Jerdonase, a novel serine protease with kinin-releasing and fibrinogenolytic activity from Trimeresurus jerdonii venom

A novel kinin-releasing and fibrin (ogen)olytic enzyme termed jerdonase was purified to homogeneity from the venom of Trimeresurus jerdonii by DEAE Sephadex A-50 anion exchange, Sephadex G-100 (superfine) gel filtration and reverse-phase high performance liquid chromatography (RP-HPLC). Jerdonase migrated as a single band with an approximate molecular weight of 55 kD under the reduced conditions and 53 kD under the non-reduced conditions. The enzyme was a glycoprotein containing 35.8% neutral carbohydrate. The N-terminal amino acid sequence of jerdonase was determined to be IIGGDECNINEHPFLVALYDA, which showed high sequence identity to other snake venom serine proteases. Jerdonase catalyzed the hydrolysis of BAEE, S-2238 and S-2302, which was inhibited by phenymethylsulfonyl fluoride (PMSF), but not affected by ethylenediaminetetraacetic acid (EDTA). Jerdonase preferentially cleaved the Aalpha-chain of human fibrinogen with lower activity towards Bbeta-chain. Moreover, the enzyme hydrolyzed bovine low-molecular-mass kininogen and releasing bradykinin. In conclusion, all results indicated that jerdonase was a multifunctional venom serine protease.

Effect of adrenalectomy and corticosterone replacement on prepulse inhibition and locomotor activity in mice

1 Stress is a risk factor in psychiatric illnesses such as schizophrenia. The aim of the present study was to investigate the effect of different circulating levels of the adrenal steroid corticosterone (CORT) on locomotor hyperactivity and prepulse inhibition of acoustic startle, two behavioural animal models of aspects of schizophrenia. 2 Male C57BL/6J mice (n = 10 per group) were anaesthetised with isoflurane and sham-operated or adrenalectomised (ADX). ADX mice were implanted with 50 mg pellets consisting of 100% cholesterol, or 2, 10 or 50 mg of CORT mixed with cholesterol. CORT pellet implantation dose dependently increased plasma CORT levels 3 weeks after surgery. Starting 1 week after surgery, mice were tested for prepulse inhibition after injection of saline or 5 mg kg(-1) of haloperidol. 3 In intact mice and in mice implanted with 10 mg of CORT, haloperidol treatment significantly increased prepulse inhibition (average values from 38 - 42 to 52%). Similar results were observed when testing the mice for amphetamine-induced locomotor hyperactivity (5 mg kg(-1)). In contrast, there was no significant effect of haloperidol in mice implanted either with cholesterol or 2 or 50 mg of CORT. 4 These results in behavioural animal models of schizophrenia suggest an important role of the stress hormone CORT in modulating dopaminergic activity in this illness.

Glycine uptake regulates hippocampal network activity via glycine receptor-mediated tonic inhibition

Functional glycine receptors (GlyRs) are enriched in the hippocampus, but their role in hippocampal function remains unclear. Since the concentration of ambient glycine is determined by the presence of powerful glycine transporter (GlyT), we blocked the r

Inhibition of the predatory activity of the copepod Mesocyclops notius by Microcystis spp.

The feeding rate of the copepod Mesocyclops notius on two species of cladocerans (Daphnia carinata and Ceriodaphnia cornuta) decreased with increasing environmental concentration of 64-122 mum colonial Microcystis spp. Rate of copepod feeding on Moina micrura was unaffected by the presence of Microcystis spp. Mesocyclops notius rarely preyed on Diaphanosoma brachyurum.

A Simple, Universal Colorimetric Assay for Endonuclease/Methyltransferase Activity and Inhibition Based on an Enzyme-Responsive Nanoparticle System

An enzyme responsive nanoparticle system that uses a DNA-gold nanoparticle (AuNP) assembly as the substrate has been developed for the simple, sensitive, and universal monitoring of restriction endonucleases in real time. This new assay takes advantage of the palindromic recognition sequence of the restriction nucleases and the unique optical properties of AuNPs and is simpler than the procedure previously described by by Xu et al. (Angew. Chem. Int. Ed. Engl. 2007, 46, 3468-3470). Because it involves only one type of ssDNA modified AuNPs, this assay can be directed toward most of the endonucleases by simply changing the recognition sequence found within the linker DNA. In addition, the endonuclease activity could be quantitatively analyzed by the value of the reciprocal of hydrolysis half time (t(1/2)(-1). Furthermore, our new design could also be applied to the assay of methyltransferase activity since the methylation of DNA inhibits its cleavage by the corresponding restriction endonuclease, and thus, this new methodology can be easily adapted to high-throughput screening of methyltransferase inhibitors.

Digestive gut structure and activity of protease, amylase, and alkaline phosphatase in Calanus sinicus during summer in the Yellow Sea and the East China Sea

Calanus sinicus aggregate at the depth of 40-60 m (ambient temperature is 16 degreesC) in the waters of the continental shelf of the Yellow Sea during summer. in animals found in near shore regions, there are changes in digestive gut cells structure, digestive enzyme activity (protease, amylase), and tissue enzyme (alkaline phosphatase (ALP)), which may represent adaptations by this cold-water animal to a sharp seasonal increase in temperature of 6-23 degreesC. The activities of the digestive enzymes (protease and amylase) are very low in animals at stations near the estuary of Yangtse River, whereas they are relatively high in animals at stations in the central Yellow Sea, During summer, B-cells of the intestine and the villi intestinalis disappear in animals that do not feed at stations near the estuary of the Yangtse River. Respiration rates were undetectable or quite low during summer in C. sinicus from stations near the estuary of the Yangtse River, whereas they were relatively high at stations in the central Yellow Sea. Based upon the morphological characteristics of the digestive gut structure, enzyme levels, respiration rates, and the distribution of C. sinicus, we concluded that C. sinicus might be dormant during summer in the near shore areas of the East China Sea while remaining active in the central Yellow Sea. (C) 2002 Elsevier Science B.V. All rights reserved.

Factors affecting the protease activity of venom from jellyfish Rhopilema esculentum Kishinouye

In this paper, the effects of some chemical and physical factors such as temperature, pH values, glycerol, and divalent metal cations on the protease activity of venom from jellyfish, Rhopilema esculentum Kishinouye, were assayed. Protease activity was dependent on temperature and pH values. Zn2+, Mg2+, and Mn2+ in sodium phosphate buffer (0.02 M, pH 8.0) could increase protease activity. Mn2+ had the best effects among the three metal cations and the effect was about 20 times of that of Zn2+ or Mg2+ and its maximal protease activity was 2.3 x 10(5) U/mL. EDTA could increase protease activity. PMSF had hardly affected protease activity. O-Phenanthroline and glycerol played an important part in inhibiting protease activity and their maximal inhibiting rates were 87.5% and 82.1%, respectively. (c) 2005 Elsevier Ltd. All rights reserved.

MYC activity mitigates response to rapamycin in prostate cancer through eukaryotic initiation factor 4E-binding protein 1-mediated inhibition of autophagy.

Loss of PTEN and activation of phosphoinositide 3-kinase are commonly observed in advanced prostate cancer. Inhibition of mammalian target of rapamycin (mTOR), a downstream target of phosphoinositide 3-kinase signaling, results in cell cycle arrest and apoptosis in multiple in vitro and in vivo models of prostate cancer. However, single-agent use of mTOR inhibition has limited clinical success, and the identification of molecular events mitigating tumor response to mTOR inhibition remains a critical question. Here, using genetically engineered human prostate epithelial cells (PrEC), we show that MYC, a frequent target of genetic gain in prostate cancers, abrogates sensitivity to rapamycin by decreasing rapamycin-induced cytostasis and autophagy. Analysis of MYC and the mTOR pathway in human prostate tumors and PrEC showed selective increased expression of eukaryotic initiation factor 4E-binding protein 1 (4EBP1) with gain in MYC copy number or forced MYC expression, respectively. We have also found that MYC binds to regulatory regions of the 4EBP1 gene. Suppression of 4EBP1 expression resulted in resensitization of MYC-expressing PrEC to rapamycin and increased autophagy. Taken together, our findings suggest that MYC expression abrogates sensitivity to rapamycin through increased expression of 4EBP1 and reduced autophagy.

In vivo inhibition of elevated myocardial beta-adrenergic receptor kinase activity in hybrid transgenic mice restores normal beta-adrenergic signaling and function.

BACKGROUND: The clinical syndrome of heart failure (HF) is characterized by an impaired cardiac beta-adrenergic receptor (betaAR) system, which is critical in the regulation of myocardial function. Expression of the betaAR kinase (betaARK1), which phosphorylates and uncouples betaARs, is elevated in human HF; this likely contributes to the abnormal betaAR responsiveness that occurs with beta-agonist administration. We previously showed that transgenic mice with increased myocardial betaARK1 expression had impaired cardiac function in vivo and that inhibiting endogenous betaARK1 activity in the heart led to enhanced myocardial function. METHODS AND RESULTS: We created hybrid transgenic mice with cardiac-specific concomitant overexpression of both betaARK1 and an inhibitor of betaARK1 activity to study the feasibility and functional consequences of the inhibition of elevated betaARK1 activity similar to that present in human HF. Transgenic mice with myocardial overexpression of betaARK1 (3 to 5-fold) have a blunted in vivo contractile response to isoproterenol when compared with non-transgenic control mice. In the hybrid transgenic mice, although myocardial betaARK1 levels remained elevated due to transgene expression, in vitro betaARK1 activity returned to control levels and the percentage of betaARs in the high-affinity state increased to normal wild-type levels. Furthermore, the in vivo left ventricular contractile response to betaAR stimulation was restored to normal in the hybrid double-transgenic mice. CONCLUSIONS: Novel hybrid transgenic mice can be created with concomitant cardiac-specific overexpression of 2 independent transgenes with opposing actions. Elevated myocardial betaARK1 in transgenic mouse hearts (to levels seen in human HF) can be inhibited in vivo by a peptide that can prevent agonist-stimulated desensitization of cardiac betaARs. This may represent a novel strategy to improve myocardial function in the setting of compromised heart function.