牛脑皮层G_i的纯化及其与AC的偶联

用1％胆酸钠和20％饱和度的硫酸铵抽提牛脑皮层细胞膜得到含G蛋白和腺苷酸环化酶（AC）的制剂,通过Sepharose6B柱将两者分开,再将含G蛋白的级分用庚胺-Sepharose4B疏水柱、羟基磷灰石柱将其它亚型的G蛋白（主要是Gs和Go）从抑制型G蛋白（Gi）中除去,获得纯化的高活力的Gi,其GTP结合活力为17．6nmol／mg,比细胞膜Gi活力提高50倍;并具有较高的产率,从1g膜蛋白中可获得0．66mg的Gi,同时可获得无G蛋白污染的AC和少量的Gs蛋白．SDS-PAGE显示分子量为41000和36000的两条蛋白带,证实是Gi的α基和β亚基．进一步用重建脂酶体的方法检测Gi对AC的抑制作用,结果显示Gi对AC活力的抑制达40％左右,表明CAMP信息跨膜转导通路中Gi与AC之间具有较好偶联功能．     

Cloning of a Novel Rat Gene, DB83, That Encodes a Putative Membrane Protein

Using a partial cDNA sequence and a 5'-RACE technique, we isolateda novel cDNA from rat liver referred to as DB83. DB83 had fourhydrophobic trans-membrane domains and one N-myristoylationsite as well as multiple possible phosphorylation sites. Thedb83 gene was highly expressed in the liver and significantlyin brain, lungs and kidneys. We suggest that DB83 is a tissue-specificputative membrane protein.     

绿色荧光蛋白gfp基因在钝顶螺旋藻(Spirulina platensis)A9藻株中的表达

将钝顶螺旋藻(Spirulina platensis)A9藻株在24℃培养,经2 mmol/L的EDTA预处理24 h;采用功率300 W的超声波处理70 s获得单细胞样品,以本实验室构建的携带gfp基因的质粒p215t转化A9藻株单细胞藻液,利用Amp作为选择标记,使单细胞在平板上再生长出单藻落,获得17株具有Amp抗性的转化藻株,转化率3.73‰。在390 nm紫光激发下,生长30天的转化藻丝体发出稳定绿色荧光;培养45天后具有绿色荧光的藻丝出现断裂、具有荧光藻丝长度缩短的现象。实验结果初步表明:报告基因gfp在螺旋藻中得到稳定有效的表达,可以采用单细胞再生形成单藻落技术进行螺旋藻的基因克隆。     

Dlmo基因编码一个32kDa的蛋白

为了获取研究Dlmo（果蝇LMO基因的简称）的功能信息,使用耦联网织红细胞体外翻译系统将Dlmo基因进行体外转录翻译得到32kDa的蛋白产物。该蛋白产物与抗人类LMO2蛋白的多抗抗体发生免疫沉淀,并得到了32kDa的阳性带。为了证实Dlmo基因在体外和体内翻译能得到同一大小蛋白,从2～4小时果蝇胚盘中分别抽提核和胞浆提取物进行Western印迹分析,免疫血清可以识别胞浆提取物中的32kDa蛋白,而在核提取物中未曾见到,证实体外和体内翻译产物相同。免疫组织化学的分析在0～4小时胚盘外周胞浆中有Dlmo基因的阳性染色信号,结果证实,Dlmo与人类LMO不同,其表达产物是一个胞浆蛋白。 Abstract：In order to gain some insight into a possible function of Dlmo gene,we used the TNT coupled reticulocyte lysate systems as an in vitro translation system to detect the Drosoplila protein. We found a 32kDa protein product.To demonstrate that the DLMO protein has the same size in vivo as in vitro we studied nuclear and cytoplasmic extracts from 2-4h embryos in Western blots. The immuno serum recognizes a 32kDa protein in the cytoplasm that is not present in the nucleus.The products were immune precipitated with the polyclonal anti-LMO2 antibody raised against the human protein and seen a positive band of 32kDa.Using immuno histochemical analysis was seen a positive staining in the basal cytoplasm of blastoderm embryos at 4 hours. This result confirms that the DLMO is a cytoplasmprotein, not like human LMO protein.     

The Hansenula polymorpha ATG25 Gene Encodes a Novel Coiled-Coil Protein that is Required for Macropexophagy

We have isolated the Hansenula polymorpha ATG11 and ATG25 genes, which are both required for glucose-induced selective peroxisome degradation (macropexophagy). ATG11 was identified before in other yeast species and shown to be involved in the Cvt pathway in Saccharomyces cerevisiae and glucose-induced micropexophagy in Pichia pastoris. Our data indicate that HpATG11 is required for macropexophagy. ATG25 represents a novel gene that encodes a 45 kDa coiled-coil protein. We show that this protein co-localizes with Atg11 on a small structure, which most likely represents the pre-autophagosomal structure (PAS). Cells of a constructed ATG25 deletion strain (atg25) displayed relatively slow, continuous degradation of peroxisomes by microautophagy during growth on methanol in the presence of excess nitrogen that also continued after induction of selective peroxisome degradation. This suggests that the processes of selective and non-selective autophagy are dysregulated in atg25 cells.     

Exchange of Guanine Nucleotides Between Tubulin and GTP-Binding Proteins That Regulate Adenylate Cyclase: Cytoskeletal Modification of Neuronal Signal Transduction

Tubulin, the primary constituent of microtubules, is a GTP-binding proteins with structural similarities to other GTP-binding proteins. Whereas microtubules have been implicated as modulators of the adenylate cyclase system, the mechanism of this regulation has been elusive. Tubulin, polymerized with the hydrolysis-resistant GTP analog, 5'-guanylylimidodiphosphate [Gpp(NH)p], can promote inhibition of synaptic membrane adenylate cyclase which persists subsequent to washing. Tubulin with Gpp(NH)p bound was slightly less potent than free Gpp(NH)p in the inhibition of adenylate cyclase, but tubulin without nucleotide bound had no effect on the enzyme. A GTP-binding protein from the rod outer segment (transducin), with Gpp(NH)p bound, was also without effect on adenylate cyclase. Tubulin (regardless of the nucleotide bound to it) did not alter the activity of the adenylate cyclase catalytic unit directly. When tubulin was polymerized with the hydrolysis-resistant photoaffinity GTP analog, [32P]P3(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP), and this protein was added to synaptic membranes, AAGTP was transferred from tubulin to the inhibitory GTP-binding protein, Gi. This transfer was blocked by prior incubation of the membranes with Gpp(NH)p or covalent binding of AAGTP to tubulin prior to exposure of that tubulin to membranes. Incubation of membranes with Gpp(NH)p subsequent to incubation with tubulin-AAGTP results in a decrease in AAGTP bound to Gi and a compensatory increase in AAGTP bound to the stimulatory GTP-binding protein, Gs. Likewise, persistent inhibition of adenylate cyclase by tubulin-Gpp(NH)p could be overridden by the inclusion of 100 microM Gpp(NH)p in the assay inhibition. Whereas Gpp(NH)p promotes persistent inhibition of synaptic membrane adenylate cyclase without incubation at elevated temperatures, tubulin [with AAGTP or Gpp(NH)p bound] requires 30 s incubation at 23 degrees C to effect adenylate cyclase inhibition. Photoaffinity experiments yield parallel results. These data are consistent with synaptic membrane tubulin regulating neuronal adenylate cyclase by transferring GTP to Gi and, subsequently, to Gs.     

Adenylate Cyclase in Silkworm: Regulation by Calcium and a Calcium-binding Protein

Homogenates of silkworm pupal fat body were separated into particulate and supernatant fractions by centrifugation. The particulate fraction was further washed with EGTA. Adenylate cyclase activity of the washed particulate fraction was stimulated 2-fold by the addition of supernatant fraction in the presence of low concentrations of Ca²⁺. The activating factor in supernatant was heat-stable, non-dialyzable and trypsin-sensitive, and shown to be a Ca²⁺-dependent regulator protein. For the activation of adenylate cyclase by the regulator protein, the optimum concentrations of free Ca²⁺ were in a range of 2 µm, and higher concentrations of Ca²⁺ were inhibitory.     

The Abnormal Spindle-like,Microcephaly-associated (ASPM) Gene Encodes a Centrosomal Protein

Homozygous mutations in the abnormal spindle-like, microcephaly-associatedASPM gene are the leading cause of autosomal recessive primary microcephaly. ASPM isthe putative human ortholog of the Drosophila melanogaster abnormal spindles gene(asp), which is essential for mitotic spindle function. Here, we report thatdownregulation of endogenous ASPM by siRNA decreases protein levels of endogenousBRCA1. ASPM localizes to the centrosome in interphase and to the spindle poles fromprophase through telophase. These findings indicate that ASPM may be involved inmitotic spindle function, possibly, through regulation of BRCA1.     

Distribution of Protein I, a Synapse-Specific Phosphoprotein, and Adenylate Cyclase in the Rat Spinal Cord

The longitudinal and transverse distributions of the synapse-specific phosphoprotein Protein I and adenylate cyclase in the rat spinal cord were studied. Protein I was found to be enriched in all cervical and midlumbar (L3-L5) segments, and sparse in midthoracic and sacral segments. Adenylate cyclase activity was high in all cervical and lumbosacral segments, and low in mid-thoracic segments. Cross sectionally, both Protein I and adenylate cyclase were more enriched in the dorsal half than in the ventral half in the various segments studied. The similar topographical distributions of Protein I and adenylate cyclase in the spinal cord support the idea that adenylate cyclase may be intimately associated with Protein I in the nervous system, and could thereby regulate the state of in vivo phosphorylation of Protein I through formation of cyclic AMP.     

日本血吸虫信号转导蛋白Sjwnt-4基因的克隆、表达及功能分析

由Wnt基因家族产物与其它相关基因产物构成的Wnt信号通路,是细胞发育和生长调节的一个关键途径,对动物的发育特别是生殖系统的发育起重要的调节作用。在人类和小鼠中,Wnt4蛋白是性腺分化过程中主要调节因子,在胚胎发育中起着关键作用。利用RACE技术从日本血吸虫19d童虫中首次扩增到一个Wnt家族基因,序列分析表明该基因的完整编码框含1311bp,编码436个氨基酸,理论分子量49.6kD。同源性分析结果表明,该基因的氨基酸序列具有典型Wnt家族蛋白特征,与日本三角涡虫、人Wnt4的氨基酸序列相似性分别达43%、37%,推测为血吸虫的Wnt4基因,命名为Sjwnt4(GenBank登陆号DQ643829)。实时定量PCR分析显示该基因在14d童虫、19d童虫、31d虫体、44d雌虫及44d雄虫中均有表达,其中19d童虫中的表达量明显高于其它发育阶段,44d雌虫中的表达量明显高于雄虫。构建了该基因的原核表达载体pGEX-4T-2-Sjwnt4,应用大肠杆菌系统进行了表达,表达蛋白以包涵体形式存在,Western印迹显示表达产物能被日本血吸虫成虫粗抗原免疫血清所识别。Sjwnt4基因及其表达产物的获得,为探索Wnt信号通路对血吸虫发育、生殖的调节提供了重要基础。     

Protein Clusters in Phosphotyrosine Signal Transduction

Signal transduction systems based on tyrosine phosphorylation are central to cell–cell communication in multicellular organisms. Typically, in such a system, the signal is initiated by activating tyrosine kinases associated with transmembrane receptors, which induces tyrosine phosphorylation of the receptor and/or associated proteins. The phosphorylated tyrosines then serve as docking sites for the binding of various downstream effector proteins. It has long been observed that the cooperative association of the receptors and effectors produces higher-order protein assemblies (clusters) following signal activation in virtually all phosphotyrosine signal transduction systems. However, mechanistic studies on how such clustering processes affect signal transduction outcomes have only emerged recently. Here we review current progress in decoding the biophysical consequences of clustering on the behavior of the system, and how clustering affects how these receptors process information.     

The Epstein-Barr Virus BARF1 Gene Encodes a Novel, Soluble Colony-Stimulating Factor-1 Receptor

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus associated with infectious mononucleosis and several tumors. The BARF1 gene is transcribed early after EBV infection from the BamHI A fragment of the EBV genome. Evidence shown here indicates that the BARF1 protein is secreted into the medium of transfected cells and from EBV-carrying B cells induced to allow lytic replication of the virus. Expression cloning identified colony-stimulating factor-1 (CSF-1) as a ligand for BARF1. Computer-assisted analyses indicated that subtle amino acid sequence homology exists between BARF1 and c-fms, the cellular proto-oncogene that is the receptor for CSF-1. Recombinant BARF1 protein was found to be biologically active, and it neutralized the proliferative effects of human CSF-1 in a dose-dependent fashion when assayed in vitro. Since CSF-1 is a pleiotropic cytokine best known for its differentiating effects on macrophages, these data suggest that BARF1 may function to modulate the host immune response to EBV infection.     

An Auxin-Regulated Gene of Arabidopsis thaliana Encodes a DNA-Binding Protein

We have isolated a single-copy gene from the plant Arabidopsis thaliana, called dbp, which encodes a lysine-rich, DNA-binding protein. The Dbp protein has a molecular weight and a composition resembling histone H1. When the dbp gene was expressed in bacteria, the protein product bound DNA nonspecifically. The dbp gene is expressed constitutively in all parts of the plant but is induced five times above this basal level in apical zones. In vitro hormone-depletion experiments showed that the expression in the shoot apex could be induced by exogenous auxin. In situ hybridizations in the root apex indicated that the expression of dbp is enhanced in the region of cell division.     

Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein

Background

Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.

Methodology/Principal Findings

Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.

Conclusions/Significance

The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.     

Study of the Functional Organization of a Novel Adenylate Cyclase Signaling Mechanism of Insulin Action

In this study we continued decoding the adenylate cyclase signaling mechanism that underlies the effect of insulin and related peptides. We show for the first time that insulin signal transduction via an adenylate cyclase signaling mechanism, which is attended by adenylate cyclase activation, is blocked in the muscle tissues of the rat and the mollusk Anodonta cygnea in the presence of: 1) pertussis toxin, which impairs the action of the inhibitory GTP-binding protein (G_i); 2) wortmannin, a specific blocker of phosphatidylinositol 3-kinase; and 3) calphostin C, an inhibitor of different isoforms of protein kinase C. The treatment of sarcolemmal membrane fraction with cholera toxin increases basal adenylate cyclase activity and decreases the sensitivity of the enzyme to insulin. We suggest that the stimulating effect of insulin on adenylate cyclase involves the following stages of hormonal signal transduction cascade: receptor tyrosine kinase → G_iprotein (βγ) → phosphatidylinositol 3-kinase → protein kinase C (ζ?) → G_sprotein → adenylate cyclase → cAMP.