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.

Distribution of rDNA in the nucleus of Giardia lamblia - Detection by Ag-I silver stain

OBJECTIVE: To determine whether rDNA of Giardia lamblia forms a nucleolus organizer region (NOR)-like structure and is in a very primitive state. STUDY DESIGN: G lamblia was used as the experimental animal, with Euglena gracilis as the control. The distribution was demonstrated indirectly by the modified Ag-I silver technique, which can specifically indicate the NOR under both light and electron microscopes. RESULTS: In the ultrathin sections of silver-stained Euglena cells, all the silver grains were concentrated in the fibrosa of the nucleolus, while no grains found in the cytoplasm, nucleoplasm, condensed chromosomes or pars granulosa of the nucleus. In the silver-stained Giardia cells, no nucleolus was found; a few silver grains were scattered in the nucleus but were not concentrated in any specific region. CONCLUSION: The distribution of silver grains in G lamblia showed that the transcription of rDNA occurs inside the nucleus, though no nucleolus is present. It is possible that chromosomes are in a very primitive state in diplomonad cells; as each chromosome has few prRNA genes, the transcription is independent of a nucleolus. These results imply that the rDNA of Giardia does not form a NOR-like structure and seems to represent a very primitive state in the evolution of the nucleolus.

Effects of Pallas' viper (Agkistrodon halys pallas) venom on blood coagulation and characterization of a prothrombin activator

The action of Pallas' viper (Agkistrodon halys pallas) venom on blood coagulation was examined in vitro and a strong anticoagulant effect was observed. This action was abolished after treatment with a specific inhibitor of phospholipase A(2) activity (p-bromophenacyl bromide), revealing a procoagulant action in low concentrations of treated venom (around 1 mu g/ml). The effect of the venom an haemostasis was further characterized by measuring its ability to activate purified blood coagulation factors. It is concluded that A. halys pallas venom contains prothrombin activation activity. A prothrombin activator (aharin) was purified from the venom by Sephadex G-75 gel filtration and ion-exchange chromatography on a Mono-Q column. It consisted of a single polypeptide chain, with a mol. wt of 63,000. Purified aharin possessed no amidolytic activity on chromogenic substrates. It did not act on other blood coagulation factors, such as factor X and plasminogen, nor did it cleave or clot purified fibrinogen. The prothrombin activation activity of aharin was readily inhibited by ethylenediamine tetracetic acid (a metal chelator), but specific serine protease inhibitors such as diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride had no effect on it. These observations suggest that, like those prothrombin activators from Echis carinatus and Bothrops atrox venoms, the prothrombin activator from A. halys pallas venom is a metalloproteinase. (C) 1998 Elsevier Science Ltd. All rights reserved.

Effect of age and breeding season on the developmental capacity of oocytes from unstimulated and follicle-stimulating hormone-stimulated rhesus monkeys

Effects of age and season on the developmental capacity of oocytes from unstimulated and FSH-stimulated rhesus monkeys were examined, Immature cumulus-oocyte complexes were matured in vitro in modified CMRL-1066 medium containing 20% bovine calf serum and

The mastoparanogen from wasp

Mastoparans are a family of small peptides identified from the venom of hymenopteroid insects. Although they have been characterized as early as 1979, and so far are recognized as a leading biomolecule in potential drug therapy, their precursors, mastopar

Molecular dynamics research of G249 and S249 substitutions of p53 protein.

,The molecular dynamics research of the core domain of p53 protein crystal structure shows that besides the stability in biochemistry this domain also shows a high stability in molecular mechanics. Based on that work, the residue R249 was substituted with amino acids Gly and Ser respectively, and molecular dynamics researches were performed separately. The results show that these substitutions cause a relax tendency between loop2 and 3 domains, leading to an alteration of the whole conformation of p53 core domain and ruining its stability. The results visually explains the mechanism of p53 changes in immunological and biochemical reactions, which are caused by 249 residue substitutions from 3-D structure variations.

Maturation of rhesus monkey oocytes in chemically defined culture media and their functional assessment by IVF and embryo development

This study compared success of in-vitro maturation of rhesus monkey oocytes in protein-free versus serum-containing culture systems, assessed by embryo development subsequent to IVF. Four media were tested: (i) modified Connaught Medical Research Laborato

Two families of antimicrobial peptides with multiple functions from skin of rufous-spotted torrent frog, Amolops loloensis

There are around 27 species of Amolops amphibian distributed in South-east of Asia. Seven antimicrobial peptides (AMPs) belonging to two different families were purified from skin of rufous-spotted torrent frog, Amolops loloensis, and designated brevinins

Cytogenetics, morphology and evolution of four subspecies of the Giant Sheep argali (Ovis ammon) of Asia

Dalai-lamae (Ovis ammon dalai-lamae), Gobi (O. a. darwini), Kara Tau (O. a. nigrimontana) and Tibetan (O. a. hodgsoni) argali share a 2n = 56 diploid chromosome number and a karyotype consisting of 2 pairs of biarmed and 25 pairs of acrocentric autosomes, a large acrocentric X and a minute Y chromosome. The Giemsa-banding patterns of the largest pair of biarmed chromosomes were identical to those of the largest biarmed chromosomes in all wild sheep and domestic sheep of the genus Ovis. The banding patterns of the second pair of biarmed chromosomes (metacentric) were identical to the third pair of biarmed chromosomes in Ovis with 2n = 54 and to the third largest pair of chromosomes in the 2n = 52 karyotype of Siberian snow sheep (O. nivicola). The G-banded karyotypes of dalai-lamae, darwini, hodgsoni and nigrimontana are consistent with all subspecies of argali (O. ammon), except that the Y chromosome is acrocentric instead of metacentric as typical of the argaliform wild sheep and Ovis. The Dalai-lamae and Tibetan argali specimens exhibit the light-colored, long-haired ruffs and body coloration typical of argalis from the Tibetan Plateau. The Gobi argali, from the extreme western Gobi, is similar to the dark phase argali.

Energy substrate requirement for in vitro maturation of oocytes from unstimulated adult rhesus monkeys

The energy substrates lactate, pyruvate, and glucose were evaluated for supporting in vitro cytoplasmic maturation of rhesus monkey oocytes. A total of 321 cumulus-oocyte complexes (COCs) aspirated from greater than or equal to 1000 mum diameter follicles

Amphibian tachykinin precursor

The precursor of amphibian tachykinin has not been found although more than 30 tachykinins have been isolated from amphibians since 1964. In this report, two tachykinin-like peptides are identified from the skin secretions of the frog, Odorrana grahami. T

Cloning of cDNAs encoding C-type lectins from Elapidae snakes Bungarus fasciatus and Bungarus multicinctus

A number of C-type lectins with various biological activities have been purified and characterized from Viperidae snake venoms. In contrast, only a few reports could be found in literature concerning the C-type lectins in Elapidae snake venoms. Based on t

Cryopreservation of rhesus macaque (Macaca mulatta) spermatozoa and their functional assessment by in vitro fertilization

Although spermatozoa from several species of nonhuman primates have been cryopreserved, there has been no report of success with rhesus macaque spermatozoa as judged by functional assays. Two Tris-egg yolk freezing media. TEST and TTE. which have: been successfully used for cynomolgus macaque (Macaca fascicularis) spermatozoa, were compared for cryopreservation of spermatozoa From four rhesus macaques (Macaca mulatta). The postthaw motility (percentage and duration) of spermatozoa cryopreserved in TTE was much higher than that for spermatozoa cryopreserved in TEST. The function of sperm cryopreserved in TTE was evaluated by in vitro fertilization or oocytes collected from gonadotropin-stimulated prepubertal rhesus macaques. Of the inseminated oocytes. 82 +/- 13% were fertilized and 63 +/- 22 and 39 +/- 21% of the resulting zygotes developed into morulae and blastocysts. respectively. These results indicate that rhesus macaque spermatozoa can be effectively cryopreserved in TTE medium. This finding will facilitate the application of in vivo and in vitro assisted reproductive technologies in this species. (C) 2001 Academic Press.

The first report of kininogen from invertebrates

The hornet possesses highly toxic venom, which is rich in toxin, enzymes, and biologically active peptides. Several bradykinin-like peptides, vespakinins, have been found in wasp venoms since 1970s, but the mode of biosynthesis of these peptides is unknow