivity and specificity of the two procedures were comparable and the geometric mean antibody titer detected with ABC-ELISA was higher than with standard ELISA. The ABC-ELISA is a sensitive, specific, simple, and convenient method for diagnosing hydatidosis.