The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily, and it plays a key role in the process of protecting cells, facilitating the folding of nascent peptides and responding to stress. The cDNA of bay scallop Argopecten irradians HSP70 (designated AIHSP70) was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE). The full length of AIHSP70 cDNA was 2651 bp in length, having a 5' untranslated region (UTR) of 96 bp, a 3' UTR of 575 bp, and an open reading frame (ORF) of 1980 bp encoding a polypeptide of 659 amino acids with an estimated molecular mass of 71.80 kDa and an estimated isoelectric point of 5.26. BLAST analysis revealed that the AIHSP70 gene shared high identity with other known HSP70 genes. Three classical HSP signature motifs were detected in AIHSP70 by InterPro, analysis. 3-D structural prediction of AIHSP70 showed that its N terminal ATPase activity domain and,C terminal substrate-binding domain shared high similarity with that in human heat shock protein 70. The results indicated that the AIHSP70 was a member of the heat shock protein 70 family. A semi-quantitive RT-PCR method was used to analyse the expression of AIHSP70 gene after the treatment of naphthalin which is one kind of polycyclic aromatic hydrocarbon (PAH) and the challenge of bacteria. mRNA expression of AIHSP70 in scallop was up-regulated significantly after the stimulation of naphthalin and increased with increasing naphthalin concentration. A clearly time-dependent expression pattern of AIHSP70 was observed after the scallops were infected by Vibrio anguillarum, and the mRNA expression reached a maximum level at 8 h and lasted to 16 h, and then dropped progressively. The results indicated that AIHSP70 could play an important role in mediating the environmental stress and immune response in scallop. (c) 2006 Elsevier Ltd. All rights reserved.

皱纹盘鲍热休克蛋白70基因的克隆、表达及应用

热休克蛋白70是热休克蛋白家族中最重要的一类家族成员，它具有“分子伴侣”、细胞保护、抗凋亡以及肿瘤免疫治疗等独特复杂的生物学功能，它的研究已成为生命科学领域研究的热点之一。采用RACE方法从皱纹盘鲍中克隆到一种HSP70基因的全长cDNA序列，该序列与其它物种的HSP70基因高度同源。克隆得到的皱纹盘鲍HSP70基因的全长cDNA序列为2631bp，开放阅读框为1968bp，另外其5'-和3'-非翻译区长度分别是90和573bp，其中3'-非翻译区具有AATAAA mRNA加尾信号及PolyA寡聚腺苷酸。开放阅读框编码655个氨基酸，包含N端包含的382个氨基酸的ATP酶结合域（约45kDa）、由161个氨基酸编码的底物肽结合结构域（18kDa）及112个氨基酸编码的C端结构域（10kDa）。构建了含有皱纹盘鲍HSP70的表达载体，以期得到体外表达的重组热激蛋白70，为制备抗体，通过Western blot杂交或酶联免疫等方法检测HSPs蛋白表达情况作准备。 为了研究皱纹盘鲍HSP70的转录表达，采用半定量RT-PCR方法，对HSP70不同组织及热休克、鳗弧菌感染下的转录表达情况分析。试验结果表明，采用热应激及鳗弧菌处理皱纹盘鲍，均能导致皱纹盘鲍肌肉中HSP70 mRNA转录水平明显增加，并在处理后96 h恢复至正常水平；与肌肉组织不同的是，鳃组织在温度及鳗弧菌处理后12 h后达到最高值，并一直维持相对较高的表达水平。在温度和鳗弧菌处理下，皱纹盘鲍HSP70上调表达表明HSP70的响应没有胁迫特异性，可能参与了皱纹盘鲍的免疫反应。 以皱纹盘鲍近交和杂交群体为材料，研究了不同群体的生长速率和HSP70的表达量的差异。试验结果表明，在较低的处理温度下，皱纹盘鲍近交群体HSP70的表达量高于杂交群体，随着处理温度的升高，在接近致死温度时，杂交群体的HSP70表达量要高于近交群体；不同群体达到最大HSP70表达量时的温度（Tpeak）也不同，近交群体的Tpeak为26C，低于杂交群体（28C）；近交群体的最大HSP70表达量高于杂交群体。以上试验结果表明杂交群体的HSP70的表达变化幅度较近交群体的变化幅度小，具有更宽的温度适应性。可能提供了关于杂交能够缓解皱纹盘鲍内在遗传压力的证据。 用荧光定量PCR方法分析不同壳色皱纹盘鲍在热激胁迫下不同恢复时间和南方与北方越冬的皱纹盘鲍在不同程度温度胁迫下的HSP70变化。在南方与北方越冬的皱纹盘鲍由于其越冬环境的温度明显不同，表现出明显的热响应差异。具体表现如下几个方面：首先，两个群体在非胁迫的正常条件下，其HSP70的初始表达量明显不同。南方越冬群体在其正常生长温度下（16C）的表达量高于北方群体的相应的表达量（12C）；其次，随着热激温度的增高，两个群体HSP70的表达量升高的幅度也不同。第三，两个群体HSP70表达量达到最高值后，随着温度的升高，表达量开始下降，但两个群体的表达量下降幅度也有所不同。结果表明在南方的适用后，体内可能聚集了较多的热休克蛋白，可能更有利于渡过北方的高温季节。

四种海藻热休克蛋白70（HSP70）基因的克隆与表达分析

热休克蛋白70是热休克蛋白家族中重要的成员，参与新生蛋白的折叠、转运、重折叠变性蛋白、协助降解变性蛋白和抗逆环境胁迫等功能。海带和裙带菜是浅海潮下带典型的褐藻，孔石莼和浒苔是潮间带典型的绿藻，四种大型海藻均有重要的经济价值和生态价值。随着潮汐变化固着藻类生境理化因素变化剧烈，藻类面临着严重的环境胁迫，因此研究藻类抗逆机理有着重要的意义。 本研究采用同源克隆法配合RACE-PCR，克隆了海带、孔石莼、裙带菜和浒苔HSP70基因的全序列（分别命名为LJHSP70、UPHSP70、QDHSP70和EPHSP70）。利用生物信息学方法分析了四种藻类HSP70结构特征、同源性关系和进化地位。获得的海带HSP70基因全序列长为2918 bp，5’非翻译区为248 bp，3’非翻译区为696 bp，开放阅读框为1974 bp，编码657个氨基酸，预测的分子量为72.03 kDa，等电点为4.97。获得的裙带菜HSP70基因全序列长为3243 bp，5’非翻译区为248 bp，3’非翻译区为1021 bp，开放阅读框为1974 bp，编码657个氨基酸，预测的分子量为72.03 kDa，等电点为4.96。获得的孔石莼HSP70基因全序列长为2283 bp，5’非翻译区为65 bp，3’非翻译区为247 bp，开放阅读框为1971 bp，编码656个氨基酸，预测的分子量为71.13 kDa，等电点为5.04。获得的浒苔HSP70基因全序列长为2265 bp，5’非翻译区为65 bp，3’非翻译区为217 bp，开放阅读框为1983 bp，编码660个氨基酸，预测的分子量为71.39 kDa，等电点为5.03。四种海藻HSP70氨基酸序列均含有四肽重复序列GGMP，具有三个典型的HSP70签名基序。细胞质定位的HSP70 C-末端特征基序为EEID或EEVD，并且N-端氨基酸序列保守性高于C-端。海带和裙带菜HSP70蛋白同源性为98%，孔石莼和浒苔HSP70蛋白同源性为96%，四种海藻HSP70蛋白序列与陆地植物和其他藻类HSP70蛋白序列同源性为70-80%。 利用荧光定量RT-PCR技术对不同胁迫条件处理的海带和孔石莼HSP70 mRNA的表达水平进行定量分析。不同热激温度（5-40 ℃）处理组中，30 ℃处理组的海带HSP70 mRNA表达量最高是10 ℃处理组的海带HSP70 mRNA表达量的3倍，而35 ℃或40 ℃处理组的海带HSP70表达量却低于25 ℃或30 ℃处理组的海带HSP70 mRNA表达量。25 ℃不同热激时间（0-12 h）处理组中，海带HSP70 mRNA表达量呈先上升后下降趋势。热激1 h后海带HSP70 mRNA表达量迅速上升，热激7 h后mRNA表达量达到最大，是对照组表达量的4倍。不同盐度（0‰-45‰）胁迫处理组中，0‰或5‰盐度处理组的海带HSP70 mRNA表达量是30‰盐度处理组海带HSP70 mRNA表达量的3倍。35‰、40‰和45‰盐度处理组之间HSP70 mRNA表达量较低且无显著差异。 不同热激温度（5-40℃）处理组中，20 ℃或25 ℃处理的孔石莼HSP70 mRNA表达量较低，而5 ℃、35 ℃、或40 ℃处理组的孔石莼HSP70 mRNA的表达量是25 ℃处理组孔石莼HSP70 mRNA表达量的2倍以上。30 ℃不同热激时间（0-12 h）处理组中，孔石莼HSP70 mRNA表达量也呈先上升后下降趋势。热激5 h后孔石莼HSP70 mRNA表达量达到最大，是对照组的3.5倍。不同盐度（0‰-45‰）胁迫处理组中，0‰或5‰盐度处理组的孔石莼HSP70 mRNA表达量是30‰盐度处理组孔石莼HSP70表达量的3倍。30‰、40‰和45‰盐度处理组孔石莼HSP70 mRNA表达量较低，且无显著差异。不同紫外线照射时间（0-4.0 h）和不同干燥时间（0-4.0 h）处理组中，孔石莼HSP70 mRNA表达量都在3 h后达到最高值，之后表达量维持在较高水平。 为进一步研究藻类HSP70的生物学功能，将海带HSP70基因的开放阅读框区域克隆到表达载体pEASY-E2中，并转化到大肠杆菌BL21(DE3)pLysS。将阳性重组子培养于含有AMP（100 U/mL）的LB培养基，IPTG诱导表达，SDS-PAGE电泳鉴定。经5 h诱导，其表达量达到平台期，继续培养HSP70表达量并不显著增高。5 mM IPTG诱导海带HSP70蛋白表达量高于1 mM IPTG诱导蛋白表达量。

Bromophenols coupled with nucleoside bases and brominated tetrahydroisoquinolines from the red alga Rhodomela confervoides

Three new bromophenols C-N coupled with nucleoside base derivatives (1-3) and three new brominated 1,2,3,4-tetrahydroisoquinolines (5-7, together with a new brominated tyrosine derivative (4, have been isolated from polar fractions of an ethanolic extract of the red alga Rhodomela confervoides. By spectroscopic and chemical methods including HRMS and 2D NMR data, their structures were determined as 7-[3-bromo-2-(2,3-dibromo-4,5-dihydroxybenzyl)-4,5-dihydroxybenzyl]-3,7-dihydro-1H-purine-2,6-dione (1), 7-(2,3-dibromo-4,5-dihydroxybenzyl)-3,7-dihydro-1H-purine-2,6-dione (2, 9-[3-bromo-2-(2,3-dibromo-4,5-dihydroxybenzyl)-4,5-dihydroxybenzyl]adenine (3), (-)-8S-(3-bromo-5-hydroxy-4-methoxy)phenylalanine (4), (-)-3S-8-bromo-6-hydroxy-7-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (5), methyl (-)-3S-8-bromo-6-hydroxy-7-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (6), and methyl (-)-3S-6-bromo-8-hydroxy-7-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (7). Compounds 5-7 were semisynthesized by using 4 as the starting material.

Evaluation of HER2, p53, bcl-2, topoisomerase II-alpha, heat shock proteins 27 and 70 in primary breast cancer and metastatic ipsilateral axillary lymph nodes.

BACKGROUND: Breast cancer is a heterogeneous disease. Predictive biological markers (BM) of responsiveness to therapy need to be identified. Evaluation of BM is mainly done at the primary site. However, in the adjuvant therapy of breast cancer, the main goal is control of micrometastases. It is still unknown whether heterogeneity in the expression of BM between the primary site and its micrometastases exists. OBJECTIVE: To evaluate the expression of some BM with potential predictive value from the primary breast cancer site and metastatic ipsilateral axillary lymph nodes. PATIENTS AND METHODS: Focality (percentage of positive cells) and intensity staining scores were evaluated for each marker. Freshly cut sections (4 microm) from embedded blocks of breast cancer fixed in formalin or bouin were put onto superfrost slides (Menzel-Gläser). Protein expression was evaluated immunohistochemically (IHC) using monoclonal antibodies against: topo II-alpha (clone KiS1, 1 microg/ml, Roche) with a trypsine pre-treatment (P); HSP27 (clone G3.1, 1/60, Biogenex), HSP70 (clone BRM.22, 1/80, Biogenex) and HER2 (clone CB11, 1/40, Novocastra; without P); p53 (clone D07, 1/750, Dako) and bcl-2 (clone 124, 1/60, Dako) with citrate buffer as P. RESULTS: Overall, the percentage of discordant marker status in the primary tumour and its metastatic lymph nodes was 2% for HER2, 6% for p53, 15% for bcl-2, 19% for topoisomerase II-alpha, 24% for HSP27 and 30% for HSP70. For the subgroup of patients with positive BM in the primary tumour, the percentage of discordance was 6% for HER2, 7% for p53, 14% for bcl-2, 19% for HSP70, 21% for topoisomerase II-alpha and 36% for HSP27. For the subgroup of patients with positive BM in the lymph nodes, the percentage of discordance was 9% for bcl-2, 15% for HER2 and p53, 21% for topoisomerase II-alpha, 22% for HSP27 and 25% for HSP70. CONCLUSIONS: 1) No biological marker had 100% concordant results. 2) Although some discordant cases might be explained by the limitations of the IHC technique, future studies aiming to evaluate the predictive value of BM in the adjuvant therapy of breast cancer should take into account a possible difference in BM expression between the primary and the metastatic sites.

WHO guidelines for antimicrobial treatment in children admitted to hospital in an area of intense Plasmodium falciparum transmission: prospective study.

OBJECTIVES: To assess the performance of WHO's "Guidelines for care at the first-referral level in developing countries" in an area of intense malaria transmission and identify bacterial infections in children with and without malaria. DESIGN: Prospective study. SETTING: District hospital in Muheza, northeast Tanzania. PARTICIPANTS: Children aged 2 months to 13 years admitted to hospital for febrile illness. MAIN OUTCOME MEASURES: Sensitivity and specificity of WHO guidelines in diagnosing invasive bacterial disease; susceptibility of isolated organisms to recommended antimicrobials. RESULTS: Over one year, 3639 children were enrolled and 184 (5.1%) died; 2195 (60.3%) were blood slide positive for Plasmodium falciparum, 341 (9.4%) had invasive bacterial disease, and 142 (3.9%) were seropositive for HIV. The prevalence of invasive bacterial disease was lower in slide positive children (100/2195, 4.6%) than in slide negative children (241/1444, 16.7%). Non-typhi Salmonella was the most frequently isolated organism (52/100 (52%) of organisms in slide positive children and 108/241 (45%) in slide negative children). Mortality among children with invasive bacterial disease was significantly higher (58/341, 17%) than in children without invasive bacterial disease (126/3298, 3.8%) (P<0.001), and this was true regardless of the presence of P falciparum parasitaemia. The sensitivity and specificity of WHO criteria in identifying invasive bacterial disease in slide positive children were 60.0% (95% confidence interval 58.0% to 62.1%) and 53.5% (51.4% to 55.6%), compared with 70.5% (68.2% to 72.9%) and 48.1% (45.6% to 50.7%) in slide negative children. In children with WHO criteria for invasive bacterial disease, only 99/211(47%) of isolated organisms were susceptible to the first recommended antimicrobial agent. CONCLUSIONS: In an area exposed to high transmission of malaria, current WHO guidelines failed to identify almost a third of children with invasive bacterial disease, and more than half of the organisms isolated were not susceptible to currently recommended antimicrobials. Improved diagnosis and treatment of invasive bacterial disease are needed to reduce childhood mortality.

Distribution of important and word-cued autobiographical memories in 20-, 35-, and 70-year-old adults.

For word-cued autobiographical memories, older adults had an increase, or bump, from the ages 10 to 30. All age groups had fewer memories from childhood than from other years and a power-function retention for memories from the most recent 10 years. There were no consistent differences in reaction times and rating scale responses across decades. Concrete words cued older memories, but no property of the cues predicted which memories would come from the bump. The 5 most important memories given by 20- and 35-year-old participants were distributed similarly to their word-cued memories, but those given by 70-year-old participants came mostly from the single 20-to-30 decade. No theory fully accounts for the bump.

Densidad de siembra y fertilización nitrogenada en trigo : 1-cultivares Marcos Juárez INTA y Norkinpan 70

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