Molecular cloning and sequence analysis of alboaggregin B

Green pit viper (Trimeresurus albolabris) venom contains a variety of C-type lectin-like proteins (CLPs) causing platelet aggregation and consumptive thrombocytopenia in biting victims. Alboaggregin B (AL-B), a heterodimeric glycoprotein (Gp) Ib-binding protein, was purified from the venom, but there is no reported cDNA sequence and the platelet agglutination mechanism is poorly understood. The full-length AL-B beta clone was obtained from T. albolabris venom gland cDNA library. AL-B alpha was, later, derived using 3'-RACE based on the conserved sequence. In this study, purified AL-B dimer agglutinated human platelets with the EC(50) of 180 nM and was completely inhibited by anti GpIb antibody. MALDI ToF mass spectroscopy found no glycosylation. The peptide mass fingerprints were matched with deduced amino acid sequences of cloned genes. AL-B alpha and beta contained 156 and 146 amino acid, respectively, including 23-residue signal peptides. AL-B beta showed the conserved hydrophilic patches, putative sites for GpIb binding. Furthermore, there was another conserved motif (SRTY) exclusively in platelet-agglutinating AL-B, TSV-GPIb-BP and Mamushigin. We propose that these three CLPs may function as bivalent adhesive proteins linking two GpIb molecules on adjacent platelets.     

Change of platelet activation markers using flow cytometry in patients with hematology/oncology disorders after transfusion

In spite of the frequent need of platelet transfusions, there is limited information on the association of platelet activation markers, in transfused patients with hematology/oncology disorders, with platelet function using flow cytometry. The goal of this study was to evaluate the changes of PAC-1 binding and CD62P expression, with or without agonists in patients after transfusions. Twenty-eight whole blood samples were obtained from 24 patients admitted to the department of Hematology & Oncology and transfused with platelets; these samples were compared to 30 healthy controls. Whole blood samples, either with or without agonists, such as 20 µM adenosine diphosphate (ADP) or 100 µM thrombin receptor activating peptide (TRAP), were stained with the fluorescein conjugated monoclonal antibodies PAC-1 or CD62P. Then, the percent expression for each marker was analysed using flow cytometry. ADP and TRAP induced an increased percentage of CD62P expression and PAC-1 binding after platelet transfusions compared to the samples studied before transfusion, and these findings were lower than those of the healthy controls. However, the expression of platelets without the agonists was not significantly changed, despite the transfusions. Therefore, agonist-induced platelet activation markers, studied by flow cytometry, appear to be more useful for the evaluation of platelet function after transfusions than platelet activation markers without agonists.     

下肢深静脉血栓形成患者血小板活化状态的变化及意义

为探讨下肢深静脉血栓形成(DVT)思考血小板活化状态的变化及其临床意义，用流式细跑术(PCM)，以单克隆抗体为探针，对35例下肢DVT患者(DVT组)及3l例健康人(对照组)血小板活化标记物溶酶体颗粒糖蛋白(CD63)、血小板表面选择素(CD62p)及凝血酶敏感蛋白(TSD)进行了检测。结果显示，DVT组3种血小板活化标志物阳性表达率均高于对照组(P均＜0．001)，溶栓治疗后3种血小板活化标志物的阳性表达率均呈降低趋势。提示DVT患者体内血小板活化亢进；FCM可作为活化血小板的良好检测手段。     

Current status and future prospects for platelet function testing in the diagnosis of inherited bleeding disorders

Platelets play a crucial role in haemostasis by preventing bleeding at the site of vascular injury. Several defects in platelet morphology and function have been identified and described over the years. Although a range of methodologies is available to assess platelet function, a significant proportion of subjects with bleeding symptoms and normal coagulation parameters still appear to have normal results on platelet function testing. This might suggest that the reason for bleeding is multifactorial and is due to a combination of several minor defects in platelet function and/or other parts of the haemostatic system or might indicate that the currently available platelet function tests do not provide optimal diagnostic power. This review will summarize the established platelet function tests used for diagnosing inherited platelet abnormalities in adults and children, and discuss the newly developed methodologies as well as unmet challenges and potential areas for further improvement in this field.     

Molecular cloning and sequence analysis of putative chicken prolactin cDNA

A cDNA library was prepared from mRNA isolated from anterior pituitary glands of incubating bantam hens, in which prolactin mRNA levels were predicted to be very high. Nine clones, representing abundant mRNA species, were identified and shown to contain homologous sequences. Two clones, of 871 bp and 580 bp, were analysed by DNA sequencing. The shorter clone was found to be a truncated cDNA product but otherwise identical to the longer clone. The 871 bp cDNA, PRL101, contains an open reading frame capable of encoding a polypeptide of 229 amino acids. This putative polypeptide has a high degree of homology to mammalian prolactins (approximately 70%), strongly suggesting that PRL101 encodes chicken preprolactin. The protein was predicted to have a 30 amino acid signal sequence which would be cleaved off to give a mature protein of 199 amino acids. The peptide sequence also had a 26% homology to chicken growth hormone, which is related to prolactin. This similarity confirms the conclusion that PRL101 is a chicken prolactin cDNA clone. An abundant mRNA of approximately 880 b was detected in poly(A)+ RNA from pituitary glands probed with PRL101. Analysis of chicken genomic DNA showed that there is one copy of the prolactin gene in the genome. PRL101 hybridized strongly to genomic DNA from closely related galliforms (quail and turkey) and less strongly to DNA from more distantly related species (duck and ring dove).     

Molecular cloning and sequence analysis of chum salmon gonadotropin cDNAs.

cDNAs encoding alpha and beta subunits of salmon gonadotropins, sGTHI and sGTHII, have been isolated from the cDNA library prepared from salmon pituitary mRNA. sGTHI alpha, sGTHI beta, and sGTHII beta cDNAs encode polypeptides of 114, 137, and 142 amino acids, including signal peptides of 22, 24, and 23 amino acids, respectively. The deduced amino acid sequence for sGTHI alpha revealed rather high homology (66-69%) to mammalian alpha chains, whereas sGTHI beta and sGTHII beta show lower homology (30%) to each other and to mammalian beta subunits. The existence of two distinct beta-subunit cDNAs in the teleost suggests that divergence of the GTH gene took place earlier than divergence of teleosts from the main line of evolution leading to tetrapods.     

Molecular cloning and cDNA sequence analysis of coho salmon stanniocalcin.

Stanniocalcin (STC) (formerly known as both teleocalcin and hypocalcin) is an anti-hypercalcemic, glycoprotein hormone that is produced by the corpuscles of Stannius (CS), endocrine glands that are confined to bony fishes. The hormone has a unique amino acid sequence and exists as a disulfide-linked homodimer in the native state. In previous studies, we have described the purification and characterization of two salmon STCs, and examined the regulation of hormone secretion in response to calcium using both in vitro and in vivo model systems. This report describes the molecular cloning and cDNA sequence analysis of a coho salmon STC messenger RNA (mRNA) from a salmon CS lambda gt10 cDNA library. The STC mRNA in salmon is approximately 2 kilobases in length and encodes a primary translation product of 256 amino acids. The first 33 residues comprise the prepro region of the hormone, whereas the remaining 223 residues make up the mature form of the hormone. One N-linked, glycosylation consensus sequence was identified in the protein coding region as well as an odd number of half cysteine residues, the latter of which would allow for interchain bonding or dimerization of monomeric subunits. In addition, three sites were identified in the mature protein core of STC (two dibasic, one tribasic) that may be acted upon by endopeptidases to produce truncated forms of the hormone. In support of this latter possibility, Western blot analysis revealed multiple molecular weight forms of sTC within salmon glands.     

原发性高血压患者血小板活化状态研究

目的 :探讨血小板活化与原发性高血压 (EH)的关系。方法 :观察 36例EH患者 (EH组 )和 2 5例正常人 (对照组 )的血小板α颗粒膜蛋白 14 0 (GMP 14 0 )在血小板膜表面的分子数和血浆中的含量 ,并同时测定了血浆血栓烷B2 (TxB2 )。结果 :EH组患者血小板膜表面GMP 14 0分子数和血浆中GMP 14 0浓度、血浆TxB2 浓度均显著高于对照组。未发现GMP 14 0与TxB2 在EH患者异常变化中的线性相关性。结论 :GMP 14 0与TxB2 反映了血小板活化在EH中的两种不同的重要作用     

Molecular cloning and nucleotide sequence analysis of complementary DNA for bullfrog prolactin

A prolactin cDNA was cloned from a cDNA expression library constructed from total RNA of bullfrog (Rana catesbeiana) adenohypophyses by immunoscreening with antiserum against bullfrog prolactin. The cDNA clone thus obtained contained a 249 bp insert. Using this clone as a probe, plaque hybridizations were performed and two additional clones obtained. These clones had a polyadenylation site different from that of the first obtained clone, suggesting that the 3'-untranslated sequence was heterogeneous in length. The longest clone contained 830 bp, which encoded part of the signal peptide and the entire sequence of mature prolactin. The deduced amino acid sequence was in good accord with that determined by direct protein sequencing of purified bullfrog prolactin. The length of the bullfrog prolactin mRNA was estimated by Northern blot analysis to be about 1.0 kb. Homologies of prolactin nucleotide and amino acid sequences between bullfrog and other vertebrates were 64 and 65% for man, 66 and 68% for pig, 61 and 52% for rat, 69 and 74% for chicken, and 50 and 35% for salmon respectively. Highly conserved regions reported for mammalian prolactins also existed in bullfrog prolactin. Homologies of nucleotide and amino acid sequences between prolactin and GH of bullfrog origin were 49 and 25% respectively. Using the cDNA, the content of prolactin mRNA in the pituitary glands of metamorphosing tadpoles was measured. Prolactin mRNA levels rose at the mid-climax stage, suggesting that the increase in plasma and pituitary prolactin levels known to occur at the climax stage accompanies the increase in prolactin synthesis.     

Molecular cloning and sequence analysis of a chondroitin sulfate proteoglycan cDNA.

We report the identification and DNA sequence of a chondroitin sulfate proteoglycan core protein cDNA. A cDNA clone, pPG1, was selected from a rat yolk sac tumor poly(A)+RNA-derived cDNA library by using synthetic oligonucleotides predicted from the NH2-terminal peptide sequence of the mature chondroitin sulfate proteoglycan. The resulting sequence analysis demonstrated that the 874-base-pair pPG1 clone contained the complete coding region of the mature proteoglycan core protein as well as 5' and 3' flanking sequences. The 104 amino acid proteoglycan core protein sequence reveals that the core protein is composed of three regions, the most striking of which is the central 49 amino acid region composed of alternating serine and glycine residues. This region clearly functions as the acceptor site for the attachment of chondroitin sulfate side chains. The serine-glycine repeat region is flanked by a 14 amino acid NH2-terminal region identical to the NH2-terminal sequence of the proteoglycan obtained by amino acid sequencing and a 41 amino acid COOH-terminal region. RNA transfer blot hybridizations of poly(A)+ mRNA from rat yolk sac tumor cells with nick-translated pPG1 reveal a single mRNA of approximately equal to 1300 nucleotides. The possibility of detecting mRNAs and genomic sequences for other proteoglycans with a serine-glycine repeat by using this cDNA clone is discussed.     

Molecular cloning, sequence analysis and expression of the snake follicle-stimulating hormone receptor

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) control gonadal function in mammalian and many non-mammalian vertebrates through the interaction with their receptors, FSHR and LHR. Although the same is true for some reptilian species, in Squamata (lizards and snakes) there is no definitive evidence for the presence of either two distinct gonadotropins or two distinct gonadotropin receptors. Our aim was to characterize the gonadotropin receptor(s) of the Bothrops jararaca snake. Using a cDNA library from snake testis and amplification of the 5'-cDNA ending, we cloned a cDNA related to FSHR. Attempts to clone a cDNA more closely related to LHR were unsuccessful. Expression of FSHR mRNA was restricted to gonadal tissues. The snake FSHR is a G protein-coupled receptor with 673 amino acids, and the aminoterminal domain with 346 amino acids consists of a nine leucine-rich repeat-containing subdomain (LRR) flanked by two cysteine-rich subdomains. The beta-strands in the LRR are conserved with exception of the third, a region that may be important for FSH binding. In contrast with mammalian, avian and amphibian FSHRs, the snake FSHR presents amino acid deletions in the carboxyterminal region of the extracellular domain which are also seen in fish and lizard FSHRs. cAMP assays with the recombinant protein transiently expressed in HEK-293 cells showed that the snake FSHR is more sensitive to human FSH (hFSH) than to human chorionic gonadotropin. Phylogenetic analysis indicated that the squamate FSHRs group separately from mammalian FSHRs. Our data are consistent with the apparently unique gonadotropin-receptor system in Squamata reptilian subgroup. Knowledge about the snake FSHR structure may help identify structural determinants for receptor function.     

细粒棘球绦虫DNA聚合酶δ小亚基EgPolD2基因的克隆及序列分析

目的从新疆株细粒棘球绦虫原头蚴中克隆DNA聚合酶δ小亚基EgPolD2,进行序列测定和生物信息学分析。方法设计EgPolD2特异性引物,用RT-PCR方法从细粒棘球绦虫原头蚴cDNA中克隆EgPolD2基因,然后将其克隆至pMD18-T载体,测序并进行生物信息学分析,半定量反转录PCR分析其在细粒棘球绦虫原头蚴和囊泡中的表达情况。结果 EgPolD2基因开放阅读框为1 218bp,编码405个氨基酸,等电点为4.74。NCBI保守功能区分析软件预测EgPolD2蛋白第113～378个氨基酸为MPP_PolD2_C结构域。登陆GenBank进行Blast比对,EgPolD2与曼氏血吸虫SmPolD2基因同源性为32.1%,与线虫和人等其他物种PolD2基因同源性在18.3%～22.8%之间。进化树分析表明EgPolD2与SmPolD2相聚集,与其他物种同源性较低。半定量RT-PCR显示,EgPolD2在细粒棘球绦虫原头蚴和囊泡中均有表达,且表达水平差异无统计学意义(P>0.05)。结论本实验成功克隆出细粒棘球绦虫EgPolD2新基因,为进一步研究抗细粒棘球绦虫药物靶标奠定了基础。     

Are platelets activated after a rapid,one‐step density gradient centrifugation? Evidence from flow cytometric analysis

This procedure describes the preparation of platelets from whole blood of healthy donors and pregnancy‐induced hypertensive (PIH) patients by a rapid, one‐step density gradient centrifugation, and the direct immunofluorescence staining of obtained platelets (CD63). Platelets are relatively fragile structures. Consequently, for the investigation of their biochemical properties it is recommended to isolate them by a simple method that does not damage their functional parameters and induce their activation. During platelet activation, several changes occur at the platelet surface. CD63 is the receptor for a lysosomal glycoprotein expressed in activated platelets. Currently, flow cytometry (fluorescence‐activated cell sorting) is the most sensitive method to detect increased surface exposure of activation antigens on the platelet surface. The present technical note describes that compared with other whole blood flow cytometric techniques, our one‐step density‐gradient centrifugation method using OptiPrep^TM can also prevent artificial, sample manipulation‐related platelet activation.     

Development and Evaluation of a Molecular Hepatitis A Virus Assay for Serum and Stool Specimens

Hepatitis A virus (HAV) is an emerging public health concern and there is an urgent need for ways to rapidly identify cases so that outbreaks can be managed effectively. Conventional testing for HAV relies on anti-HAV IgM seropositivity. However, studies estimate that 10–30% of patients may not be diagnosed by serology. Molecular assays that can directly detect viral nucleic acids have the potential to improve diagnosis, which is key to prevent the spread of infections. In this study, we developed a real-time PCR (RT-PCR) assay to detect HAV RNA for the identification of acute HAV infection. Primers were designed to target the conserved 5′-untranslated region (5′-UTR) of HAV, and the assay was optimized on both the Qiagen Rotor-Gene and the BD MAX. We successfully detected HAV from patient serum and stool samples with moderate differences in sensitivity and specificity depending on the platform used. Our results highlight the clinical utility of using a molecular assay to detect HAV from various specimen types that can be implemented in hospitals to assist with diagnostics, treatment and prevention.     

Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene.

Genomic DNA clones that coded for the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) (DHFR-TS) activities from a pyrimethamine-sensitive strain of Plasmodium falciparum were isolated and sequenced. The deduced DHFR-TS protein contained 608 amino acids (71,682 Da). The coding region for DHFR-TS contained no intervening sequences and had a high A + T content (75%). The DHFR domain, in the amino-terminal portion of the protein, was joined by a 94-amino acid junction sequence to the TS domain in the carboxyl-terminal portion of the protein. The TS domain was more conserved than the DHFR domain and both P. falciparum domains were more homologous to eukaryotic than to prokaryotic forms of the enzymes. Predicted secondary structures of the DHFR and TS domains were nearly identical to the structures identified in other DHFR and TS enzymes.     

Molecular cloning, sequence analysis and expression of putative chicken insulin-like growth factor-I cDNAs

A cDNA library was constructed from mRNA isolated from the liver of a 5-week-old female broiler chicken; at this age the level of insulin-like growth factor-I (IGF-I) mRNA was expected to be high. Three clones, of sizes 2.3, 0.86 and 0.2 kb, were isolated by using a homologous human (IGF-I) probe. The DNA sequence of these clones has been determined and the potential amino acid sequence deduced. The sequence of the mature chicken IGF-I peptide shows a high degree of homology with IGF-I from other species, providing evidence for the identity of these clones. Alternative splicing of the chicken IGF-I mRNA has been found in the region potentially encoding the leader peptide. This may give rise to two forms of prepeptide, differing in the length and nature of their leader peptide. The 0.86 kb cDNA has been used as a probe to Northern blots of chicken mRNA. A major band of approximately 0.65-0.85 kb was seen, plus several minor bands of larger molecular weights. Analysis of genomic Southern blots shows that there is one copy of the chicken IGF-I gene.     

Flow cytometric analysis of platelets from childrenwith the Wiskott-Aldrich syndrome reveals defects in platelet development, activation and structure

The pathophysiology of platelet dysfunction in the Wiskott-Aldrich immune deficiency syndrome (WAS) remains unclear. Using flow cytometry, we have characterized the functional properties of platelets from 10 children with WAS. Patients with WAS had thrombocytopenia, small platelets, increased platelet-associated IgG and reduced platelet-dense granule content. Levels of reticulated 'young' platelets were normal in the WAS patients. Although the mean numbers of platelet glycoprotein (GP) Ib, GPIIbIIIa and GPIV molecules per platelet appeared lower in WAS patients than in healthy controls, analysis of similar-sized platelets revealed the mean number of GPIb molecules per platelet to be comparable in patients and normal controls. Surface GPIIbIIIa and GPIV expression was, however, significantly lower on the WAS platelets than on normal platelets. Compared with normal platelets, WAS platelets showed a reduced ability to modulate GPIIbIIIa expression following thrombin stimulation. In addition, thrombin- and ADP-induced expression of CD62P and CD63 was defective in WAS platelets. Phallacidin staining of the WAS platelets revealed less F-actin content than in normal platelets. Together, these data suggest that the reduced platelet number and function in WAS reflects, at least in part, a defect in bone marrow production as well as an intrinsic platelet abnormality.     

1株汉坦病毒的分离及其S基因片段的克隆和序列分析

目的对浙江省肾综合征出血热(Hemorrhagic Fever with Renal Syndrome,HFRS)疫区-龙泉,新分离到的毒株的S基因片段进行克隆并进行序列测定及分析,以确定毒株的型别和基因变异程度,为进一步研究病毒进化和变异提供了有利条件。方法采用直接免疫荧光检测疫区鼠肺HV抗原。将HV抗原阳性的鼠肺标本接种Vero-E6细胞,分离病毒。参考GenBank发表的汉坦病毒核蛋白基因序列,设计合成一对引物,提取分离株细胞培养物的总RNA,应用逆转录聚合酶链反应(RT-PCR)方法扩增新毒株S片段基因,并克隆入载体,纯化后进行核苷酸序列测定及分析。结果成功分离到的新毒株S片段的全基因序列共1 700个核苷酸,只有一个开放读码框架,共编码429个氨基酸。新毒株与HTN型毒株比较,核苷酸同源率为85.7%~91.9%。与SEO型毒株比较,核苷酸同源率为71.2%~75%,表明该毒株为HTN型毒株。结论浙江HFRS疫区新分离汉坦病毒株可以定型为HTN型毒株,可能为新的亚型。     

Molecular cloning and nucleotide sequence of human glucocerebrosidase cDNA.

Mutations in the human glucocerebrosidase gene cause Gaucher disease. A cDNA clone containing the entire human glucocerebrosidase coding region from normal cells has been isolated using lambda gt11 expression libraries. The complete nucleotide sequence, a restriction map, and a hydropathy profile are presented. Hybridization to chromosome-specific DNA localizes the human glucocerebrosidase gene to chromosome 1. The likely precursor protein is 515 amino acids long. The NH2-terminal 19 amino acids constitute a leader sequence that is cleaved from the mature protein. The predicted molecular weight of the mature protein is 55,384, without glycosylation or carboxyl-terminal processing.