Identification of Pseudomonas bacteriophage PaP3 lysozyme

The complete genome of bacteriophage PaP3 was sequenced in a previous study by our laboratory; however, the PaP3 lysozyme gene could not be identified by homology search. In this study, based on bioinformatic analysis of its secondary structure, we have determined that the protein encoded by the p02 gene of PaP3 is likely to be a lysin. To confirm the function of the p02 gene, a recombinant expression plasmid was constructed by inserting the p02 gene into a pQE-31 plasmid; the recombinant construct was cloned and expressed in Escherichia coli JM109. The lytic activity of the expressed, purified product was observed by gel diffusion assay. The result showed that the recombinant plasmid successfully expressed 6 × his-tagged p02 protein. The expressed product had a growth inhibitory effect on Staphylococcus aureus but not on Pseudomonas aeruginosa or E. coli. However, it retained lytic activity against peptidoglycan from cell walls of P. aeruginosa and E. coli. Therefore, it is supposed that this lysozyme requires the help of holin or other punching proteins to exert lytic effects on live gram-negative bacteria. The results suggest that the p02 protein of PaP3 is a new member of the lysozyme family, which is not completely host-specific and might serve as an anti-staphylococcal agent.     

一株假单胞菌(Pseudomonas alcaliphila MBR)好氧还原亚硒酸钠为红色单质硒

【目的】本实验室保藏的一株异化硝酸盐还原菌(Pseudomonas alcaliphila MBR),其能够在好氧环境下以有机碳源为电子供体,把易溶解、高毒性亚硒酸钠还原成为红色单质硒,本文对该菌株还原亚硒酸盐的特征进行了研究。【结果】结果表明该菌株可以在pH为6-11环境中生长,对亚硒酸钠有较强抗性,其MIC(minimal inhibitory concentration)可高达50 mmol/L。在5天时间内,菌体以柠檬酸钠为电子供体,把2 mmol/L亚硒酸钠完全还原为红色单质硒并主要积累于胞外。硝酸盐和还原型谷胱甘肽对菌体还原亚硒酸钠具有促进作用,初步确定菌体对亚硒酸钠的还原是细胞膜或细胞质中的某些物质催化的结果。【结论】本项研究为应用Pseudomonas alcaliphila MBR于生物反应器提供了重要基础。     

A review of current large‐scale mouse knockout efforts

After the successful completion of the human genome project (HGP), biological research in the postgenome era urgently needs an efficient approach for functional analysis of genes. Utilization of knockout mouse models has been powerful for elucidating the function of genes as well as finding new therapeutic interventions for human diseases. Gene trapping and gene targeting are two independent techniques for making knockout mice from embryonic stem (ES) cells. Gene trapping is high‐throughput, random, and sequence‐tagged while gene targeting enables the knockout of specific genes. It has been about 20 years since the first gene targeting and gene trapping mice were generated. In recent years, new tools have emerged for both gene targeting and gene trapping, and organizations have been formed to knock out genes in the mouse genome using either of the two methods. The knockout mouse project (KOMP) and the international gene trap consortium (IGTC) were initiated to create convenient resources for scientific research worldwide and knock out all the mouse genes. Organizers of KOMP regard it as important as the HGP. Gene targeting methods have changed from conventional gene targeting to high‐throughput conditional gene targeting. The combined advantages of trapping and targeting elements are improving the gene trapping spectrum and gene targeting efficiency. As a newly‐developed insertional mutation system, transposons have some advantages over retrovirus in trapping genes. Emergence of the international knockout mouse consortium (IKMP) is the beginning of a global collaboration to systematically knock out all the genes in the mouse genome for functional genomic research. genesis 48:73–85, 2010. © 2010 Wiley‐Liss, Inc.     

Study of the restitution of action potential duration using the artificial neural network

It is widely accepted that the APD (action potential duration) restitution plays a key role in the initializing and maintaining of the reentry arrhythmias. The Luo-Rudy II models paced with different protocols showed that the current APD had a complex relation with the previous APDs and diastole intervals (DIs). This relation could not be accurately described by a single exponential function. We used an artificial neural network to formularize this relation. The results suggested that back-propagation (BP) network could predict the current APD from the information of the first three previous beats. This would help provide a target for potential anti-arrhythmic therapies.     

Growth hormone receptor deficiency in mice results in reduced systolic blood pressure and plasma renin, increased aortic eNOS expression, and altered cardiovascular structure and function

To study the role of the growth hormone receptor (GHR) in the development of cardiovascular structure and function, female GHR gene-disrupted or knockout (KO) and wild-type (WT) mice at age 18 wk were used. GHR KO mice had lower plasma renin levels (12 +/- 2 vs. 20 +/- 4 mGU/ml, P < 0.05) and increased aortic endothelial NO synthase (eNOS) expression (146%, P < 0.05) accompanied by a 25% reduction in systolic blood pressure (BP, 110 +/- 4 vs. 147 +/- 3 mmHg, P < 0.001) compared with WT mice. Aldosterone levels were unchanged, whereas the plasma potassium concentration was elevated by 14% (P < 0.05) in GHR KO. Relative left ventricular weight was 14% lower in GHR KO mice (P < 0.05), and cardiac dimensions as analyzed by echocardiography were similarly reduced. Myograph studies revealed a reduced maximum contractile response in the aorta to norepinephrine (NE) and K(+) (P < 0.05), and aorta media thickness was decreased in GHR KO (P < 0.05). However, contractile force was normal in mesenteric arteries, whereas sensitivity to NE was increased (P < 0.05). Maximal acetylcholine-mediated dilatation was similar in WT and GHR KO mice, whereas the aorta of GHR KO mice showed an increased sensitivity to acetylcholine (P < 0.05). In conclusion, loss of GHR leads to low BP and decreased levels of renin in plasma as well as increase in aortic eNOS expression. Furthermore, GHR deficiency causes functional and morphological changes in both heart and vasculature that are beyond the observed alterations in body size. These data suggest an important role for an intact GH/IGF-I axis in the maintenance of a normal cardiovascular system.     

Disruption of the Fatty Acid Δ<Superscript>6</Superscript>-Desaturase Gene in the Oil-Producing Fungus <Emphasis Type="Italic">Mortierella isabellina</Emphasis> by Homologous Recombination

The γ-linolenic acid-producing fungus Mortierella isabellina 6-22 is an important industrial strain. To clarify the biosynthetic pathways for polyunsaturated fatty acids in this strain, a disruption vector pD4MI6, including 5′ and 3′ regions of the fatty acid Δ⁶-desaturase open reading frame as homologous recombination elements and the Escherichia coli hygromycin B (HmB) phosphotransferase gene (hph) as selectable marker, was successfully constructed. Following transformation of pD4MI6 into the hygromycin B-sensitive recipient strain M. isabellina 6-22-4, a Δ⁶-desaturase gene-defective mutant strain was selected that was unable to produce γ-linolenic acid as determined by gas chromatography and molecular analysis. The morphology and physiology of the mutant, such as colony shape, color, and growth rate, were changed dramatically compared with that of strain M. isabellina 6-22-4.     

心电、呼吸信号采集分析系统的研制

设计一种用于移动监护系统的生理信息采集及预处理装置。该装置以ARM为核心,包括低功耗的双路心电信号放大、滤波、抗基线漂移电路。实现了心电信号的采集、预处理、简单分析及从心电信号中提取呼吸信号等功能。     

Visfatin levels do not change after the oral glucose tolerance test and after a dexamethasone-induced increase in insulin resistance in humans

INTRODUCTION, MATERIAL AND METHODS: Visfatin is a cytokine, mainly expressed in visceral fat, that exerts insulin-mimicking effects in rodents through activation of an insulin receptor, although the binding-site is distinct from that of insulin. However, the mechanisms that regulate visfatin synthesis are still not fully understood. In particular, it is not clear whether short-term glucose-induced hyperglycaemia and hyperinsulinaemia as well as a glucocorticoid-induced increase in insulin resistance are reflected in appreciable alterations in serum visfatin levels in humans. In order to investigate this we measured serum visfatin, glucose and insulin concentrations during a 75.0 gram oral glucose tolerance test (OGTT) [Study 1], as well as before and after oral administration of dexamethasone [Study 2]. Study 1 included 17 subjects (2 males), aged 35.7 +/- 15.6 (mean +/- SD) years of BMI 35.2 +/- 9.3 kg/m(2). Blood samples were taken before (0 minutes) and at 60 and 120 minutes after glucose administration. Study 2 included 20 subjects (4 males, 5 subjects with type 2 diabetes), aged 42.1 +/- 17.2 years of BMI 36.7 +/- 8.38 kg/m(2) who underwent screening for Cushing's disease/syndrome. Dexamethasone was administered at a dose of 0.5 mg every 6 hours for 48 hours. Fasting serum concentrations of visfatin, glucose and insulin were assessed before (D0) and after 48 hours of dexamethasone administration (D2). Insulin resistance was assessed according to the HOMA method in non-diabetic individuals (n = 15). RESULTS: In Study 1 two subjects were found to have impaired glucose tolerance and one subject was found to have diabetes mellitus. Glucose administration resulted in a highly significant increase in insulin (from 11.4 +/- 7.2 microU/mL at 0 min to 98.9 +/- 68.6 microU/mL at 60 min and 72.6 +/- 45.1 microU/mL at 120 minute of OGTT, p < 0.001 for 60 and 120 minutes in comparison to baseline). However, there was no change in serum visfatin concentrations (84.6 +/- 11.6 ng/mL at 0 minutes, 82.6 +/- 12.7 ng/mL at 60 minutes and 81.1 +/- 14.5 ng/mL at 120 minutes of OGTT, p = ns). All subjects in Study 2 achieved suppression of cortisol concentrations below 50 nmo/l. Dexamethasone administration resulted in an increase in fasting insulin (from 11.5 +/- 6.9 to 16.9 +/- 7.6 microU/mL; p = 0.011) and an increase in HOMA (from 2.73 +/- 1.74 to 4.02 +/- 2.27; p = 0.015), albeit without a significant change in serum visfatin concentrations (61.1 +/- 19.8 vs. 68.3 +/- 19.4 ng/mL, p = ns). In neither Study 1 nor Study 2 was there any significant correlation between serum visfatin and age, BMI or HOMA. CONCLUSIONS: There is a striking difference between the marked rise in insulin concentrations and the lack of change in visfatin concentrations during the oral glucose tolerance test. This implies that it is highly unlikely that visfatin is involved in the short-term regulation of glucose homeostasis in human subjects. Dexamethasone administration (4 mg/48 hours) induces an increase in insulin resistance, although without significant change in serum visfatin concentrations. Therefore in contrast to the in vitro data, short term glucocorticoid administration does not result in appreciable changes in serum levels of this adipocytokine. Furthermore, the results of our study do not support the notion that glucocorticoid-induced insulin resistance is likely to be related to changes in serum concentrations of visfatin.     

Constitutive activation of smoothened (SMO) in mammary glands of transgenic mice leads to increased proliferation, altered differentiation and ductal dysplasia

The hedgehog signaling network regulates pattern formation, proliferation, cell fate and stem/progenitor cell self-renewal in many organs. Altered hedgehog signaling is implicated in 20-25% of all cancers, including breast cancer. We demonstrated previously that heterozygous disruption of the gene encoding the patched-1 (PTCH1) hedgehog receptor, a negative regulator of smoothened (Smo) in the absence of ligand, led to mammary ductal dysplasia in virgin mice. We now show that expression of activated human SMO (SmoM2) under the mouse mammary tumor virus (MMTV) promoter in transgenic mice leads to increased proliferation, altered differentiation, and ductal dysplasias distinct from those caused by Ptch1 heterozygosity. SMO activation also increased the mammosphere-forming efficiency of primary mammary epithelial cells. However, limiting-dilution transplantation showed a decrease in the frequency of regenerative stem cells in MMTV-SmoM2 epithelium relative to wild type, suggesting enhanced mammosphere-forming efficiency was due to increased survival or activity of division-competent cell types under anchorage-independent growth conditions, rather than an increase in the proportion of regenerative stem cells per se. In human clinical samples, altered hedgehog signaling occurs early in breast cancer development, with PTCH1 expression reduced in approximately 50% of ductal carcinoma in situ (DCIS) and invasive breast cancers (IBC). Conversely, SMO is ectopically expressed in 70% of DCIS and 30% of IBC. Surprisingly, in both human tumors and MMTV-SmoM2 mice, SMO rarely colocalized with the Ki67 proliferation marker. Our data suggest that altered hedgehog signaling may contribute to breast cancer development by stimulating proliferation, and by increasing the pool of division-competent cells capable of anchorage-independent growth.