Nitration of protein kinase G-Iα modulates cyclic nucleotide crosstalk via phosphodiesterase 3A: Implications for acute lung injury

Phosphodiesterase 3A (PDE3A) selectively cleaves the phosphodiester bond of cAMP and is inhibited by cGMP, making it an important regulator of cAMP–cGMP signaling crosstalk in the pulmonary vasculature. In addition, the nitric oxide–cGMP axis is known to play an important role in maintaining endothelial barrier function. However, the potential role of protein kinase G-Iα (PKG-Iα) in this protective process is unresolved and was the focus of our study. We describe here a novel mechanism regulating PDE3A activity, which involves a PKG-Iα–dependent inhibitory phosphorylation of PDE3A at serine 654. We also show that this phosphorylation is critical for maintaining intracellular cAMP levels in the pulmonary endothelium and endothelial barrier integrity. In an animal model of acute lung injury (ALI) induced by challenging mice with lipopolysaccharide (LPS), an increase in PDE3 activity and a decrease in cAMP levels in lung tissue was associated with reduced PKG activity upon PKG-Iα nitration at tyrosine 247. The peroxynitrite scavenger manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin prevented this increase in PDE3 activity in LPS-exposed lungs. In addition, site-directed mutagenesis of PDE3A to replace serine 654 with alanine yielded a mutant protein that was insensitive to PKG-dependent regulation. Taken together, our data demonstrate a novel functional link between nitrosative stress induced by LPS during ALI and the downregulation of barrier-protective intracellular cAMP levels. Our data also provide new evidence that PKG-Iα is critical for endothelial barrier maintenance and that preservation of its catalytic activity may be efficacious in ALI therapy.     

Human papillomavirus DNA and liquid-based cervical cytology cotesting in screening and follow-up patient groups

OBJECTIVE: To compare the use of human papillomavirus (HPV) DNA and cervical cytology cotesting in screening and follow-up of patients with previous cervical abnormalities and to assess the significance of a positive HPV DNA test result in re-screening of cytologically normal cases. STUDY DESIGN: Cellular samples collected in liquid-based fixative were used for both cervical cytology and HPV DNA testing. The cervical cytology slides were manually screened by cytotechnologists followed by rapid re-screening by pathologists. The HPV DNA tests were performed using hybrid capture test kits. Statistical analyses of cervical cytology results and HPV DNA tests for high- and low-risk HPV from both patient groups were carried out. RESULTS: The prevalence of HPV DNA-positive cases was higher in younger patients. There was a poor correlation between cervical cytology results and HPV DNA tests for the screening group (kappa = 0.23), but a fair to good correlation was obtained for the follow-up group (kappa = 0.51). The false negative fraction of cytology negative/HPV DNA positive cases (0.1317), as compared with cytology negative/HPV DNA negative cases (0.0056), was statistically significant (p = 0.000001). CONCLUSION: The prevalence of HPV DNA decreased with increasing age in both the screening and follow-up patient groups. Virus clearance was delayed in the follow-up group as compared with the screening group. There was a poor correlation between cervical cytology and HPV DNA tests in the screening group but a fair to good correlation in the follow-up patient group. Cotesting of HPV DNA and cervical cytology increases the sensitivity and decreases the false negative fraction, suggesting that cotesting could be used to increase the interval of screening.     

TUNEL法检测乳酸杆菌对小鼠宫颈鳞癌的凋亡诱导作用

目的应用脱氧核苷酸转移酶介导的dUTP缺口末端标记法———TUNEL法,检测乳酸杆菌(Lactoba-cillus,LB)对小鼠宫颈鳞癌的凋亡诱导作用。方法建立615近交系小鼠U14移植瘤动物模型,并随机分组,TUNEL法检测各组细胞凋亡指数(apoptosis index,AI)。结果乳酸杆菌实验组的AI为(11.25±4.06)%,与对照组的差异有非常显著性(P<0.01),与抗癌药顺铂组的差异有显著性(P<0.05)。结论乳酸杆菌可诱导U14移植瘤细胞凋亡,这可能是其抑癌机制之一。     

Cloning and expressing DBT (dibenzothiophene) monooxygenase gene (dszC) from Rhodococcus sp. DS-3 in Escherichia coli

Dibenzothiophene (DBT) monooxygenase (DszC)catalysis,the first and also the key step in the microbial DBT desulfurization,is the conversion of DBT to DBT sulfone (DBTO2).In this study,dszC of a DBT-desulfiaizing bacterium Rhodococcus sp.DS-3 was cloned by PCR.The sequence cloned was 99% homologous to Rhodococcus erythropolis IGTS8 that was reported in the Genebank.The gene dszC could be overexpressed effectively after being inserted into plasmid pET28a and transformed into E.coli BL21 strain.The expression amount of DszC was about 20% of total supernatant at low temperature.The soluble DszC in the supematant was purified by Ni2+ chelating His-Tag resin column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to electronics purity.Only one band was detected by Western-blotting,which is for the antibody released in mouse against purified DszC in the expression product of BL21 (DE3,paC5) and Rhodococcus sp.DS-3.The activity of purified DszC was 0.36 U.DszC can utilize the organic compound such as DBT and methyl-DBT,hut not DBT derivates such as DBF,which has no sulfur or inorganic sulfur.     

Isolation and Phylogenetic Analysis of Halophilic Archaeon AJ6

Halophilic archaeon A J6 was isolated and purified from the Altun Mountain National Nature Reserve of the Xinjiang Uygur Autonomous Region.Strain AJ6 is a Gram-negative rod whose size is 0.2-0.6 by 1.6-4.2 μm,wherein a few cells are globular.The optimum salt concentration for its growth is 20% NaC1 and 0.6% Mg2+,and the optimum pH is 6.0-7.0.Morphological,physiological,and biochemical characteristics of strain AJ6 were observed.The 16S rRNA encoding gene (16S rDNA)sequence of strain A J6 was amplified by PCR,and its nucteotide sequence was determined subsequently."Clustalw"and"PHYLIP"software bags were used to analyze the 16S rDNA sequence;the homology was compared,and then the phylogenetic tree was established.The results indicate that strain AJ6 is a novel species of the genus Natrinema.The GenBank accession number of the 16S rDNA sequences of strain AJ6 is AY277584.     

人IFN-β启动子基因报告质粒的构建及SARS-CoV3a和7a蛋白对IFN-β诱生作用的初步研究

3a蛋白和7a蛋白是SARS-CoV的非结构蛋白,分别主要由SARS基因组中ORF 3a 和ORF 7a所编码。在体内和体外感染病毒的细胞中均发现了有3a蛋白的表达。首先将pGL3-Control载体进行了改构,除去了SV40启动子基因,获得了pGL3-Enhancer载体,将获得的IFN-β启动子基因连入载体中,构建了带有人IFN-β启动子基因的荧光素酶报告质粒IP-21,并且通过实验证明所构建的质粒在干扰素的诱导剂NDV的作用下能够表达荧光素酶活性,照度计检测荧光素酶活性增强,从而验证了所构建的重组质粒的有效性。为了观察SARS-CoV非结构蛋白3a和7a对干扰素诱生途径的影响,将IP-21瞬转入稳定表达有3a和7a蛋白的CHO细胞,通过荧光素酶的信号强弱对3a和7a的作用进行分析,结果表明SARS-CoV的3a和7a蛋白能够刺激荧光素酶的表达,即在体外的细胞实验中,3a和7a能有效地激活IFN-β的启动子部分。此结果对进一步研究3a和7a的功能以及对SARS-CoV的致病机制的进一步研究有一定意义。     

北京生物技术产业发展研究

生物技术及其相关产业是北京重点发展的高新技术领域之一.“九五”以来北京在该领域取得了稳步发展,具有研发、人才和临床优势.概述了近年来北京生物技术领域在研究、产业、政策等领域的发展现状,提出了目前北京在该领域发展面临的主要问题,并以此为基础为北京市该领域未来发展提出了相关建议.     

Cloning and expressing DBT (dibenzothiophene) monooxygenase gene (dszC) from Rhodococcus sp. DS-3 in Escherichia coli

Dibenzothiophene (DBT) monooxygenase (DszC) catalysis, the first and also the key step in the microbial DBT desulfurization, is the conversion of DBT to DBT sulfone (DBTO₂). In this study, dszC of a DBT-desulfurizing bacterium Rhodococcus sp. DS-3 was cloned by PCR. The sequence cloned was 99% homologous to Rhodococcus erythropolis IGTS8 that was reported in the Genebank. The gene dszC could be overexpressed effectively after being inserted into plasmid pET28a and transformed into E. coli BL21 strain. The expression amount of DszC was about 20% of total supernatant at low temperature. The soluble DszC in the supernatant was purified by Ni²⁺ chelating His-Tag resin column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to electronics purity. Only one band was detected by Western-blotting, which is for the antibody released in mouse against purified DszC in the expression product of BL21 (DE3, paC5) and Rhodococcus sp. DS-3. The activity of purified DszC was 0.36 U. DszC can utilize the organic compound such as DBT and methyl-DBT, but not DBT derivates such as DBF, which has no sulfur or inorganic sulfur. __________ Translated from Acta Scientiarum Naturalium Universitatis Nankaiensis, 2005, 38(6): 1–6 [译自: 南开大学学报 (自然科学版), 2005, 38(6): 1–6]     

拔节期与抽穗期玉米抗纹枯病相关QTL的初步定位

以玉米自交系R15(抗)×478(感)的F_2分离群体为作图群体,构建了包含146个SSR标记位点的遗传连锁图谱,覆盖玉米基因组1666 cM,平均图距11．4 cM。通过麦粒嵌入法对229个F_(2：4)家系进行人工接种纹枯病菌,于玉米拔节期和抽穗期进行纹枯病的抗性鉴定。应用复合区间作图法分析两个时期的抗病QTL及遗传效应。结果共检测到17个抗性QTL,其中以拔节期病情指数为指标共检测到9个QTL,分别位于第1、2、3、4、5、6、和10染色体上,可解释的表型变异为3．72%-9．26%;以抽穗期的病情指数为指标共在7条染色体上检测到10个抗玉米纹枯病的QTL,分布于第2、3、4、5、6、8和9染色体上。单个QTL可解释的表型变异为4．27%-9．27%。两个时期共检测出2个共同QTL,它们分别位于第2染色体的bnlgl662-bnlg1940区间和第6染色体的umc1006-umc1723区间。定位结果表明两个时期检测出的抗性QTL的差异表达与玉米不同发育时期基因的时空表达有密切关系,从而反映在纹枯病的抗性位点差异性上．这为玉米抗病选育提供新的信息。     

Effects of Taurine on Glial Cells Apoptosis and Taurine Transporter Expression in Retina Under Diabetic Conditions

Taurine, a ß-aminosulfonic acid, has been reported to reduce the risk of a number of diseases, including cardiovascular disease, diabetes, and also perhaps to reduce neurodegeneration in the elderly. The transport of taurine is known to be mediated by taurine transporter (TauT). The purpose of this study is to examine the effects of taurine on glial cells apoptosis and on TauT expression in retina of diabetic rats and retinal glial cells cultured with high glucose. TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining analysis showed that the number of TUNEL-positive cells in taurine treated diabetic rats was significantly lower than those of untreated diabetic rats over the 8-, and 12-week time courses, respectively (all P < 0.001). No TUNEL-positive cells were observed in retina of control groups and taurine treated control groups. In cultured retinal glial cells, the apoptosis in high glucose-treated cells was significantly increased vs the control. When the cells were incubated with high glucose and taurine at 0.1, 1.0 and 10 mmol/l, the percentage of apoptosis was significantly decreased to 16.4, 5.7 and 7.6% respectively (all P < 0.05). With supplementation of taurine in diet and culture medium, higher expression of TauT in retina of diabetic rats and cultured retinal glial cells under diabetic conditions were detected by western-blotting (P < 0.05). Taken together, our data suggest that diabetes or high glucose induced retinal glial cells apoptosis can be inhibited by taurine, and that taurine reverses the diabetes-induced or high glucose-induced decrease in TauT expression.