Characterization of a fibrinogen-clotting enzyme from Trimeresurus stejnegeri venom, and comparative study with other venom proteases

Trimeresurus stejnegeri venom, which contains TSV-PA (a specific plasminogen activator sharing 60-70% sequence homology with venom fibrinogen-clotting enzymes), also possesses fibrinogen-clotting activity in vitro. A fibrinogen-clotting enzyme (stejnobin) has been purified to homogeneity by gel filtration and ion-exchange chromatography on a Mono-Q column. It is a single-chain glycoprotein with a mol. wt of 44,000. The NH2-terminal amino acid sequence of stejnobin shows great homology with venom fibrinogen-clotting enzymes and TSV-PA. Like TSV-PA, stejnobin was able to hydrolyse several chromogenic substrates. Comparative study of substrate specificities of stejnobin and other venom proteases purified in our laboratory was carried out on five chromogenic substrates. Stejnobin clotted human fibrinogen with a specific activity of 122 NIH thrombin-equivalent units/mg protein. However, stejnobin did not act on other blood coagulation factors, such as factor X, prothrombin and plasminogen. Diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride inhibited its activity, whereas ethylenediamine tetracetic acid had no effect on it, indicating that it is a serine protease. Although stejnobin showed strong immunological cross-reaction with polyclonal antibodies raised against TSV-PA, it was interesting to observe that, unlike the case of TSV-PA, these antibodies did not inhibit the amidolytic and fibrinogen-clotting activities of stejnobin. (C) 1998 Elsevier Science Ltd. All rights reserved.

Characterization of a thrombin-like enzyme from the venom of Trimeresurus jerdonii

From the venom of Trimeresurus jerdonii, a distinct thrombin-like enzyme, called jerdonobin. was purified by DEAF A-25 ion-exchange chromatography, Sephadex G-75 gel filtration, and fast protein liquid chromatography (FPLC). SDS-PAGE analysis of this enzyme shows that it consists of a single polypeptide chain with a molecular weight of 38,000. The NH2-terminal amino acid sequence of jerdonobin has great homology with venom thrombin-like enzymes documented. Jerdonobin is able to hydrolyze several chromogenic substrates. The enzyme directly clots fibrinogen with an activity of 217 NIH units/mg, The fibrinopeptides released, identified by HPLC consisted of fibrinopeptide A and a small amount of fibrinopepide B. The activities of the enzyme were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB). However, metal chelator (EDTA) had no effect on it. indicating it is venom serine protease. (C) 2000 Elsevier Science Ltd. All rights reserved.

Molecular characterization of a weak fibrinogen-clotting enzyme from Trimeresurus jerdonii venom

A fibrinogen-clotting enzyme designed as jerdonobin-II was isolated from the venom of Trimeresurus jerdonii. It differed in molecular weight and N-terminal sequence with the previously isolated jerdonobin, a thrombin-like enzyme from the same venom. The enzyme consists of a single polypeptide chain with molecular weights of 30,000 and 32,000 under non-reducing and reducing conditions, respectively. Jerdonobin-II showed weak fibrinogen clotting activity and its activity unit on fibrinogen was calculated to be less than one unit using human thrombin as standard. The precursor protein sequence of jerodonobin-II was deduced from cloned cDNA sequence. The sequence shows high similarity (identity = 89%) to TSV-PA, a specific plasminogen activator from venom of T stejnegeri. Despite of the sequence similarity, jerdonobin-II was found devoid of plasminogen activating effect. Sequence alignment analysis suggested that the replacement of Lys(239) in TSV-PA to Gln(239) in jerdonobin-II might play an important role on their plasminogen activating activity difference. (C) 2005 Elsevier Ltd. All rights reserved.

GENETIC DIVERSITY IN THE CHINESE PANGOLIN (MANIS-PENTADACTYLA) - INFERRED FROM RESTRICTION ENZYME ANALYSIS OF MITOCHONDRIAL DNAS

Two different forms of Chinese pangolins can be recognized according to the color of their scales, i.e., brown and dusky. We analyzed mitochondrial DNA (mtDNA) purified from the livers of seven dusky and six brown Chinese pangolins from the same locality, using cleavage patterns from 19 restriction enzymes. From the 19 6-bp recognition enzymes used, 51-56 sites were observed. By combining the cleavage patterns for each enzyme, the 13 samples were classified into four restriction types: two in dusky and two in brown Chinese pangolins. The estimated number of nucleotide substitutions per site in dusky and brown types is 0.002, and that between dusky and brown types is 0.012. Divergence between brown and dusky forms began 0.6 Myr ago, provided the mean rate of sequence divergence is 0.02 per Myr in mtDNA. Our results suggest that there is considerable divergence in Chinese pangolins, and brown and dusky Chinese pangolins may be quite different forms or, at least, belong to different maternal groups.

PHYLOGENY OF RHESUS-MONKEYS (MACACA-MULATTA) AS REVEALED BY MITOCHONDRIAL-DNA RESTRICTION ENZYME ANALYSIS

Mitochondrial DNA, purified from 36 samples of 23 local populations which are widely distributed in Vietnam, Burma, and 10 provinces of China, has been analyzed to model the phylogeny of rhesus monkeys. The 20 local populations of China may represent nearly all major populations in China. Using 20 restriction endonucleases of 6-bp recognition, we observed a total of 50-61 sites in the various samples. By combining the cleavage patterns for each enzyme, the 36 samples were classified into 23 restriction types, each of which was found exclusively in the respective population from which samples were obtained By combining the earlier study of Indian rhesus monkeys, phylogenetic trees, which have been constructed on the basis of genetic distance, indicate that rhesus monkeys in China, Vietnam, India, and Burma can be divided into seven groups. Integrating morphological and geographical data, we suggest that rhesus monkeys in China, Vietnam, and Burma may be classified into six subspecies-M. m. mulatta, M. m. brevicaudus, M. m. lasiotis, M. m. littoralis, M. m. vestita, and M. m. tcheliensis-and rhesus monkeys in India may be another valid subspecies. M. m. tcheliensis is the most endangered subspecies in China. Divergence among subspecies may have begun 0.9-1.6 Ma. The radiation of rhesus monkeys in China may have spread from the southwest toward the east. The taxonomic status of the Hainan monkey and the Taiwan monkey require further investigation.

PHYLOGENY OF THE SLOW LORISES (GENUS NYCTICEBUS) - AN APPROACH USING MITOCHONDRIAL-DNA RESTRICTION ENZYME ANALYSIS

Mitochondrial DNA polymorphisms in 15 specimens of three species of slow lorises-Nycticebus coucang, N. intermedius, and N. pygmaeus-were analyzed in order to study the evolutionary relationships among the species. Eight restriction types were observed in the samples. Phylogenetic trees constructed on the basis of genetic distances showed that the slow lorises sort into two clusters: four types of N. coucang and three types of N. intermedius plus one type of N. pygmaeus. Our results suggest that there are two valid species in the genus Nycticebus-N. coucang and N. pygmaeus-and that N. intermedius should be included within N. pygmaeus. Divergence between the two species may have begun 2.7 Ma (million years ago). Evolution of gross morphology, chromosomes, and mitochondrial DNA in the slow lorises appears to be concordant.

Detection of usual and atypical aldehyde dehydrogenase alleles by mismatch amplification mutation assay

The genotypes of liver mitochondrial high-affinity aldehyde dehydrogenase-2 (ALDH2) are strongly associated with the drinking behavior and the alcohol liver diseases, since the individuals with atypical ALDH(2)(2) allele have higher levels of acetaldehyde in their plasma. The atypical ALDH(2)(2) allele has a nucleotide base transition (G-->A) in its exon 12. Based on this point mutation, we developed a rapid, reliable and inexpensive method, mismatch amplification mutation assay (MAMA), for the determination of human ALDH2 usual and atypical alleles. Two pairs of primers were designed for the amplification of the usual ALDH(2)(1) allele and the atypical ALDH(2)(2) allele, respectively. If the sample for the detection was heterozygous, it could be amplified by both of the primers. The product of polymerase chain reaction (PCR) of ALDH2 exon 12 could be easily screened by electrophoresis on a 2% agarose gel. The results of the MAMA method were further confirmed by sequencing. In the total of fifty samples from unrelated healthy Chinese Han people from Wuhan, China, the frequency of atypical ALDH(2)(2) allele was found to be 12%.

Lack of genetic association between the angiotensin-converting enzyme gene insertion/deletion polymorphism and longevity in a Han Chinese population

Introduction. The insertion/deletion polymorphism of the angiotensin-converting enzyme (ACE) gene has been reported to associate with human longevity. However, little information is available in a Han Chinese longevity Population. Therefore, we investigat

CORRELATION OF ZONA-BINDING WITH OOCYTE MATURATION AND SPERM MOTILITY IN RHESUS-MONKEYS BY HEMIZONA ASSAY

The hemizona assay (HZA) in Rhesus monkeys was employed to study the correlation of zona-binding ability with sperm motility or with naturally developing oocytes at various maturational stages. Oocytes from unstimulated ovaries were retrieved within 2 hr from monkeys sacrificed for vaccine production (in reproductive season, but with their menstrual cycles not determined). Oocytes were divided into four groups based on their morphological maturation: 1) Oocytes surrounded by more than one cumulus layer (MC); 2) Oocytes retaining intact germinal vesicle nuclei (GV); 3) Oocytes with germinal vesicle breakdown showing distinct perivitelline space (PVS); and 4) Oocytes extruding the first polar body (PB1). The mean numbers of sperm bound to hemizona for PBI, PVS, GV, and MC groups were 132.9 +/- 12.0, 71.5 +/- 10.1, 36.1 +/- 4.0, and 20.1 +/- 2.9 (Mean +/- SE), respectively. The four groups showed significant differences from each other in sperm/egg binding ability (P < 0.01). The number of bound sperm significantly increased with oocyte maturation. The present study also showed that zona-binding ability was also affected by sperm motility. For sperm with 67.7% motility and sperm with 31.2% motility, the average numbers of bound sperm were 43.5 +/- 2.2 and 25.3 +/- 2.9 (Mean +/- SE), respectively. There was significantly higher binding ability for sperm with higher motility (P < 0.01). The results suggest that: 1)The rhesus monkey model can serve as a very sensitive model for studying sperm/egg interaction by HZA; 2) Sperm motility positively correlated with sperm/egg binding; and 3) Sperm/egg binding ability increases with oocyte maturation. The binding ability is highest when oocytes matured to the PB1 stage, which is also the best opportunity for fertilization. This is strong evidence for the ''zona maturation'' hypothesis. (C) 1994 Wiley-Liss, Inc.

High prevalence of HIV-1 and hepatitis C virus coinfection among injection drug users in the southeastern region of Yunnan, China

The southeastern region of Yunnan province is a key site for drug trafficking and HIV-1 infection spread from the west of Yunnan and Laos to southeastern China. To investigate the prevalence of HIV-1 infection and hepatitis C virus (HCV) coinfection among injection drug users (IDUs) in southeastern Yunnan, three cohorts of 285 addicts, including 242 IDUs and 43 oral drug users, living in the cities of Gejiu and Kaiyuan and the county of Yanshan were studied. HIV-1 and HCV infections were detected by enzyme-linked immunosorbent assay and/or polymerase chain reaction. Data on the age, sex, risk behavior, drug use history, employment, ethnic background, and marriage status were obtained by interview. The overall prevalence of HIV-1 infection was 71.9%. The rate of HCV coinfection among 138 HIV-1-infected IDUs was 99.3%. Most HIV-infected IDUs were 20 to 35 years old (86.7%) and were ethnic Han (75.9%), suggesting that the epidemic in Yunnan is no longer confined to non-Han ethnic minorities, HIV prevalence in female IDUs (81.2%) was significantly higher than in male IDUs (68.2%) (p <.05). The prevalence of HIV infection reached 68.4% after 1 year of injection drug use. Needle/syringe sharing is the major high risk factor for the spread of HIV-1 and HCV infections. Large-scale educational campaigns are urgently needed to reduce the spread of HIV and HCV infection in these regions.

An improved microtiter assay for evaluating anti-HIV-I neutralizing antibodies from sera or plasma

Background: The anti-HIV-1 neutralizing antibody assay is widely used in AIDS vaccine research and other experimental and clinical studies. The vital dye staining method applied in the detection of anti-HIV-1 neutralizing antibody has been used in many laboratories. However, the unknown factor(s) in sera or plasma affected cell growth and caused protection when the tested sera or plasma was continuously maintained in cell culture. In addition, the poor solubility of neutral red in medium (such as RPMI-1640) also limited the use of this assay. Methods: In this study, human T cell line C8166 was used as host cells, and 3-(4,5-Dimethyl-2-thiazolyl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT) instead of neutral red was used as vital dye. In order to avoid the effect of the unknown factor( s), the tested sera or plasma was removed by a washout procedure after initial 3 - 6 h culture in the assay. Result: This new assay eliminated the effect of the tested sera or plasma on cell growth, improved the reliability of detection of anti-HIV-1 neutralizing antibody, and showed excellent agreement with the p24 antigen method. Conclusion: The results suggest that the improved assay is relatively simple, highly duplicable, cost-effective, and well reliable for evaluating anti-HIV-1 neutralizing antibodies from sera or plasma.

利用EIA生物测试法快速定量筛选环境样品中的二噁污染物

将采集的环境样品分作 3份 ,1份用于高分辨色质联用多离子检测测定二的毒性当量浓度 TEQ,另 2份分别用 7-乙氧基 -异吩唑酮 -脱乙基酶 ( EROD)活力诱导法和酶免疫法 ( Enzyme Immuno Assay,EIA)进行生物测试 .结果表明 EIA和EROD这 2种生物试验方法均具有较好的准确性和很宽的线性范围 .比较 Micro- EROD分析结果与化学分析结果以及 EIA分析结果与化学分析结果 ,发现 Micro- EROD生物试验所测得的 TEQ值均高于化学分析法的 TEQ值

Effect of water temperature on the growth performance and digestive enzyme activities of Chinese longsnout catfish (Leiocassis longirostris Gunther)

The present study was carried out to investigate the influence of water temperature on the growth performance and digestive enzyme (pepsin, trypsin and lipase) activities of Chinese longsnout catfish. Triplicate groups of Chinese longsnout catfish (35.6 +/- 0.48 g, mean +/- SE) were reared at different water temperatures (20, 24, 28 and 32 degrees C). The feeding rate (FR), specific growth rate (SGR) and feed efficiency ratio (FER) were significantly affected by water temperatures and regression relationships between water temperature and FI, SGR as well as FER were expressed as FR=-0.016T2+0.91T-10.88 (n=12, R2=0.8752), SGR=-0.026T2+1.39T-17.29 (n=12, R2=0.7599) and FER=-0.013T2+0.70T-8.43 (n=12, R2=0.7272). Based on these, the optimum temperatures for FR, SGR and FER were 27.66, 26.69 and 26.44 degrees C respectively. The specific activities of digestive enzymes at 24 or 28 degrees C were significantly higher than that at 20 or 32 degrees C. In addition, there was a significant linear regression between FR or SGR and specific activities of pepsin and lipase, which indicated that pepsin and lipase played important roles in regulating growth through nutrient digestion in Chinese longsnout catfish.