兰州兴隆山土壤微生物的分布及其相关特性分析

目的以兰州兴隆山不同区域的土壤微生物为研究对象,分析比较土壤微生物数量与土壤酶活性之间的相关性。方法利用不同方法测定土壤理化性质、微生物数量以及土壤相关酶活特性;采用三区划线法进行土壤微生物的分离与纯化,通过16S rDNA和ITS方法进行优势菌株鉴定。结果兰州兴隆山土壤中微生物菌群数量由多到少依次为细菌、放线菌、真菌。通过分离纯化后,对其中的2株优势菌进行了鉴定,初步推断X2为萎缩芽胞杆菌属(Bacillus atrophaeus)细菌,Z2为栎生青霉属(Penicillium glandicola)真菌。从酶活特性可知,阳面的土壤过氧化氢酶活性比阴面高;随着海拔高度的增加,过氧化氢酶活性呈现增加趋势;阳面的土壤脱氢酶活性总体比阴面高,并且随着海拔梯度的升高,土壤脱氢酶活性也在不断升高。从相关性分析可知,不同海拔土样间微生物数量与酶活性之间表现出明显的相关性。结论兰州兴隆山土壤微生物数量丰富,且细菌数量居多;不同阴、阳面土壤微生物的层次分布以及活性也各有不同。以上研究可为兰州兴隆山土壤生态系统演替等提供参考依据,并为土壤生态环境的治理做铺垫。     

Membrane estrogen signaling enhances tumorigenesis and metastatic potential of breast cancer cells via estrogen receptor-α36 (ERα36)

Protein kinase C (PKC) signaling can be activated rapidly by 17β-estradiol (E(2)) via nontraditional signaling in ERα-positive MCF7 and ERα-negative HCC38 breast cancer cells and is associated with tumorigenicity. Additionally, E(2) has been shown to elicit anti-apoptotic effects in cancer cells counteracting pro-apoptotic effects of chemotherapeutics. Supporting evidence suggests the existence of a membrane-associated ER that differs from the traditional receptors, ERα and ERβ. Our aim was to identify the ER responsible for rapid PKC activation and to evaluate downstream effects, such as proliferation, apoptosis, and metastasis. RT-PCR, Western blot, and immunofluorescence were used to determine the presence of ER splice variants in multiple cell lines. E(2) effects on PKC activity were measured with and without ER-blocking antibodies. Cell proliferation was determined by [(3)H]thymidine incorporation, and cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, (MTT) whereas apoptosis was determined by DNA fragmentation and TUNEL. Quantitative RT-PCR and sandwich ELISA were used to determine the effects on metastatic factors. The role of membrane-dependent signaling in cancer cell invasiveness was examined using an in vitro assay. The results indicate the presence of an ERα splice variant, ERα36, in ERα-positive MCF7 and ERα-negative HCC38 breast cancer cells, which localized to plasma membranes and rapidly activated PKC in response to E(2), leading to deleterious effects such as enhancement of proliferation, protection against apoptosis, and enhancement of metastatic factors. These findings propose ERα36 as a novel target for the development of therapies that can prevent progression of breast cancer in the primary tumor as well as during metastasis.     

曲霉产角质蛋白酶发酵条件的初步研究

曲酶F_(23)是一株角质蛋白酶分泌型菌株。我们研究了各种发酵条件如碳源、氮源等对产酶能力的影响,确定了适宜的发酵条件。摇瓶发酵培养基以1％的玉米浆为氮源,1％的玉米粉为碳源,0.3％的羽毛粉为诱导物及各种无机盐和维生素等。最适发酵pH范围为6.5～7.5,最适发酵温度为28℃～32℃,转速为180rpm,发酵102h左右酶活性达238ku/ml。     

膜生物膜法在水污染控制及资源回收中的研究进展

传统以达标排放为核心目标的废水处理工艺往往以高能耗、高物耗换取污染物削减,形成了"减排污染物、增排温室气体"的尴尬局面,并不符合可持续发展理念。作为一种新型的膜处理技术,膜生物膜法可利用无泡曝气的方式将气态电子供体(甲烷、氢气)或受体(氧气)提供给附着在膜表面的微生物,从而驱动水体中的污染物去除,并产生一些极具回收潜力的物质,最终实现污染物削减、节能减排及资源回收三大目标的有机整合。本文系统介绍了膜生物膜的传质过程及其去除污染物的微观机制,探讨了膜生物膜法在水处理资源回收方面的研究前景,梳理了膜生物膜反应器在水污染控制方面的实验研究和中试应用现状,并总结了膜生物膜法面临的挑战及发展趋势。     

A novel methodology for characterizing strand-break termini and damaged bases in plasmid DNA exposed to ionizing radiation

We have developed a de novo methodology to characterize radiation damage in DNA. An enzyme system consisting of the 3'-->5' exonuclease snake venom phosphodiesterase (SVPD) and calf intestine alkaline phosphatase (CIAP) was used to examine the 3' termini of strand-break sites. In this study, we hypothesized that the strand-break termini can be divided into two categories: CIAP-independent SVPD sites and CIAP-dependent SVPD sites. The former consists of strand-break termini that can be recognized directly and digested by SVPD without CIAP pretreatment, whereas the latter includes the termini that cannot be digested by SVPD without CIAP pretreatment. In addition, the apparent radiation-chemical yield (G value) can be estimated using the level of intact 2'-deoxynucleotides produced during a 15-min incubation with SVPD. The G value for total strand breaks in fully dried DNA irradiated with (60)Co gamma-rays was estimated to be 0.1 micromol/J. Moreover, the G values of CIAP-dependent and CIAP-independent SVPD sites were estimated to be 0.078 and 0.024 micromol/J, respectively. These values suggest that 3'-phosphate termini are more likely to be produced than 3' termini without phosphate. Furthermore, piperidine-treated irradiated plasmid DNA was also treated with the same enzyme system to examine the piperidine-labile sites. As a result of the treatment, the G value of the CIAP-dependent SVPD sites increased to 0.16 micromol/J, whereas no significant increase was seen in the G value of the CIAP-independent SVPD sites. This observation implies that most piperidine-labile damaged bases can be eliminated to form apurinic/apyrimidinic sites, which are completely removed by piperidine treatment to form 3' phosphate termini, and that prompt CIAP-independent SVPD sites are piperidine resistant.     

Engineering, cloning, and functional characterization of recombinant LIM mineralization protein-1 containing an N-terminal HIV-derived membrane transduction domain

Short peptide sequences known as protein transduction domains have become increasingly prevalent as tools to internalize molecules that would otherwise remain extracellular. Here, we determine whether a purified recombinant mammalian intracellular osteogenic factor delivered by a HIV-derived TAT-peptide tag is indeed capable of intracellular localization in a form accessible to interaction with other proteins. We engineered and bacterially expressed a TAT-fusion-cDNA construct of a known osteogenic factor, LIM mineralization protein-1 (LMP-1) involved in the bone morphogenetic protein (BMP) pathway that has the potential to serve as an enhancer of BMP-2 efficacy.The expressed recombinant protein contains an N-terminal (His)₆-tag, a hemagglutinin(HA)-tag, and an 11-amino acid HIV-derived TAT-membrane transduction domain and was purified to homogeneity by Sephacryl S-100 molecular exclusion and Ni²⁺-affinity chromatography. The purified TAT–LMP-1 protein was chemically labeled with fluorescein, and its time and concentration dependent entry into rabbit blood cells was monitored by flow cytometry. We demonstrate the accumulation of TAT-tagged LMP-1 both in cytoplasmic and nuclear compartments. By performing affinity pull-down assays, we confirm our earlier findings that the recombinant TAT–LMP-1, when used as molecular bait to identify the intracellular binding proteins, interacts with Smurf1, a known binding partner of LMP-1. We also show potentiation of BMP-2 activity using the purified TAT–LMP-1 in mouse muscle C2C12 cells by assaying a heterologous luciferase-reporter construct containing multiple copies of a BMP-responsive sequence motif. Finally, we also confirm the biological activity of the purified TAT–LMP-1 by showing enhancement of BMP-2 induced increase of alkaline phosphatase mRNA and protein by RT-PCR and enzyme activity, respectively.     

Alpha-phenyl-N-tert-butylnitrone (PBN) prevents light-induced degeneration of the retina by inhibiting RPE65 protein isomerohydrolase activity

α-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5-12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC(50) = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with K(i) = 53 μm. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-α-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.     

Lectin-like binding of Bacillus thuringiensis var. kurstaki lepidopteran-specific toxin is an initial step in insecticidal action

The two delta-endotoxins comprising the Bacillus thuringiensis var. kurstaki HD1 insecticidal protein crystal were separated. The lepidopteran-specific protoxin was activated in vitro and its mechanism of action investigated. Toxicity towards Choristoneura fumiferana CF1 cells was specifically inhibited by preincubation of the toxin with N-acetylgalactosamine and N-acetylneuraminic acid. The lectins soybean agglutinin and wheat germ agglutinin, which bind N-acetylgalactosamine, also inhibited toxicity. Since N-acetylneuraminic acid is not known to occur in insects, these results suggest that the toxin may recognise a specific plasma membrane glycoconjugate receptor with a terminal N-acetylgalactosamine residue.     

Mitochondrial,sarcoplasmic membrane integrity and protein degradation in heart and skeletal muscle in exercised rats

Several different exercise regimens varied in the severity of tissue damage induced. Therefore, this study investigated the effects of a single bout of exercise versus endurance training in heart and skeletal muscles with different predominant fiber types on indices of mitochondrial, endoplasmic reticulum (ER) integrity and protein degradation. Male Wistar rats performed different treadmill exercise protocols: exhaustive, maximal exhaustive, eccentric, training and exhaustive exercise after training. The maximal and eccentric exercises resulted in a significant loss of integrity of the sarcoplasmic and ER muscle, while no changes were observed in cardiac muscle. Mitochondrial membrane fluidity measured by the fluorescence polarization method was significantly increased post-acute exercises in heart and oxidative muscles. Regular exercise can stabilize and preserve the viscoelastic nature of mitochondrial membranes in both tissues. The highest increase in carbonyl content was obtained in heart after exhaustive exercise protocol, from 1+/-0.1 to 3.6+/-0.14 nmol mg protein(-1), such increase were not found after regular exercise with values significantly decreased. Nitrate heart levels showed attenuated generation of nitric oxide after training. Muscle protein oxidation was produced in all exhaustive exercises including eccentric exercise.     

Carbon nanotube aqueous sol-gel composites: enzyme-friendly platforms for the development of stable biosensors

A new type of composite material based on carbon nanotubes and an aqueous sol-gel process has been developed. The electrochemical characteristics of these composites were investigated and compared to composites made with an alkoxy silane sol-gel process. The use of carbon nanotubes, as the conductive part of the composite, facilitated fast electron transfer rates. The feasibility of this type of composite for the development of biosensors was demonstrated using l-amino acid oxidase. The stability of the enzyme was increased when it was encapsulated in the aqueous sol-gel, and the sensor retained more that 50% of its response after 1 month of testing.