心脏特异新基因Lrrc10的分子克隆与特性分析

采用表达序列标签(EST)介导的基因克隆和表达谱分析,从小鼠心脏克隆了一个心脏特异新基因Lrrc10（GenBank Acc No. AF527781）.该基因cDNA全长为1 410 bp,定位于小鼠染色体10D2,在基因组中无内含子.Lrrc10的最大开放阅读框编码的假想蛋白由274个氨基酸组成,含有7个亮氨酸重复基序.同源性检索未发现有整体同源性的已知基因.EST数据库中支持该基因cDNA序列的全部18条EST均来自小鼠心脏组织.对小鼠的不同组织cDNA的RT-PCR检测证实该基因主要在心脏中强表达,在肺低表达,而在其他组织中不表达或表达很弱.因此该基因是心脏特异的富亮氨酸重复超家族新成员.     

Characterization of AmiBA2446, a Novel Bacteriolytic Enzyme Active against Bacillus Species

There continues to be a need for developing efficient and environmentally friendly treatments for Bacillus anthracis, the causative agent of anthrax. One emerging approach for inactivation of vegetative B. anthracis is the use of bacteriophage endolysins or lytic enzymes encoded by bacterial genomes (autolysins) with highly evolved specificity toward bacterium-specific peptidoglycan cell walls. In this work, we performed in silico analysis of the genome of Bacillus anthracis strain Ames, using a consensus binding domain amino acid sequence as a probe, and identified a novel lytic enzyme that we termed AmiBA2446. This enzyme exists as a homodimer, as determined by size exclusion studies. It possesses N-acetylmuramoyl-l-alanine amidase activity, as determined from liquid chromatography-mass spectrometry (LC-MS) analysis of muropeptides released due to the enzymatic digestion of peptidoglycan. Phylogenetic analysis suggested that AmiBA2446 was an autolysin of bacterial origin. We characterized the effects of enzyme concentration and phase of bacterial growth on bactericidal activity and observed close to a 5-log reduction in the viability of cells of Bacillus cereus 4342, a surrogate for B. anthracis. We further tested the bactericidal activity of AmiBA2446 against various Bacillus species and demonstrated significant activity against B. anthracis and B. cereus strains. We also demonstrated activity against B. anthracis spores after pretreatment with germinants. AmiBA2446 enzyme was also stable in solution, retaining its activity after 4 months of storage at room temperature.     

多发性骨髓瘤细胞中一个新的Alu超基因家族成员的克隆与分析

根据Genebank收录的多发性骨髓瘤细胞株（ARH－77）表达上调EST AF497797设计引物,采用半定量RT－PCR证实了多发性骨髓瘤患者及正常人骨髓细胞中该expressed sequence tages(EST)存在表达差异。用EST AF497797作探针筛选人胚肾cDNA文库,获得cDNA克隆经测序并用生物信息学方法对该序列进行了初步分析。AF497797在多发性骨髓瘤患者骨髓中确有较高的表达,而在正常人骨髓细胞中低表达。获得的全长cDNA克隆序列长1248bp(Genebank登录号：AY094612）。生物信息学分析显示该片段全长cDNA编码44个氨基酸的蛋白产物且可能属于Alu家族成员。基因AY094612为一个在多发性骨髓瘤中表达上调的新基因,其改变可能与多发性骨髓瘤的发生与发展有关。     

富亮氨酸重复超家族新成员LRRC4的克隆与在脑瘤中的表达分析

在染色体7q31-32多种肿瘤杂合性丢失（loss of heterozygosity,LOH）高频区,采用表达序列标签（expressed sequence tag,EST）介导的定位候选克隆策略获得了一个定位于人染色体7q31-32的新基因（GenBank 登录号: AF196976）.该基因编码653个氨基酸,蛋白质理论pI/m:6.58/72.7 ku.它包含七个典型的LRR、一个IgC2样结构域.此外,它还包含一个N端信号肽、一个C端跨膜区.其结构特征表明它是富亮氨酸重复（leucine-rich repeat,LRR）超家族的新成员.经过人类基因组命名委员会的同意,将该基因命名为LRRC4.此外,通过序列相似性匹配还获得了定位于小鼠6号染色体的LRRC4的同源基因（GenBank 登录号: AF290542）.RNA印迹和RT-PCR检测发现LRRC4在正常人脑组织相对特异表达,而在多种原发性脑瘤表达明显下调或缺失.综合考虑LRRC4基因的序列特征及表达谱,提示LRRC4基因可能在神经系统中发挥重要作用.     

一株海洋细菌的初步鉴定及其产黑色素相关新基因(簇)的分离

对分离自近海沼泽地大米草根际的一株供试海洋细菌MWYL1进行了形态观察、生理特性检测以及16SrDNA序列分析。实验结果表明:该菌株属于海洋单胞菌属(Marinomonas),革兰氏染色呈阴性,直杆状,好氧生长,适于28℃生长。基于16SrDNA序列的Blast分析表明,菌株MWYL1与Marinomonas pontica和Marinomonas dokdonensis的序列相似性分别为97%和95%。通过基因组fosmid文库的构建,直接分离到一个产生黑色素的克隆,进一步亚克隆和测序后获得与黑色素产生相关的功能新基因(簇),并且对其进行了生物信息学的初步分析。     

Unusual N-Prenylation in Diazepinomicin Biosynthesis: The Farnesylation of a Benzodiazepine Substrate Is Catalyzed by a New Member of the ABBA Prenyltransferase Superfamily

The bacterium Micromonospora sp. RV115, isolated from a marine sponge, produces the unusual metabolite diazepinomicin, a prenylated benzodiazepine derivative. We have cloned the prenyltransferase gene dzmP from this organism, expressed it in Escherichia coli, and the resulting His₈-tagged protein was purified and investigated biochemically. It was found to catalyze the farnesylation of the amide nitrogen of dibenzodiazepinone. DzmP belongs to the ABBA prenyltransferases and is the first member of this superfamily which utilizes farnesyl diphosphate as genuine substrate. All previously discovered members utilize either dimethylallyl diphosphate (C₅) or geranyl diphosphate (C₁₀). Another putative diazepinomicin biosynthetic gene cluster was identified in the genome of Streptomyces griseoflavus Tü4000, suggesting that the formation of diazepinomicin is not restricted to the genus Micromonospora. The gene cluster contains a gene ssrg_00986 with 61.4% identity (amino acid level) to dzmP. The gene was expressed in E. coli, and the purified protein showed similar catalytic properties as DzmP. Both enzymes also accepted other phenolic or phenazine substrates. ABBA prenyltransferases are useful tools for chemoenzymatic synthesis, due to their nature as soluble, stable biocatalysts. The discovery of DzmP and Ssrg_00986 extends the isoprenoid substrate range of this superfamily. The observed prenylation of an amide nitrogen is an unusual biochemical reaction.     

Junctional Adhesion Molecule,a Novel Member of the Immunoglobulin Superfamily That Distributes at Intercellular Junctions and Modulates Monocyte Transmigration

Tight junctions are the most apical components of endothelial and epithelial intercellular cleft. In the endothelium these structures play an important role in the control of paracellular permeability to circulating cells and solutes. The only known integral membrane protein localized at sites of membrane–membrane interaction of tight junctions is occludin, which is linked inside the cells to a complex network of cytoskeletal and signaling proteins. We report here the identification of a novel protein (junctional adhesion molecule [JAM]) that is selectively concentrated at intercellular junctions of endothelial and epithelial cells of different origins. Confocal and immunoelectron microscopy shows that JAM codistributes with tight junction components at the apical region of the intercellular cleft. A cDNA clone encoding JAM defines a novel immunoglobulin gene superfamily member that consists of two V-type Ig domains. An mAb directed to JAM (BV11) was found to inhibit spontaneous and chemokine-induced monocyte transmigration through an endothelial cell monolayer in vitro. Systemic treatment of mice with BV11 mAb blocked monocyte infiltration upon chemokine administration in subcutaneous air pouches. Thus, JAM is a new component of endothelial and epithelial junctions that play a role in regulating monocyte transmigration.     

Cloning, Expression Analysis, and Chromosomal Localization of BH-Protocadherin (PCDH7), a Novel Member of the Cadherin Superfamily

We have identified a novel member of the cadherin superfamily. Among the members of the superfamily, this protein exhibited the highest overall homology with protocadherin-1 (46–49% identity). Its mRNA was predominantly expressed in the brain and heart. Hence, we named the gene BH-protocadherin (BH-Pcdh) (HGMW-approved symbol PCDH7). BH-Pcdh has an extracellular domain consisting of seven repeats of the cadherin motif (EC 1 to 7). EC2 of BH-Pcdh is unique in having a 55-amino-acid insertion in the middle of the motif. There are three isoforms of BH-Pcdh, denoted -a, -b, and -c, which have different cytoplasmic tails and a 47-amino-acid deletion in the EC2–3 region of BH-Pcdh-c. While only a 9.0-kb message was detected in normal tissues, 4.5- and 9.0-kb mRNA species were seen in the human lung carcinoma cell line A549. Furthermore, only the 4.5-kb mRNA was detected in HeLa cell S3 and human gastric cancer cell lines MKN28 and KATO-III. Southern blot analysis indicated that the BH-Pcdh gene is likely to be conserved among various vertebrates. The BH-Pcdh gene was localized to human chromosome 4p15. Interestingly, 4p15 is a region of loss of heterozygosity in some head and neck squamous cell carcinomas.     

Identification and Characterization of a Novel Staphylococcal Emetic Toxin

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have superantigenic and emetic activities, which cause toxic shock syndrome and staphylococcal food poisoning, respectively. Our previous study demonstrated that the sequence of SET has a low level of similarity to the sequences of other SEs and exhibits atypical bioactivities. Hence, we further explored whether there is an additional SET-related gene in S. aureus strains. One SET-like gene was found in the genome of S. aureus isolates that originated from a case of food poisoning, a human nasal swab, and a case of bovine mastitis. The deduced amino acid sequence of the SET-like gene showed 32% identity with the amino acid sequence of SET. The SET-like gene product was designated SElY. In the food poisoning and nasal swab isolates, mRNA encoding SElY was highly expressed in the early log phase of cultivation, whereas a high level of expression of this mRNA was found in the bovine mastitis isolate at the early stationary phase. To estimate whether SElY has both superantigenic and emetic activities, recombinant SElY was prepared. Cell proliferation and cytokine production were examined to assess the superantigenic activity of SElY. SElY exhibited superantigenic activity in human peripheral blood mononuclear cells but not in mouse splenocytes. In addition, SElY exhibited emetic activity in house musk shrews after intraperitoneal and oral administration. However, the stability of SElY against heating and pepsin and trypsin digestion was different from that of SET and SEA. From these results, we identified SElY to be a novel staphylococcal emetic toxin.     

富亮氨酸重复超家族新成员LRRC4的IgC2结构域蛋白的原核表达和纯化

富亮氨酸重复超家族新成员LRRC4基因是新克隆的脑瘤相关基因,采用多聚酶链式反应（PCR）方法获得长约500bp含IgC2结构域的DNA序列,扩增产物克隆至pGEX-4T-2质粒中,构建GST融合表达质粒,在大肠杆菌中诱导表达融合蛋白,经包涵体沉淀,溶解,Glutathione-Sepharose亲和层析纯化获得融合蛋白,并以Western blot鉴定证实,通过IgC2结构域蛋白的纯化分离该结构域,为进一步研究该结构域及LRRC4基因的结构和功能奠定了基础。     

Isolation and Characterization of Anaerobic Ethylbenzene Dehydrogenase, a Novel Mo-Fe-S Enzyme

The first step in anaerobic ethylbenzene mineralization in denitrifying Azoarcus sp. strain EB1 is the oxidation of ethylbenzene to (S)-(-)-1-phenylethanol. Ethylbenzene dehydrogenase, which catalyzes this reaction, is a unique enzyme in that it mediates the stereoselective hydroxylation of an aromatic hydrocarbon in the absence of molecular oxygen. We purified ethylbenzene dehydrogenase to apparent homogeneity and showed that the enzyme is a heterotrimer (alphabetagamma) with subunit masses of 100 kDa (alpha), 35 kDa (beta), and 25 kDa (gamma). Purified ethylbenzene dehydrogenase contains approximately 0.5 mol of molybdenum, 16 mol of iron, and 15 mol of acid-labile sulfur per mol of holoenzyme, as well as a molydopterin cofactor. In addition to ethylbenzene, purified ethylbenzene dehydrogenase was found to oxidize 4-fluoro-ethylbenzene and the nonaromatic hydrocarbons 3-methyl-2-pentene and ethylidenecyclohexane. Sequencing of the encoding genes revealed that ebdA encodes the alpha subunit, a 974-amino-acid polypeptide containing a molybdopterin-binding domain. The ebdB gene encodes the beta subunit, a 352-amino-acid polypeptide with several 4Fe-4S binding domains. The ebdC gene encodes the gamma subunit, a 214-amino-acid polypeptide that is a potential membrane anchor subunit. Sequence analysis and biochemical data suggest that ethylbenzene dehydrogenase is a novel member of the dimethyl sulfoxide reductase family of molybdopterin-containing enzymes.     

蕲蛇蛇毒中一个新的类凝血酶的分离纯化与表征

使用DEAE－Sephadex A-50、POROS 50HS及POROS 20PI等色谱分离技术,从蕲蛇粗毒中分离提纯得到1个具有凝血活力的组分,经SDS－PAGE和PAGE检测均为一条带,非还原条件下相对分子质量24.3kDa,还原条件下相对分子质量33.0kDa;其凝血活力为91.0NIH u/mg。该组分不具有激活因子ⅩⅢ的活性,肝素不影响该组分的凝血活性,EDTA部分抑制其活性,苯甲基磺酰氟（PMSF）则会产生不可逆抑制作用,该酶N－末端氨基酸序列为VIGGNGXDINEHRFLVAFF,经分析表明该酶是一种新的类凝血酶。     

Identification of a Deubiquitinating Enzyme as a Novel AGS3-Interacting Protein

Activator of G protein Signaling 3 (AGS3) is a receptor-independent G protein activator that has been implicated in multiple biological events such as brain development, neuroplasticity and addiction, cardiac function, Golgi structure/function, macroautophagy and metabolism. However, how AGS3 is regulated is little known. We demonstrate here that AGS3 interacts with a ubiquitin specific protease USP9x, and this interaction is at least partially mediated through the C-terminal G protein regulatory domain of AGS3. Knockdown of USP9x causes a moderate reduction in the level of AGS3. In contrast, overexpression of either USP9x or its deubiquitinating domain UCH increases the amount of AGS3, whereas expression of the mutant UCH domain that lacks deubiquitinating activity does not have the same effect. As previously observed in AGS3 knockdown cells, the localization of several marker proteins of the late Golgi compartments is disturbed in cells depleted of USP9x. Taken together, our study suggests that USP9x can modulate the level of a subpopulation of AGS3, and this modulation plays a role in regulating the structure of the late Golgi compartments. Finally, we have found that levels of AGS3 and USP9x are co-regulated in the prefrontal cortex of rats withdrawn from repeated cocaine treatment. In conjunction with the above data, this observation indicates a potential role of USP9X in the regulation of the AGS3 level during cocaine-induced neuroplasticity.     

肿瘤-睾丸-脑抗原家族新成员PNMA5的克隆和表达谱分析

综合运用几种生物信息学数据库 ,分析了人类X染色体上可能的未知基因 ,筛选获得一个新的肿瘤抗原基因并克隆鉴定了它的全长cDNA .该基因全长为 3194bp ,编码一个 4 48个氨基酸残基的蛋白质 ,它与肿瘤 睾丸 脑抗原家族成员PNMA1、PNMA2及PNMA3有很高的同源性 ,命名为PNMA5 .在 16种人正常组织中 ,PNMA5仅在睾丸与脑中表达 ;而在所检测的肝癌、胃癌、肠癌、肺癌、乳腺癌与头颈部肿瘤 ,PNMA5在肠癌中表达 .PNMA5是肿瘤 睾丸 脑抗原家族的一个新成员 .认识这些肿瘤 睾丸 脑抗原的意义在于它们在肿瘤组织的表达可能是肿瘤破坏机体免疫耐受的一种机制 ,其相关神经副肿瘤综合症可能是一些隐匿肿瘤的早期表现 .     

Identification and Characterization of the Interaction Site between cFLIPL and Calmodulin

Overexpression of the cellular FLICE-like inhibitory protein (cFLIP) has been reported in a number of tumor types. As an inactive procaspase-8 homologue, cFLIP is recruited to the intracellular assembly known as the Death Inducing Signaling Complex (DISC) where it inhibits apoptosis, leading to cancer cell proliferation. Here we characterize the molecular details of the interaction between cFLIP_L and calmodulin, a ubiquitous calcium sensing protein. By expressing the individual domains of cFLIP_L, we demonstrate that the interaction with calmodulin is mediated by the N-terminal death effector domain (DED1) of cFLIP_L. Additionally, we mapped the interaction to a specific region of the C-terminus of DED1, referred to as DED1 R4. By designing DED1/DED2 chimeric constructs in which the homologous R4 regions of the two domains were swapped, calmodulin binding properties were transferred to DED2 and removed from DED1. Furthermore, we show that the isolated DED1 R4 peptide binds to calmodulin and solve the structure of the peptide-protein complex using NMR and computational refinement. Finally, we demonstrate an interaction between cFLIP_L and calmodulin in cancer cell lysates. In summary, our data implicate calmodulin as a potential player in DISC-mediated apoptosis and provide evidence for a specific interaction with the DED1 of cFLIP_L.     

Site-Directed Mutagenesis Evidence for Arginine-384 Residue at the Active Site of Maize Branching Enzyme II

Essential arginine residues are suggested to be located at the active sites of maize branching enzymes (BE) based on the evidence that two arginine residues are conserved in all BE from various species and that as little as one arginine residue is located at the active site of maize BE by phenylglyoxal (PGO) modification. To determine the exact location of the active-site arginine residue in BE, we employed peptide mapping and site-directed mutagenesis approaches. A single trypsin-digested, [¹⁴C]PGO-labeled peptide was purified from maize BEII by two rounds of HPLC separation, but we failed to obtain amino acid sequencing information. Site-directed mutagenesis was then used to create one mutant (arginine-384 to alanine-384), R384A. Immunoblotting result showed that BEII protein was expressed at a similar level in the wild type and the R384A mutant. However, BE activity in the R384A mutant was only 1.4% of the wild type. These results support the conclusion that the conserved arginine-384 residue is important in BEII catalysis.     

Identification and Characterization of a Novel Human Phosphatidylinositol 4-kinase

The extensive sequence homology that exists among the catalyticdomains of phosphatidylinositol 3- and 4-kinases allowed usto clone a novel human gene encoding a putative phosphatidylinositolkinase, NPIK. Among other known phosphatidylinositol 3- and4-kinases, NPIK was most closely related to yeast PIK1 phosphatidylinositol4-kinase. Several forms of NPIK cDNAs were isolated, and expressionof NPIK message was detected in a wide variety of tissues. Fluorescencein situ hybridization and radiation hybrid analyses assignedthe NPIK gene to human chromosome 1. Recombinant NPIK proteincatalyzed a conversion from phosphatidylinositol to phosphatidylinositol4-phosphate. The catalytic activity of NPIK was augmented byTriton X-100, and was reduced in the presence of adenosine.Using green .uorescent protein system we determined that NPIKis localized in the cytoplasm. Taken together, the data suggestthat NPIK may play a pivotal role in regulating the synthesisof phosphatidylinositol 4-phosphate at the site(s) accessiblefrom cytoplasm.