Distribution of the CCR5delta32 allele (gene variant CCR5) in Rondônia, Western Amazonian region, Brazil

Since around 1723, on the occasion of its initial colonization by Europeans, Rondonia has received successive waves of immigrants. This has been further swelled by individuals from northeastern Brazil, who began entering at the beginning of the twentieth century. The ethnic composition varies across the state according to the various sites of settlement of each wave of immigrants. We analyzed the frequency of the CCR5Δ32 allele of the CCR5 chemokine receptor, which is considered a Caucasian marker, in five sample sets from the population. Four were collected in Porto Velho, the state capital and the site of several waves of migration. Of these, two, from the Hospital de Base were comprised of HB Mothers and HB Newborns presenting allele frequencies of 3.5% and 3.1%, respectively, a third from the peri-urban neighborhoods of Candelária/Bate-Estaca (1.8%), whereas a fourth, from the Research Center on Tropical Medicine/CEPEM (0.6%), was composed of malaria patients under treament. The fifth sample (3.4%) came from the inland Quilombola village of Pedras Negras. Two homozygous individuals (CCR5Δ32/CCR5Δ32) were detected among the HB Mother samples. The frequency of this allele was heterogeneous and higher where the European inflow was more pronounced. The presence of the allele in Pedras Negras revealed European miscegenation in a community largely comprising Quilombolas.     

血清miR-21 及AFP 在大鼠肝癌发生过程中的动态表达比较分析

目的:分析血清miR-21在肝癌发生过程中的表达水平并将其与传统肝癌血清标志物甲胎蛋白(AFP)比较,探索其成为肝癌早期诊断血清标志物的可能性。方法:二乙基亚硝胺(DENA)腹腔注射诱导建立大鼠肝癌模型,建模过程中收集造癌各个阶段的血清。Realtime-PCR检测血清miR-21的表达情况,ELISA法检测血清AFP水平。结果:与正常组及纤维化期大鼠相比,miR-21在肝硬化期、肝癌早期、肝癌晚期的大鼠血清均有不同程度上调(P<0.05),AFP在肝癌早、晚期的大鼠血清明显上调(P<0.05);与肝硬化期大鼠比较,肝癌早、晚期大鼠体内的miR-21表达显著上调(P<0.05),AFP在肝癌早期、肝癌晚期均显著上调(P<0.05)。结论:血清miR-21参与了肝癌发生的过程,对于肝癌发生的各个阶段均有很大的指示作用,可能作为肝癌预防和早期诊断的一个潜在标志物。     

Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY

Bacteriophage T4 UvsY is a recombination mediator protein that promotes assembly of the UvsX-ssDNA presynaptic filament. UvsY helps UvsX to displace T4 gene 32 protein (gp32) from ssDNA, a reaction necessary for proper formation of the presynaptic filament. Here we use DNA stretching to examine UvsY interactions with single DNA molecules in the presence and absence of gp32 and a gp32 C-terminal truncation (*I), and show that in both cases UvsY is able to destabilize gp32-ssDNA interactions. In these experiments UvsY binds more strongly to dsDNA than ssDNA due to its inability to wrap ssDNA at high forces. To support this hypothesis, we show that ssDNA created by exposure of stretched DNA to glyoxal is strongly wrapped by UvsY, but wrapping occurs only at low forces. Our results demonstrate that UvsY interacts strongly with stretched DNA in the absence of other proteins. In the presence of gp32 and *I, UvsY is capable of strongly destabilizing gp32-DNA complexes in order to facilitate ssDNA wrapping, which in turn prepares the ssDNA for presynaptic filament assembly in the presence of UvsX. Thus, UvsY mediates UvsX binding to ssDNA by converting rigid gp32-DNA filaments into a structure that can be strongly bound by UvsX.     

Enhanced CREB and DARPP-32 phosphorylation in the nucleus accumbens and CREB, ERK, and GluR1 phosphorylation in the dorsal hippocampus is associated with cocaine-conditioned place preference behavior

Environment-induced relapse is a major concern in drug addiction because of the strong associations formed between drug reward and environment. Cocaine-conditioned place preference is an ideal experimental tool to examine adaptations in the molecular pathways that are activated upon re-exposure to an environment previously paired with drug reward. To better understand the mechanism of cocaine-conditioned place preference we have used western blot analysis to examine changes in phosphorylation of cAMP-response element binding protein (CREB), dopamine- and cyclic AMP-regulated phosphoprotein 32 (DARPP-32), extracellular signal-regulated kinase (ERK) and GluR1, key molecular substrates altered by cocaine, in the nucleus accumbens (NAc) and dorsal hippocampus (DHC) of C57BL/6 mice. Our studies revealed that re-exposing mice to an environment in which they were previously given cocaine resulted in increased levels of Ser133 phospho-CREB and Thr34 phospho-DARPP-32 with a corresponding decrease in Thr75 phospho-DARPP-32 in the NAc. In DHC there were increased levels of phospho-CREB, Thr183/Tyr185 phospho-ERK, and Ser845 phospho-GluR1. These data suggest that the formation of contextual drug reward associations involves recruitment of the DHC-NAc circuit with activation of the DARPP-32/CREB pathway in the NAc and the ERK/CREB pathway in the DHC.     

耐甲氧西林葡萄球菌3种检测方法的实验比较及临床应用

目的 通过对200株临床分离的葡萄球菌耐苯唑西林的检测,比较3种表型检测法的阳性率并对其临床实用性进行评价。方法 采用美国NCCLS 2004年制订的头孢西丁-纸片扩散法、VITEK32微生物鉴定仪检测及苯唑西林盐琼脂法测试mecA介导的葡萄球菌耐药。结果 200株被检测的葡萄球菌中,头孢西丁-纸片扩散法检出耐苯唑西林的阳性率为68％（136／200）;苯唑西林盐琼脂法的阳性率为67．5％（135／200）;VITEK32仪的阳性率为68．5％（137／200）;且耐甲氧西林凝固酶阴性葡萄球菌（MRCNS）的耐药率（86．2％）高于耐甲氧西林金黄色葡萄球菌（26．23％）。3种检测方法结果经χ^2检测差异无显著性。结论 3种方法操作都简便,但各有优缺点,VITEK32仪器检测葡萄球菌耐笨唑西林费用高,一般小型医院不具备条件;盐琼脂筛选法判读直观,但要掌握好孵育时间;NCCLS2004版的纸片扩散法较经济实用,不仅可检测被检菌是否耐笨唑西林,同时还可获得大环内酯-林可霉素-链阳霉素（MLSB）结果,但是当头孢西丁的判断折点在临界时应当复检,建议与盐琼脂筛选法同步检测。     

菌丝桥在日本落叶松幼苗间磷传递和植株生长中的作用

 应用四室隔网系统研究了菌丝桥在日本落叶松(Larix kaempferi)幼苗间传递磷的作用。结果表明,供体接种卷缘桩菇(Paxillus involutus)和彩色豆马勃（Pisolithus tinctorius）后,其外延菌丝可以穿过隔离层侵染受体落叶松,在供体和受体落叶松间形成了菌丝桥。供体植株接种菌根真菌后生物量明显增加,但是对受体植株没有显著的影响。菌根真菌侵染的供体和受体植株的根、地上部吸磷量均分别显著高于对照,而且供体植株根、地上部吸磷量增加的程度明显高于受体。被卷缘桩菇和彩色豆马勃侵染的受体植株体内32P的放射性强度分别是对照的10倍和6倍,两者形成菌丝桥后传递到受体植株的32P分别为供体植株体内32P的1.10%和0.22%。供体植株吸收的32P可以通过菌丝桥传递给受体,但是绝对数量十分有限,对受体植株磷营养没有产生显著的影响,但P. involutus和P. tinctorius侵染受体植株后,促进了受体落叶松对磷的吸收,这是菌丝桥形成后,真菌帮助受体植株吸收磷引起的。     

A cell-based high-throughput screen for epidermal growth factor receptor pathway inhibitors

Epidermal growth factor receptor (EGFR) is a valid drug target for development of target-based therapeutics against non-small-cell lung cancer. In this study, we established a high-throughput cell-based assay to screen for compounds that may inhibit EGFR activation and/or EGFR-mediated downstream signaling pathway. This drug screening platform is based on the characterization of an EGFR-transfected 32D cell line (32D-EGFR). The expression of EGFR in 32D cells allowed cell proliferation in the presence of either epidermal growth factor (EGF) or interleukin 3 (IL-3) and provided a system for both screening and counterscreening of EGFR pathway-inhibitory compounds. After the completion of primary and secondary screenings in which 32D-EGFR cells were grown under the stimulation of either EGF or IL-3, 9 of 20,000 compounds were found to selectively inhibit the EGF-dependent proliferation, but not the IL-3-dependent proliferation, of 32D-EGFR cells. Subsequent analysis showed that 3 compounds of the 9 initial hits directly inhibited the kinase activity of recombinant EGFR in vitro and the phosphorylation of EGFR in H1299 cells transfected with EGFR. Thus, this 32D-EGFR assay system provides a promising approach for identifying novel EGFR and EGFR signaling pathway inhibitors with potential antitumor activity.     

A proposed role for Leishmania major carboxypeptidase in peptide catabolism

Leishmaniasis is a tropical disease caused by Leishmania, eukaryotic parasites transmitted to humans by sand flies. Towards the development of new chemotherapeutic targets for this disease, biochemical and in vivo expression studies were performed on one of two M32 carboxypeptidases present within the Leishmania major (LmaCP1) genome. Enzymatic studies reveal that like previously studied M32 carboxypeptidases, LmaCP1 cleaves substrates with a variety of C-terminal amino acids—the primary exception being those having C-terminal acidic residues. Cleavage assays with a series of FRET-based peptides suggest that LmaCP1 exhibits a substrate length restriction, preferring peptides shorter than 9-12 amino acids. The in vivo expression of LmaCP1 was analyzed for each major stage of the L. major life cycle. These studies reveal that LmaCP1 expression occurs only in procyclic promastigotes—the stage of life where the organism resides in the abdominal midgut of the insect. The implications of these results are discussed.     

人类新基因C17orf32的电子克隆和编码区序列RT-PCR验证

利用生物信息学与实验验证的技术路线,成功地克隆了人类新基因C17orf32的cDNA(GenBank登记号：AY074907和TPA: BK000260),发现C17orf32的完整开放阅读框架(ORF,31～657 bp）cDNA（627 bp）与人类假定基因LOC124919 ORF(25～807 bp)的25～651位只有一个碱基不同.经RT-PCR验证并cDNA测序、人类表达序列标签(EST)数据库的BLAST检索和基因组成规律分析三方面的结果,均支持C17orf32的序列,而不支持LOC124919的编码序列.C17orf32基因组序列全长4.610 kb,含有6个外显子和5个内含子,cDNA序列全长1 679 bp, ORF横跨全部6个外显子.该基因ORF翻译起始处符合Kozak规则,ORF起始码上游同一相位有终止码,ORF后有2个加尾信号和PolyA尾.C17orf32基因的成功克隆表明,NCBI GENOME Annotation Project在2001年12月预测的人类假定蛋白XP-058865编码基因LOC124919的模式参考序列XM-058865中存在偏差,即在C17orf32基因cDNA的406与407位碱基之间错误插入一个碱基G, 从而导致在插入位点后,ORF编码125位氨基酸以后蛋白质序列的改变,出现260个氨基酸的多肽.因此,应慎重看待计算机注释的人类基因组编码序列.建立的技术路线有助于发现更多新的人类功能基因.