蝮蛇毒碱性磷脂酶A_2基因的克隆

从蝮蛇毒腺中抽提总ＲＮＡ．利用人工合成寡核苷酸引物作逆转录,以ｃＤＮＡ为模板进行体外扩增,获得磷脂酶Ａ２（简称ＰＬＡ２）基因,克隆至ｐＢＳ－ｋｓ载体中。通过对３个碱性ＰＬＡ２（简称ＢＰＬＡ２）基因单独克隆分别作ＤＮＡ全序列分析,推导ｐｒｏ－ＢＰＬＡ２由１３８个氨基酸残基构成,与已测定的部分氨基酸序列比较,基本相符。该基因成功的克隆,不仅推导出ＢＰＬＡ２的蛋白质全序列,也为进一步开展蛇毒功能肽蛋白质工程的研究工作打下了良好的基础。     

Development of a selective pseudosubstrate-based peptide inhibitor of pp60c-src protein tyrosine kinase

Summary Using a combinatorial peptide library method, we identified YIYGSFK as an efficient and specific peptide substrate for pp60^c-src protein tyrosine kinase (PTK) [Lam et al., Int. J. Pept. Protein Res., 45 (1995) 587]. Employing YIYGSFK as a template, we synthesized and evaluated a series of pseudosubstrate-based inhibitors for pp60^c-src. We found that the efficiency of a given inhibitor was highly dependent on the specific tyrosine analog used at the phosphorylation site of the substrate. One of these pseudosubstrate inhibitors, YI(2-Nal)GSFK, selectively inhibited the kinase activity of pp60^c-src, with a K_i of 24 M. This peptide inhibitor exhibited selectivity for pp60^c-src as compared to other PTKs tested, such as c-Abl and Bcr-Abl. Our results suggest that selective inhibitors for a specific PTK can be developed when the structure of a specific and efficient small peptide substrate for this PTK can be used as a template for structure modification.Abbreviations 1-Nal l-1-naphthylalanine - 2-Nal l-2-naphthylalanine - BOP benzotriazolyl-N-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate - BSA bovine serum albumin - cAPK cyclic AMP-dependent protein kinase - DIEA diisopropylethylamine - EGFR epidermal growth factor receptor - Fmoc fluorenylmethoxycarbonyl - HOBt 1-hydroxybenzotriazole - MES 2-[N-morpholino]ethanesulfonic acid - PBS phosphate-buffered salts - pCl l-p-chlorophenylalanine - pF l-p-fluorophenylalanine - PTK protein tyrosine kinase - TLC thin-layer chromatography     

西双版纳石灰岩森林的植物区系地理研究

西双版纳石灰岩森林植物区系经调查有维管束植物１５３科,６４０属,１３９４种及变种,其中,种子植物占１２９科５５８属１２６９种及变种。种子植物的分布区类型组成是热带和主产热带的科占总科数的７１．３％;热带分布属占总属数的９０．１％;热带分布种超过总种数的９０％。热带分布属中又以热带亚洲分布属最多,占总数的３５．３％;热带分布种中则以热带亚洲分布及其变型的种类占总种数的６４．５％为特点。这表明该石灰岩森林植物区系是热带性质的植物区系,属于热带亚洲区系的一部分。由于特殊的地理位置,西双版纳地区是许多典型热带植物的分布北界,同时又是几种地理成分的交汇地带,这又使该石灰岩区系带有明显热带边缘性质和多种地理成分交汇的特点。     

栝楼核糖核酸酶在RNA序列分析中的应用

栝楼核糖核酸酶（RNase TCS）对U碱基具有高度的专一性,在无脲、pH3.5、50℃时,它几乎都在-NP ↓ U-处裂解RNA.它与RNase T1,U₂和有限的碱水解一起,可用于直接的酶法RNA序列分析．     

稻茎毛眼水蝇幼虫田间分布型及抽样技术

根据14丘稻田稻茎毛眼水蝇幼虫调查资料．对其幼虫分布型进行了分析。结果表明;用I、CAm／m、Iσ四种聚集指标法测定,64．3％的田块呈随机分布．28．4%为均匀分布．7．3%为聚集分布。用Iwao平均拥挤度和Taylor幂法则测定．其田间分布型符合随机分布。用频次拟合法测验,50%的田块同时符合渡松、奈曼和负二项分布三种分布型;35．7%的田块同时符合2种分布型。根据该虫田间分布型的特点．制定了相应的序贯抽样技术。     

Morphometrics of Infective Juveniles of Steinernema spp. and Heterorhabditis bacteriophora (Nemata: Rhabditida)

Selected morphometrics of Heterorhabditis bacteriophora and seven species of Steinernema from in vivo culture were compared in relation to time of harvest. In addition, five Steinernema species were reared in vitro and their morphometrics were compared with those from in vivo culture. With in vivo culture, there was generally a negative linear relationship between body length of infective juveniles (IJ) and time of harvest. The distance from the anterior end to the excretory pore (EP) and the tail length (T) of IJ also varied with time of harvest. The E percentage (= EP/T x 100) was the least variable. Body lengths of IJ reared in vitro were much less than those of IJ reared in vivo. The study suggests that IJ harvested from in vivo culture within 1 week of emergence from cadavers are best for species identification. Infective juveniles from in vitro culture should not be used for species identification.     

A Role for Cdk2 Kinase in Negatively Regulating DNA Replication during S Phase of the Cell Cycle

Using cell-free extracts made from Xenopus eggs, we show that cdk2-cyclin E and A kinases play an important role in negatively regulating DNA replication. Specifically, we demonstrate that the cdk2 kinase concentration surrounding chromatin in extracts increases 200-fold once the chromatin is assembled into nuclei. Further, we find that if the cdk2–cyclin E or A concentration in egg cytosol is increased 16-fold before the addition of sperm chromatin, the chromatin fails to initiate DNA replication once assembled into nuclei. This demonstrates that cdk2–cyclin E or A can negatively regulate DNA replication. With respect to how this negative regulation occurs, we show that high levels of cdk2–cyclin E do not block the association of the protein complex ORC with sperm chromatin but do prevent association of MCM3, a protein essential for replication. Importantly, we find that MCM3 that is prebound to chromatin does not dissociate when cdk2– cyclin E levels are increased. Taken together our results strongly suggest that during the embryonic cell cycle, the low concentrations of cdk2–cyclin E present in the cytosol after mitosis and before nuclear formation allow proteins essential for potentiating DNA replication to bind to chromatin, and that the high concentration of cdk2–cyclin E within nuclei prevents MCM from reassociating with chromatin after replication. This situation could serve, in part, to limit DNA replication to a single round per cell cycle.     

Human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus Nef use distinct but overlapping target sites for downregulation of cell surface CD4.

Although the Nef proteins encoded by human immunodeficiency virus type 1 (HIV-1) and simian immuno-deficiency virus (SIV) are known to induce the efficient internalization and degradation of cell surface CD4, it remains unclear whether this process involves a direct interaction between Nef and CD4. Here, we report that CD4 downregulation by HIV-1 and SIV Nef requires distinct but overlapping target sites within the CD4 intracytoplasmic domain. In particular, mutation of a glutamic acid residue located at CD4 residue 405 or of arginine and methionine residues located, respectively, at residue 406 and 407 results in a mutant CD4 protein that is efficiently downregulated by HIV-1 Nef but refractory to downregulation by SIV Nef. However, both HIV-1 and SIV Nef require an isoleucine located at residue 410 and the dileucine motif found at CD4 residues 413 and 414. CD4 downregulation induced by the Nef protein encoded by HIV-2 is shown to require a CD4 target sequence that is similar to, but distinct from, that observed with SIV Nef. These data explain the previous finding that the murine CD4 protein, which has an alanine at residue 405, is refractory to downregulation by SIV, but not HIV-1, Nef (J. L. Foster, S.J. Anderson, A. L. B. Frazier, and J. V. Garcia, Virology 201:373-379, 1994). In addition, these observations provide strong genetic support for the hypothesis that the Nef-mediated downregulation of cell surface CD4 requires a direct Nef-CD4 interaction.     

Binding characterization of the iron transport receptor from the outer membrane of Escherichia coli (FepA): differentiation between FepA and FecA

The dissociation constants for the binding of ferric enterobactin with FepA and FecA are quantitated with displacement experiments. It is found that K _d for FepA is 12 times lower than the one for FecA. This indicates that FepA is an high-affinity receptor while FecA binds ferric enterobactin with a lower affinity. Monoclonal antibodies specific for binding epitopes of FepA inhibit the binding of ferric enterobactin with purified FepA. These same antibodies do not inhibit the binding of ferric enterobactin with purified FecA. This indicates that the binding epitopes in FecA and FepA are different.