Effect of NBD chloride (4-chloro-7-nitrobenzo-2-oxa-1,3-diazole) on the pyridine nucleotide transhydrogenase of Escherichia coli.

Pyridine nucleotide transhydrogenases of bacterial cytosolic membranes and mitochondrial inner membranes are proton pumps in which hydride transfer between NADP(+) and NAD(+) is coupled to proton translocation across cytosolic or mitochondrial membranes. The pyridine nucleotide transhydrogenase of Escherichia coli is composed of two subunits (alpha and beta). Three domains are recognized. The extrinsic cytosolic domain 1 of the amino-terminal region of the alpha subunit bears the NAD(H)-binding site. The NADP(H)-binding site is present in domain 3, the extrinsic cytosolic carboxyl-terminal region of the beta subunit. Domain 2 is composed of the membrane-intrinsic carboxyl-terminal region of the alpha subunit and the membrane-intrinsic amino-terminal region of the beta subunit. Treatment of the transhydrogenase of E. coli with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD chloride) inhibited enzyme activity. Analysis of inhibition revealed that several sites on the enzyme were involved. NBD chloride modified two (betaCys-147 and betaCys-260) of the seven cysteine residues present in the transhydrogenase. Modification of betaCys-260 in domain 2 resulted in inhibition of enzyme activity. Modification of residues other than cysteine residues also resulted in inhibition of transhydrogenation as shown by use of a cysteine-free mutant enzyme. The beta subunit was modified by NBD chloride to a greater extent than the alpha subunit. Reaction of domain 2 and domain 3 was prevented by NADPH. Modification of domain 3 is probably not associated with inhibition of enzyme activity. Modification of domain 2 of the beta subunit resulted in a decreased binding affinity for NADPH at its binding site in domain 3. The product resulting from the reaction of NBD chloride with NADPH was a very effective inhibitor of transhydrogenation. In experiments with NBD chloride in the presence of NADPH it is likely that all of the sites of reaction described above will contribute to the inhibition observed. The NBD-NADPH adduct will likely be more useful than NBD chloride in investigations of the pyridine nucleotide transhydrogenase.     

人嗜中性白细胞活化蛋白-1/白细胞介素-8合成基因的修正及修正基因在大肠杆菌中的表达

本文采用寡核苷酸介导的定位诱变技术修正了人嗜中性白细胞活化蛋白-1/白细胞介素-8合成基因中出现的合成错误,在该基因编码区的201位插入了原来缺失的G残基,从而使之恢复正确读框。经对修正基因进行DNA全序列测定,表明定位诱变的结果符合设计要求。在此基础上,利用原核高效表达载体pBV220在P_RP_L串联启动子的控制下在大肠杆菌中对该基因进行了表达,并测定了重组人嗜中性白细胞活化蛋白-1/白细胞介素-8的嗜中性白细胞趋化活性。本工作为开展人嗜中性白细胞活化蛋白-1/白细胞介素-8基因工程及蛋白质工程的研究奠定了基础。     

丹霞地貌山顶生态效应

首次探讨了丹霞地貌山顶生态效应现象,揭示特殊的地貌、形态所产生的特殊生态现象.测定广东省丹霞山宝珠峰和海螺峰的山顶的生态因子特征、群落结构特征和物种生态型特征,并与山脚的群落与种群以及相近非丹霞地貌区作比较.结果表明,山顶的平均温度高于山脚沟谷,平均湿度小于山脚沟谷,群落物种数和物种多样性均小于山脚沟谷.相比山顶而言,山脚沟谷的植物有很强的热带性.这些特征都有别于一般非丹霞地貌山地.另外,在山顶和山脚调查的几个种群在叶面积、比叶面积、树皮、枝下高和冠幅等方面,均出现了生态型的差异.讨论了丹霞地貌山顶对生态型研究、岛屿理论研究和适应性进化研究的科学意义.     

Strain improvement of <Emphasis Type="Italic">Trichoderma viride</Emphasis> for increased cellulase production by irradiation of electron and <Superscript>12</Superscript>C<Superscript>6+</Superscript>-ion beams

Objectives

To improve cellulase production and activity, Trichoderma viride GSICC 62010 was subjected to mutation involving irradiation with an electron beam and subsequently with a ¹²C⁶⁺-ion beam.

Results

Mutant CIT 626 was the most promising cellulase producer after preliminary and secondary screening. Soluble protein production and cellulase activities were increased mutifold. The optimum temperature, pH and culture time for the maximum cellulase production of the selected mutant were 35 °C, pH 5 and 6 days. The highest cellulase production was obtained using wheat bran. The prepared cellulases from T. viride CIT 626 had twice the hydrolytic performance with sawdust (83 %) than that from the parent strain (42.5 %). Furthermore, molecular studies demonstrated that there were some key mutation sites suggesting that some amino acid changes in the protein caused by base mutations had led to the enhanced cellulase production and activity.

Conclusions

Mutagenesis with electron and ¹²C⁶⁺-ion beams could be developed as an effective tool for improvement of cellulase producing strains.

     

Changes in the Prevalence of Rheumatic Diseases in Shantou,China, in the Past Three Decades: A COPCORD Study

This study aimed to clarify changes in the prevalence of rheumatic diseases in Shantou, China, in the past 3 decades and validate whether stair-climbing is a risk factor for knee pain and knee osteoarthritis (KOA). The World Health Organization-International League Against Rheumatism Community Oriented Program for Control of Rheumatic Diseases (COPCORD) protocol was implemented. In all, 2337 adults living in buildings without elevators and 1719 adults living in buildings with elevators were surveyed. The prevalence of rheumatic pain at any site and in the knee was 15.7% and 10.2%, respectively; both types of pain had a significantly higher incidence in residents of buildings without elevators than was reported by people who lived in buildings with elevators (14.9% vs. 10.6% and 11.32% vs. 8.82%, respectively) (both P < 0.0001). The prevalence of rheumatic pain in the neck, lumbar spine, shoulder, elbow, and foot was 5.6%, 4.5%, 3.1%, 1.4%, and 1.8%, respectively; these findings were similar to the data from the 1987 rural survey, but were somewhat lower than data reported in the urban and suburban surveys of the 1990s, with the exception of neck and lumbar pain. The prevalence of KOA, gout, and fibromyalgia was 7.10%, 1.08%, and 0.07%, respectively, and their prevalence increased significantly compared with those in previous studies from the 20^th century. There were no significant differences in the prevalence of rheumatoid arthritis (RA) (0.35%) or ankylosing spondylitis (AS) (0.31%) compared to that reported in prior surveys. The prevalence of KOA was higher in for residents of buildings without elevators than that in those who had access to elevators (16–64 years, 5.89% vs. 3.95%, P = 0.004; 16->85 years, 7.64% vs. 6.26%, P = 0.162). The prevalence of RA and AS remained stable, whereas that of KOA, gout, and fibromyalgia has increased significantly in Shantou, China, during the past 3 decades. Stair-climbing might be an important risk factor for knee pain and KOA.     

九连小蘖悬浮培养细胞生理生化研究 Ⅰ.细胞生长与培养液的电导率和过氧化物酶活性的变化

本文报道了九连小蘖细胞悬浮培养过程中,细胞生长与培养液的电导率、pH值、可溶性糖含量及过氨化物酶活性的变化。实验表明细胞生长曲线与培养液的电导率、可溶性糖含量变化的曲线恰成镜像关系。而细胞生长曲线与培养液的过氨化物酶活性变化的曲线相互平行。从而,可以通过监测培养液的电导率和过氧化物酶活性的变化来了解细胞生长状况,并可作为植物细胞培养过程中生物量增长的参考指标。     

Mechanisms of Metabonomic for a Gateway Drug: Nicotine Priming Enhances Behavioral Response to Cocaine with Modification in Energy Metabolism and Neurotransmitter Level

Nicotine, one of the most commonly used drugs, has become a major concern because tobacco serves as a gateway drug and is linked to illicit drug abuse, such as cocaine and marijuana. However, previous studies mainly focused on certain genes or neurotransmitters which have already been known to participate in drug addiction, lacking endogenous metabolic profiling in a global view. To further explore the mechanism by which nicotine modifies the response to cocaine, we developed two conditioned place preference (CPP) models in mice. In threshold dose model, mice were pretreated with nicotine, followed by cocaine treatment at the dose of 2 mg/kg, a threshold dose of cocaine to induce CPP in mice. In high-dose model, mice were only treated with 20 mg/kg cocaine, which induced a significant CPP. ¹H nuclear magnetic resonance based on metabonomics was used to investigate metabolic profiles of the nucleus accumbens (NAc) and striatum. We found that nicotine pretreatment dramatically increased CPP induced by 2 mg/kg cocaine, which was similar to 20 mg/kg cocaine-induced CPP. Interestingly, metabolic profiles showed considerable overlap between these two models. These overlapped metabolites mainly included neurotransmitters as well as the molecules participating in energy homeostasis and cellular metabolism. Our results show that the reinforcing effect of nicotine on behavioral response to cocaine may attribute to the modification of some specific metabolites in NAc and striatum, thus creating a favorable metabolic environment for enhancing conditioned rewarding effect of cocaine. Our findings provide an insight into the effect of cigarette smoking on cocaine dependence and the underlying mechanism.