应用改进的多聚组氨酸标签T7表达载体构建肺癌cDNA文库

应用噬菌体C端展示系统构建的cDNA文库缺乏开放阅读框筛选机制,文库中多数噬菌体克隆展示框外非天然短肽,给后期蛋白质的筛选带来了不便. 为实现噬菌体的ORF筛选功能,利用PCR技术对已有载体T7Select10-3b进行改造,在MCS处外源cDNA插入位点的3′端引入6聚组氨酸筛选标签,经包装后挑取成功表达的单克隆构建肺癌cDNA文库. 经镍柱亲和层析后,收集文库中表达组氨酸的克隆,利用化学发光免疫试验进行筛选效果鉴定. 结果显示,改造的新型载体可成功表达组氨酸标签,以此构建的肺癌cDNA文库经筛选后,含ORF插入的克隆由筛前的6 %提高至70 %,本研究为提高cDNA文库的质量提供了一种简便可行的方法.     

连作花生田根际土壤优势微生物的分离和鉴定

【目的】从不同连作年限的花生田根际土壤中分离优势微生物并进行鉴定,为研究花生连作后优势微生物的变化奠定基础。【方法】采用土壤稀释分离法从不同连作年限花生根际土壤中分离优势细菌、真菌和放线菌,结合菌株形态特征、培养性状、生理生化特征及16S rDNA序列分析对细菌、放线菌进行鉴定,通过形态特征、培养特征和分子鉴定方法对优势真菌进行鉴定。【结果】从连作花生田根际土壤中分离鉴定出7种优势细菌、7种优势真菌和7种优势放线菌。7种优势细菌分别为Leifsonia xyli、氯酚节杆菌(Arthrobacterchlorophenolicus)、黄色微杆菌(Microbacterium flavescens)、鞘氨醇单胞菌属(Sphingomonas sp.)、巴斯德菌属(Pasteurella sp.)、简单芽孢杆菌(Bacillus simplex)和巨大芽孢杆菌(Bacillus megaterium)。7种优势真菌分别为枝状枝孢菌(Cladosporium cladosporioides)、产紫青霉(Penicillium purpurogenum)、哈茨木霉有性型(Hypocrea lixii)、Exophiala pisciphila、微紫青霉(Penicillium janthinellum)、曲霉(Aspergillus sp.)和大丽轮枝菌(Verticillium dahliae)。7种优势放线菌分别为紫红链霉菌(Streptomyces violaceoruber)、华丽黄链霉菌(Streptomyces flaveus)、Streptomyces panaciterrae、不产色链霉菌(Streptomyces achromogenes)、假浅灰链霉菌(Streptomyces pseudogriseolus)、纤维素链霉菌(Streptomyces cellulosae)和金色链霉菌(Streptomyces aureus)。【结论】本研究是第一次系统的从连作花生根际土中分离鉴定优势微生物,种植花生后根际土壤中优势微生物的种类发生了明显变化,但变化没有规律。     

Bacteria may contribute to distant species recognition in ant–aphid mutualistic relationships

Mutualistic interactions between ant and aphid species have been the subject of considerable historical and contemporary investigations, the primary benefits being cleaning and protection for the aphids and carbohydrate‐rich honeydew for the ants. Questions remained, however, as to the volatile semiochemical factor influencing this relationship. A recent study highlighted the role of bacterial honeydew volatile compounds in ant attraction. Here, ant's ability to distantly discriminate 2 aphid species was investigated based on bacterial honeydew semiochemicals emissions using a two‐way olfactometer. Both the mutualistic aphid Aphis fabae L. and the nonmyrmecophilous aphid Acyrthosiphon pisum Harris were found to be attractive for the ant Lasius niger L. The level of attraction was similar in both assays (control vs. one of the aphid species). However, when given a choice between these 2 aphid species, ants showed a significant preference for Aphis fabae. Honeydew volatiles, mostly from bacterial origins, are known to be a key element in ant attraction. Using the same olfactometry protocol, the relative attractiveness of volatiles emitted by honeydews collected from each aphid species and by bacteria isolated from each honeydew was investigated. Again, ants significantly preferred volatiles released by Aphis fabae honeydew and bacteria. This information suggests that microbial honeydew volatiles enable ants to distantly discriminate aphid species. These results strengthen the interest of studying the occurrence and potential impact of microorganisms in insect symbioses.     

Dimers of nicotinamide adenine dinucleotide: New evidence for the structure and the involvement in an enzymatic redox process

The composition and the structure of the product from the known electrochemical dimerization of the NAD⁺ have been conclusively demonstrated. A detailed analysis of the ¹H and ¹³C nmr spectra has in fact led to the conclusion that the product contains three diastereoisomeric dimers of the 4,4′-tetrahydrobipyridyl type. Furthermore, the cytoplasmic fraction obtained from a standard mitochondrial preparation of rat liver has been shown to catalyze the oxygen uptake by the dimers. A 1 : 1 molar ratio of the reagents in the redox process is indicated by manometric data on oxygen uptake complemented by spectrophotometric analysis of the oxidized substrates, suggesting that H₂O₂ is the reduction product. NAD⁺ was identified as the oxidation product by an enzymatic method.     

The Growth-promoting Effect of Beijerinckia mobilis and Clostridium sp. Cultures on Some Agricultural Crops

New strains of Beijerinckia mobilis and Clostridium sp. isolated from the pea rhizosphere were studied with respect to their promoting effect on the growth and development of some agricultural crops. Seed soaking in bacterial suspensions followed by the soil application of the suspensions or their application by means of foliar spraying was found to be the most efficient method of bacterization. The application of B. mobilis andClostridium sp. cultures in combination with mineral fertilizers increased the crop production by 1.5–2.5 times. The study of the population dynamics of B. mobilis by the method of genetic marking showed that this bacterium quickly colonized the rhizoplane of plants and, therefore, had characteristics of an r-strategist. At the same time, Clostridiumsp. was closer to K-strategists, since this bacterium slowly colonized the econiches studied. The introduction of the bacteria into soil did not affect the indigenous soil bacterial complex. The presence of Clostridium sp. slowed down the colonization of roots by the fungal mycelium. The possible mechanisms of the plant growth–promoting activity of B. mobilisand Clostridiumsp. are discussed.     

Combined effects of cadmium and nickel on testicular xenobiotic metabolizing enzymes in rats

When male rats were given a single dose of cadmium (Cd) (3.58 mg CdCl₂·H₂O/kg, ip) 72 hr prior to sacrifice, the testicular 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EAA), 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP), and cumene hydroperoxide (CHPx) decreased significantly as compared to controls. Cd also inhibited reduced glutathione (GSH) level while increasing the lipid peroxidation (LP) level significantly. When the animals were given a single dose of nickel (Ni) (59.5 mg NiCl₂·6H₂O/kg, ip) 16 hr prior to sacrifice, significant decreases were observed in EROD and GST activities toward CDNB, EAA, EPNP, and CHPx, and GSH level. No significant alterations were noted in DCNB GST activity and LP level by Ni. For the combined treatment, rats received the single dose of Ni 56 hr after the single dose of Cd and were killed 16 hr later. In these animals, lesser depressions were observed on EROD activity and LP level than those of Cd alone. The combination of metals significantly inhibited GST activities and GSH level but not to a greater degree than noted by Cd or Ni alone. Plasma testosterone levels of Cd-, Ni-, and combination-treated rats decreased significantly compared to controls. The strongest depression was achieved by Cd alone. Cd, both alone and in combination with Ni, increased the tissue Ni uptake significantly. Ni, however, did not produce such an effect on the tissue uptake of Cd in either case. Cd treatment caused interstitial edema and coagulation necrosis in seminiferous tubules and also caused fibrinoidal necrosis in vascular endothelium. Ni treatment did not produce any pathological testicular alterations compared to controls. Combined treatment produced fewer pathological alterations (i.e., only interstitial edema) than that of Cd treatment. These results reveal that the combination of Cd and Ni does not have a synergistic effect on testicular xenobiotic metabolizing enzymes, and in contrast, Ni has an ameliorating effect on pathological disturbances caused by Cd alone in the rat testis.     

Role of the CC Chemokine receptor 9/TECK interaction in apoptosis

Chemokine receptors are members of the G protein coupled receptor (GPCR) supergene family whose expression is highly restricted to hematopoietic cells. Although the primary role of chemokine and chemokine receptor interaction is believed to be regulation of chemotaxis of leukocytes, subsequent information clearly suggests that multiple immune regulatory functions are attributed to chemokine receptor signaling. We recently showed that activation of the CC chemokine 9 receptor (CCR9), a thymus-specific chemokine receptor, led to potent cFLIP_L-independent resistance to cycloheximide-induced apoptosis and modest resistance to Fas-mediated apoptosis possibly via activation of multiple signaling components involving Akt and glycogen synthase kinase 3. The fact that these two apoptotic signals involve activation of similar arrays of death execution machinery such as caspase-8, caspase-9, or caspase-3, suggests that chemokine receptor signaling may provide a wide range of antiapoptotic activities to hematopoietic cells under certain biological conditions. GPCR is a large family of cell surface receptors, many of which are critically involved in hormonal and behavioral control. Recent observations also suggest that GPCR signaling plays a pivotal role in immune cell activation. Heterotrimeric G protein is an integral part of GPCR signaling. Thus, dissection of signaling components involved in the CCR9-mediated antiapoptosis could be a framework for cell survival mechanisms and may provide options for therapeutic interventions for neurdegenerative diseases or T cell malfunctioning.     

Morphology and Phylogeny of the Soil Ciliate Metopus yantaiensis n. sp. (Ciliophora,Metopida), with Identification of the Intracellular Bacteria

The morphology and infraciliature of a new ciliate, Metopus yantaiensis n. sp., discovered in coastal soil of northern China, were investigated. It is distinguished from its congeners by a combination of the following features: nuclear apparatus situated in the preoral dome; 18–21 somatic ciliary rows, of which three extend onto the preoral dome (dome kineties); three to five distinctly elongated caudal cilia, and 21–29 adoral polykinetids. The 18S rRNA genes of this new species and two congeners, Metopus contortus and Metopus hasei, were sequenced and phylogenetically analyzed. The new species is more closely related to M. hasei and the clevelandellids than to other congeners; both the genus Metopus and the order Metopida are not monophyletic. In addition, the digestion‐resistant bacteria in the cytoplasm of M. yantaiensis were identified, using a 16S rRNA gene clone library, sequencing, and fluorescence in situ hybridization. The detected intracellular bacteria are affiliated with Sphingomonadales, Rhizobiales, Rickettsiales (Alphaproteobacteria), Pseudomonas (Gammaproteobacteria), Rhodocyclales (Betaproteobacteria), Clostridiales (Firmicutes), and Flavobacteriales (Bacteroidetes).     

Expression and cellular localization of naturally occurring beta estrogen receptors in uterine and mammary cell lines

The protein ER-alpha has been exhaustively characterized in estrogen-sensitive tissues and cell lines. However, little is known regarding the expression and cellular distribution of the newly identified ER-beta protein. We first quantified the specific estradiol binding site content in the estrogen-responsive cell lines MCF-7 (mammary) and SHM (myometrial). In the two cell types, these sites were associated to the expression of both ER-alpha and -beta isoforms. Native ER-beta was visualized to reside inside the nucleus by means of conventional indirect immunofluorescence. The cells expressed ER-beta as a tight approximately 50 kDa triplet when resolved by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and blotted using antibodies mapping different domains of the cloned ER-beta version. When the cells were subjected to homogenization and differential centrifugation, a substantial proportion of ER-beta immunolabeling was localized at membrane subfractions. ER-beta expression and partitioning was confirmed by Ligand blotting assays using estrogen derivatives coupled to different macromolecular tags. However, ER-alpha was expressed as the major estrogen binding protein in both cell lines. Similar localization experiments were performed on HeLa cells (cervix). Though usually considered ER-negative, this cell line displayed basal significant estrogen binding capacity and co-expression of both ER isoforms. Taken as a whole, the results indicate that ER-beta could be expressed as functional estrogen binding proteins among a dominant population of ER-alpha sites in the cell lines under study.