Comparison of hybridization behavior between double and single strands of targets and the application of asymmetric PCR targets in cDNA microarray

Double stranded targets on the cDNA microarray contain representatives of both the coding and noncoding strands, which will introduce hybridization competition with probes. Here, the effect of double and single strands of targets on the signal intensity and the ratios of Cy5/Cy3 within the same slide were compared. The results show that single stranded targets can increase the hybridization efficiency without changing the Cy5/Cy3 ratio. Based on these results, a new strategy was established by generating cDNA targets with asymmetric PCR, instead of conventional PCR, to increase the sensitivity of the cDNA microarray. Furthermore, the feasibility of this approach was validated. The results indicate that the cDNA microarray system based on asymmetric PCR is more sensitive, with no decrease in the reliability and reproducibility as compared with that based on conventional symmetric PCR.     

根霉液态发酵产生纤溶酶的培养条件优化

目的:目前临床使用的溶栓药物疗效肯定,但还存在许多缺陷,而且价格昂贵,因此研制新型溶栓药物的需求迫切。方法:研究了根霉Rhizopus chinensisYY-15液体摇瓶发酵产生纤溶酶的工艺条件。采用单因素试验对液体发酵培养基的碳源、氮源、碳氮比、初始pH进行了优化;采用正交试验对发酵时间、接种量进行了研究。结果:实验范围内菌株液体发酵产纤溶酶的适宜培养基组成为:麸皮水浓度3%(w/v),豆粕浓度5%(w/v),初始培养基pH5.0。适宜培养条件为接种量6%,培养时间72h。优化条件下的摇瓶液体发酵纤溶酶产量平均达98.31 U/ml。     

Effects of Arachidonic Acid on the Lysosomal Ion Permeability and Osmotic Stability

In this study, we investigated the effects of arachidonic acid, a PLA₂-produced lipid metabolite, on the lysosomal permeability, osmotic sensitivity and stability. Through the measurements of lysosomal β-hexosaminidase free activity, membrane potential, intralysosomal pH, and lysosomal latency loss in hypotonic sucrose medium, we established that arachidonic acid could increase the lysosomal permeability to both potassium ions and protons, and enhance the lysosomal osmotic sensitivity. As a result, the fatty-acid-promoted entry of potassium ions into the lysosomes via K⁺/H⁺ exchange, which could produce osmotic imbalance across their membranes and osmotically destabilize the lysosomes. In addition, the enhancement of lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in osmotic shock. The results suggest that arachidonic acid may play a role in the lysosomal destabilization.     

Stimulation effect of lithium on the metabolic activity of liver tissue mitochondria measured by microcalorimetry

The effect of Li(I) on the metabolism of mitochondria isolated from Carassius auratus liver tissue was investigated by microcalorimetric method to provide evidence for mitochondria hypothesis of biporlar disorder (BPD) and to explore therapeutic mechanism of drug for treatment of BPD. Obvious stimulation induced by Li(I) on mitochondria metabolism was reflected by power-time (P-t) curves. The power-time curves of hepatic mitochondria metabolism without Li(I) could be divided into four parts: lag phase, active recovery phase, stationary phase, and decline phase. When Li(I) was added, the second heat peak occurred in a concentration-dependent sequence. Considering the first heat peak on the p-t curves, Li(I) in the range of therapeutic and lower concentration induced slight alterations in comparison with the characteristic heat peak observed in the control. However, Li(I) above the therapeutic concentration resulted in significant changes. Heat output increased with the concentration of Li(I), but the rate constant (k ₂) and the maximum heat power (P _max2) for the second heat peak reached maximum value in the range of therapeutic concentration. Mechanism of activation of mitoKatp was suggested and discussed.     

West Nile virus 5'-cap structure is formed by sequential guanine N-7 and ribose 2'-O methylations by nonstructural protein 5

Many flaviviruses are globally important human pathogens. Their plus-strand RNA genome contains a 5'-cap structure that is methylated at the guanine N-7 and the ribose 2'-OH positions of the first transcribed nucleotide, adenine (m(7)GpppAm). Using West Nile virus (WNV), we demonstrate, for the first time, that the nonstructural protein 5 (NS5) mediates both guanine N-7 and ribose 2'-O methylations and therefore is essential for flavivirus 5'-cap formation. We show that a recombinant full-length and a truncated NS5 protein containing the methyltransferase (MTase) domain methylates GpppA-capped and m(7)GpppA-capped RNAs to m(7)GpppAm-RNA, using S-adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m(7)GpppA- and m(7)GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2'-O methylation. Mutagenesis of a K(61)-D(146)-K(182)-E(218) tetrad conserved in other cellular and viral MTases suggests that NS5 requires distinct amino acids for its N-7 and 2'-O MTase activities. The entire K(61)-D(146)-K(182)-E(218) motif is essential for 2'-O MTase activity, whereas N-7 MTase activity requires only D(146). The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K(61)-D(146)-K(182)-E(218) motif in a WNV luciferase-reporting replicon significantly reduced or abolished viral replication in cells. Additionally, the mutant MTase-mediated replication defect could not be trans complemented by a wild-type replicase complex. These findings demonstrate a critical role for the flavivirus MTase in viral reproduction and underscore this domain as a potential target for antiviral therapy.     

Designing a conservation plan for protecting the habitat for giant pandas in the Qionglai mountain range, China

Population viability of the giant panda ( Ailuropoda melanoleuca ) is threatened by small population sizes in scattered isolated habitat areas. Designing a conservation plan for protecting and connecting the fragmented habitat will improve the chances for survival of this endangered species. For such a plan, this study assessed the overall habitat suitability for the species in the Qionglai mountain range (Sichuan, China) using Landsat TM imagery acquired in 2001, geographical data, field surveys, and information acquired in previous researches. Results show that the habitat is separated by roads and rivers, as well as by human settlements and cropland areas, into four main habitat blocks. Overlapping these four habitat blocks with the current nature reserve network reveals that only 36% of the total habitat is protected within nature reserves. Thus, the current nature reserve network is failing to preserve essential habitat for dispersal and genetic exchange. In this study, five key areas and four linkage areas were identified and suggested as nature reserves and/or corridors. These areas, together with the six currently established nature reserves in the mountain range, will form a conservation unit for facilitating the exchange of giant panda individuals among previously isolated habitat blocks. Policies recently implemented by the Chinese government, including the Natural Forest Conservation Program (NFCP) and the Grain-to-Green Program (GTGP), could aid in the formation of such a conservation unit.     

Identification of hepatitis C virus (HCV) 2a-derived epitope peptides having the capacity to induce cytotoxic T lymphocytes in human leukocyte antigen-A24+ and HCV2a-infected patients

Since virus-specific cytotoxic T lymphocytes (CTLs) play a critical role in preventing the spread of hepatitis C virus (HCV), vaccine-based HCV-specific CTL induction could be a promising strategy to treat HCV-infected patients. In this study, we tried to identify HCV2a-derived epitopes, which can induce human leukocyte antigen (HLA)-A24-restricted and peptide-specific CTLs. Peripheral blood mononuclear cells of HCV2a-infected patients or healthy donors were stimulated in vitro with HCV2a-derived peptides, which were prepared based on the HLA-A24 binding motif. As a result, three peptides (HCV2a 576-584, HCV2a 627-635, and HCV2a 1085-1094) efficiently induced peptide-specific CTLs from HLA-A24(+) HCV2a-infected patients as well as healthy donors. The cytotoxicity was exhibited by peptide-specific CD8(+) T cells in an HLA-A24-restricted manner. In addition, the HCV2a 627-635 peptide was frequently recognized by immunoglobulin G of HCV2a-infected patients. These results indicate that the identified three HCV2a peptides might be applicable to peptide-based immunotherapy for HLA-A24(+) HCV2a-infected patients.     

长江中游鱼类寄生棘头虫区系的研究

经过3年10次调查,剖检湖北省宜都、黄冈两处江段所产72种鱼类,共计766尾。收集棘头虫10种,其中包括2新种和1新组合,即蛇鮈新棘吻虫(新种)Neoechinorhynchus saurogobi sp.nov.,长江丽棘虫(新种)Brentisentis yangtzensis sp.nov.(Illiosontidae),鲤丽棘虫(新组合)B.cyprini comb.nov.。对长江中游鱼类寄生棘头虫区系的特点进行了分析和探讨。     

Gene cluster responsible for validamycin biosynthesis in Streptomyces hygroscopicus subsp. jinggangensis 5008

A gene cluster responsible for the biosynthesis of validamycin, an aminocyclitol antibiotic widely used as a control agent for sheath blight disease of rice plants, was identified from Streptomyces hygroscopicus subsp. jinggangensis 5008 using heterologous probe acbC, a gene involved in the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone of the acarbose biosynthetic gene cluster originated from Actinoplanes sp. strain SE50/110. Deletion of a 30-kb DNA fragment from this cluster in the chromosome resulted in loss of validamycin production, confirming a direct involvement of the gene cluster in the biosynthesis of this important plant protectant. A sequenced 6-kb fragment contained valA (an acbC homologue encoding a putative cyclase) as well as two additional complete open reading frames (valB and valC, encoding a putative adenyltransferase and a kinase, respectively), which are organized as an operon. The function of ValA was genetically demonstrated to be essential for validamycin production and biochemically shown to be responsible specifically for the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone in vitro using the ValA protein heterologously overexpressed in E. coli. The information obtained should pave the way for further detailed analysis of the complete biosynthetic pathway, which would lead to a complete understanding of validamycin biosynthesis.