Adaptation of Cochlear Amplification to Low Endocochlear Potential

Endocochlear potential (EP) is essential for cochlear amplification by providing the voltage source needed to drive outer hair cell (OHC) transducer current, which leads to OHC electromechanical force. An early study using furosemide to reversibly reduce EP showed that distortion product otoacoustic emissions (DPOAEs) recovered before EP. This indicated that cochlear amplification may be able to adjust to a new, lower EP. To investigate the mechanism of this adjustment, the extracellular OHC voltage, which we term local cochlear microphonic (LCM), was measured simultaneously with DPOAE and EP while using intraperitoneal (IP) and intravenous injection of furosemide to reversibly reduce EP. With IP injection, the DPOAEs recovered fully, whereas the EP was reduced, but LCM showed a similar time course as EP. The DPOAEs failed to accurately report the variation of cochlear amplification. With intravenous injection, for which both reduction and recovery of EP are known to occur relatively quickly compared to IP, the cochlear amplification observed in LCM could attain nearly full or even full recovery with reduced EP. This showed the cochlea has an ability to adjust to diminished operating condition. Furthermore, the cochlear amplifier and EP recovered with different time courses: cochlear amplification just started to recover after the EP was nearly fully recovered and stabilized. Using a Boltzmann model and the second harmonic of the LCM to estimate the mechanoelectric transducer channel operating point, we found that the recovery of cochlear amplification occurred with recentering of the operating point.     

SR-BI的分子结构及其表达调控

小鼠B族Ⅰ型清道夫受体是目前已确认的唯一真正介导细胞与高密度脂蛋白作用的膜受体,主要在肝脏和固醇生成组织中表达,并受促激素、胆固醇、饮食以及药理等因素所调控。该受体介导高密度脂蛋白-胆固醇酯的选择性吸收,是调节胆固醇逆转运的唯一靶点,在高密度脂蛋白代谢和胆固醇运输中起重要作用。该基因缺陷对不同的组织具有不同的影响。它有可能作为一个新的治疗靶点来预防和治疗动脉粥样硬化性心脑血管疾病。对其分子结构、表达调控及相关研究作了详细介绍。     

Regulation by ABA of beta-Conglycinin Expression in Cultured Developing Soybean Cotyledons

The regulation of cotyledon-specific gene expression by exogenously applied abscisic acid (ABA) was studied in developing cultured cotyledons of soybean (Glycine max L. Merr. cv Provar). When immature cotyledons were cultured in modified Thompson's medium, the addition of ABA resulted in an increased concentration of the β-subunit of β-conglycinin, one of the major storage proteins of soybean seeds. The amount of the α′-and α-subunits of β-conglycinin was relatively unaffected by the ABA treatment. When fluridone, an inhibitor of carotenoid biosynthesis that has been shown to decrease ABA levels in plant tissues, was added to the medium the level of ABA and the β-subunit decreased in the cotyledons. Increasing the concentration of sucrose in the culture medium caused an increase in the concentration of ABA and β-subunit in the cotyledons. When in vitro translation products from RNA isolated from cotyledons cultured with ABA were immunoprecipitated with antiserum against β-conglycinin, there was an increased amount of pre-β-subunit polypetide compared to the translation products from RNA isolated from control cotyledons. The pre-β-subunit polypeptide was not detected in translation products from RNA isolated from fluridone-treated cotyledons. Nucleic acid hybridization reactions showed that the level of β-subunit mRNA was higher in ABA-treated cotyledons compared to the control, and was lower in the fluridone-treated cotyledons. We have shown that exogenous ABA is able to modulate the accumulation of the β-subunit of β-conglycinin in developing cultured soybean cotyledons.     

热休克后表达受抑基因的分子克隆及其表达特性

以人成骨肉瘤细胞株ＨＯＳ－８６０３为热休克模型,在利用ＤＤｍＲＮＡ方法筛选和克隆了一个热休克后表达受抑基因的ｃＤＮＡ片段的基础上,以该片段为探针,筛选ＨＯＳ－８６０３细胞的ｃＤＮＡ文库,获得该基因的全长ｃＤＮＡ克隆（ＨＳＳＧ－１）;ＤＮＡ序列测定表明该ｃＤＮＡ全长１４５６ｂｐ,编码２７６个氨基酸,经计算机辅助分析,该ｃＤＮＡ序列尚未被报道。经ＲＮＡ斑点杂交证实,该基因的表达于多种组织细胞之中,并且     

烟草渗调蛋白基因的分离克隆及序列分析

参照国外报道的渗调蛋白基因序列,自行设计并合成了一对寡核苷酸引物,通过ＰＣＲ技术从云南地方栽培的烤烟品种“红花大金元”基因组ＤＮＡ中直接扩增出了其全编码序列。ＰＣＲ产物纯化后直接克隆到ｐＧＥＭ－Ｔ载体系统中,转化大肠杆菌ＤＨ５α,在Ｘｇａｌ平板上筛选白色菌落,经ＳｐｈＩ和ＳａｃＩ双酶切,鉴定所得的重组质粒中含有７５３ｂｐ左右的ＰＣＲ扩增片段。采用双链双脱氧法定序分析表明所克隆的渗调蛋白基因编码区序列与国外迄今所报道的两个株系的序列完全一致,提示了该基因在进化上的保守性     

Adaptation of Pseudomonas fluorescens to Low Levels of Water Activity Produced by Different Solutes

A culture of Pseudomonas fluorescens which grew at a minimal a_w of 0.97 in a glucose-salts broth adjusted to that level with NaCl, KCl, or glucose, and at a_w 0.9725 with Na₂SO₄, was adapted to grow at lower a_w levels produced by these solutes. Adaptation to growth at a low a_w produced by means of one solute did not necessarily result in growth at that a_w when another solute was employed to lower the a_w. The culture adapted to grow at a_w 0.9650 produced by addition of glucose could grow equally well in broth adjusted to that a_w with NaCl or KCl, but not Na₂SO₄. NaCl and KCl permitted growth at the lowest a_w level (0.9625), and the culture adapted in KCl did equally well in NaCl.     

microRNA分子表达与调控研究的最新进展

microRNA(miRNA)是一类分子长度为19~24nt的微小RNA,通常在转录后水平调控靶基因的降解或抑制翻译。miRNA分子在进化上高度保守,已经发现越来越多的miRNA分子参与真核生物的生长发育、生理活性、细胞增殖、组织分化、细胞凋亡、复杂疾病调控等功能。通过介绍miRNA的起源、合成、修饰、细胞表达特点,以及对真核细胞调控等的最新进展与研究方法,阐述miRNA在基因表达调节中的重要地位及应用前景。     

中华鳖Dmrt1基因的克隆、表达及在性逆转中的变化分析

在大部分脊椎动物中,Dmrt1基因在雄性性别决定和性腺分化中起重要的调控作用.本文从m RNA和蛋白水平分析Dmrt1基因的组织差异性表达、在不同发育阶段性腺中的细胞定位及在性逆转中的表达变化,研究Dmrt1基因在中华鳖性别分化中的调控作用.Rapid-amplification of c DNA ends(RACE)结果显示,Dmrt1基因c DNA序列全长2409 bp,其中5′非编码区为230 bp,3′非编码区为1072 bp,开放阅读框为1107 bp,编码368个氨基酸,具有一个高度保守的DM结构域.荧光定量PCR和免疫组化结果显示,Dmrt1在性腺分化之前的第16期雄性性腺中开始表达,先于Amh和Sox9基因表达.随着性腺的发育,Dmrt1蛋白主要定位于性腺Sertoli细胞的细胞核上,在雌性性腺发育过程中并未见其表达.此外,在雌二醇诱导的雄性转雌性性逆转胚胎性腺中,Dmrt1表达显著下调;在芳香化酶抑制剂诱导的雌性转雄性性腺中,Dmrt1表达则显著上升.上述研究表明,Dmrt1基因是中华鳖雄性特异性基因,参与雄性性腺的发育过程,可能在中华鳖早期性别决定中起重要的调控作用.     

The Responses of Leaf Water Potential and Leaf Diffusive Resistance to Abscisic Acid, Water Stress and Low Temperature in Hibiscus esculentus: The Effect of Water Stress and ABA Pre-Treatments

Intact plants of okra (Hibiscus esculentus) were chilled at6°C in the light, and leaf diffusion resistance (LDR) andleaf water potential measured. The response of the LDR of excisedleaves to fresh weight loss and, separately, exogenous abscisicacid (ABA) supply, was also studied at 6°C and 30°C.The influence of two pre-treatments upon these measurementswas studied. The two pre-treatments consisted of the impositionof a period of water stress at 30°C prior to measurement(followed by re-watering) or the spraying of the leaves withABA. It was found that plants that had been grown in a highhumidity environment continuously (designated control plants)had stomata that were very unresponsive to both water loss fromthe leaves and to exogenous ABA at both temperatures (6°Cand 30°C). Chilling the control plants resulted in rapidwilting and concomitant decline in leaf water potential. A pre-treatmentof water stress prior to chilling did not alter or reduce therate of development of chilling injury, nor did the pre-treatmentincrease the responsiveness of stomata at 6°C to water lossor exogenous ABA. However, spraying the leaves with ABA priorto chilling reduced the severity and delayed the onset of chillinginjury. Stomatal response to water stress and exogenous ABAwas increased by the spraying pre-treatment. These results arediscussed in relation to previous studies of the phenomenonof stomatal locking open at low temperature and the effect ofpre-treatments upon the development of chill-resistance. Key words: Water stress, chilling, stomata     

蝴蝶兰PhTSJT1基因的克隆及在低温胁迫下的表达分析

本研究从蝴蝶兰叶片中克隆了茎部特异基因Ph TSJT1的全长序列(GenBank登录号为MF797883),并分析了其在不同组织及低温胁迫下的表达特性。结果表明,Ph TSJT1基因全长994 bp,编码239个氨基酸,属于Class-Ⅱ谷氨酰胺酰胺基转移酶超家族成员;同源性分析表明,该蛋白与多种植物的茎部特异蛋白和铝诱导蛋白有较高的同源性,进化上与小兰屿蝴蝶兰的茎部特异蛋白亲缘关系最近;该基因在营养器官中表达水平较高,在花器官中表达水平较低;13℃/8℃(昼/夜)的低温胁迫抑制PhTSJT1基因的转录表达,并随着低温胁迫时间的延长,Ph TSJT1基因的表达水平逐渐降低,在温度恢复正常时其表达水平升高;4℃冷胁迫低温条件下,PhTSJT1基因在处理1 h时,表达水平升高,处理8 h时表达水平最高,16 h后表达水平逐渐降低。由此推测,PhTSJT1参与4℃冷胁迫的分子调控。本研究不但有助于理解热带亚热带植物的耐冷机制,也为蝴蝶兰新品种的遗传改良提供帮助。     

Molecular Cloning of Giant Panda Pituitary Prolactin cDNA and Its Expression in Escherichia coli

cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229 amino acids including a putative signal peptide of 30 amino acids and a mature protein of 199 residues with one potential N-glycosylation site. Sequence comparison indicated that panda PRL shares a high degree of identity to other known PRL sequences ranging from 98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could be recognized by antibody against human PRL. Our results would contribute to further elucidating the structural and functional characteristics of pituitary PRL and provide a basis for the production of recombinant panda prolactin for future use in the breeding of giant panda.     

Molecular Cloning of the Transglutaminase Gene from Bacillus subtilis and Its Expression in Escherichia coli

We cloned and characterized a gene, tgl, encoding transglutaminase in Bacillus subtilis. The tgl gene contained a open reading frame 735-nucleotides long that encoded a 245-residue protein with the molecular weight of 28,300. The deduced amino acid sequence had little sequence similarity with sequences of other transglutaminases from a Streptoverticillium sp. or from mammals. The -10 and -35 regions of a putative promoter resembled the consensus sequence for the σ^K-dependent promoter. In addition, a sequence similar to the consensus sequence for the GerE binding site was found upstream from this region. These findings suggested that tgl was transcribed in the mother cells during a late stage of sporulation. Evidence for this suggestion was that transglutaminase activity was detected in sporulating cells during the same stage. Transglutaminase activity was detected in Escherichia coli cells transformed with a plasmid for expression of the tgl gene.     

猪骨髓基质抗原2基因的克隆、表达及生物学活性研究

研究目的是克隆猪骨髓基质抗原2(Bone marrow stromal antigen-2,BST-2)基因并进行真核表达,获得具有抗病毒活性的重组BST-2蛋白。研究通过RT-PCR从猪瘟弱毒疫苗和聚肌胞(Poly I:C)刺激的猪肾细胞(PK-15)中扩增出猪BST-2cDNA,克隆至真核表达载体pcDNA3.1/V5-His,构建重组表达质粒pcDNA-BST-2,转染HEK293T细胞,纯化BST-2蛋白并经Bradford法定量,Western blot检测,研究BST-2抗病毒生物学活性。酶切鉴定和核酸序列测定证实pcDNA-BST-2真核表达质粒构建成功,转染HEK293T细胞后,经间接免疫荧光能够检测到绿色荧光。生物学活性测定重组BST-2蛋白具有一定的抗水泡性口炎病毒(Vesicular stomatitis virus,VSV)、H9禽流感病毒(Avian influenza virus,AIV)及猪繁殖与呼吸综合症病毒(Porcine reproductive and respiratory syn-drome virus,PRRSV)的活性。结果表明,重组BST-2具有一定抗病毒生物功能,为进一步研究重组BST-2蛋白的活性以及BST-2抗病毒药物研究奠定了基础。     

Molecular Cloning,Expression and Molecular Modeling of Chemosensory Protein from Spodoptera litura and Its Binding Properties with Rhodojaponin III

Insects stimulate specific behaviors by the correct recognition of the chemicals in the external environment. Rhodojaponin III is a botanical grayanoid diterpenid oviposition deterrent isolated from Rhododendron molle. In this study we aimed to determine whether the CSPs involved in the recognition of Rhodojaponin III. A full-length cDNA encoding chemosensory protein was isolated from the antennae of Spodoptera litura Fabricius (CSPSlit, GenBank Accession No. {"type":"entrez-nucleotide","attrs":{"text":"DQ007458","term_id":"63020521","term_text":"DQ007458"}}DQ007458). The full-length cDNA of NlFoxA is 1789 bp and has an open reading frame (ORF) of 473 bp, encoding a protein of 126 amino acids, Northern blot analysis revealed that CSPSlit mRNA was mainly expressed in the antennae, legs, wings and female abdomens. A three-dimensional model of CSPSlit was constructed using homology modeling method, and its reliability was evaluated. The active site of CSPSlit was calculated using CDOCKER program indicated that the Tyr24, Ile45, Leu49, Thr64, Leu68, Trp79 and Leu82 were responsible ligand-binding active site on identifying Rhodojaponin III in the CSPSlit. The recombinant CSPSlit protein was expressed in Escherichia coli and purified using single-step Ni-NTA affinity chromatography. Fluorescence emission spectra revealed that the CSPSlit protein had significant affinity to rhodojaponin III. These results mean that CSPSlit is critical for insects identify the Rhodojaponin III.     

Molecular Cloning and Expression Analysis of Pig Cd90

The CD90 (Thy-1) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein that transfers signals involved in many biological events including cell activation, cell migration, cell adhesion, and tumor suppression. In this study, we cloned pig CD90 cDNA and determined its complete cDNA sequence. Pig CD90 cDNA contained an open reading frame (486 bp) encoding 161 amino acids with three putative N-glycosylation sites and four well-conserved cysteine residues, which form a possible disulfide bond within the extracellular domain among mammalian species. Pig CD90 mRNA was detected in various tissues, indicating the multicellular functions of CD90 in pigs. Flow cytometry analyses demonstrated that anti-human CD90 antibody recognizes a pig CD90 on the cell surface. Moreover, immunohistochemistry analysis revealed that CD90 expression is widely diffused in several pig tissues. Further studies will be necessary to define the functional contribution of CD90 during specific infectious diseases in pigs.     

肌抑素(Myostatin)基因突变体活性区的克隆、表达及活性的研究

肌抑素Myostatin是肌肉发育的重要抑制因子,肌抑素的突变,使其抑制功能的全部或几乎全部丧失,表现为肌肉细胞的增大和肌纤维束的增加。采用PCR技术,从肌抑素天然突变的双肌牛皮尔蒙特（Piedmontese）的基因组中扩增得到肌抑素突变体的活性区,并将其亚克隆到pMD18T载体上,利用基因重组技术,构建原核表达质粒pET30a(+)/action/Myostatin,在大肠杆菌中高效表达,采用亲和层析法纯化表达产物,并将其共孵育于离体培养的绵羊肌肉细胞,检测肌抑素突变体的生化活性,结果显示肌抑素的突变体具有促进肌肉细胞增生和增殖的功能。     

Molecular Cloning and Expression of Aven Gene in Chicken

Aven was originally identified as a protein that regulates apoptosis by binding to apoptotic regulators, Bcl-xL and Apaf-1. Recently was found that Aven protein is a potent activator of ATM, critical for its DNA damage-induced activation. An Aven cDNA clone was isolated from chicken (Gallus gallus) after screening of a cerebellum cDNA library. The full-length cDNA is 1,430 nt in size, encoding for a polypeptide of 352 amino acid residues. The predicted amino acid sequence of the chicken Aven is 69, 46, 45 and 37% identical to those of zebra finch, human, xenopus and zebrafish orthologs, respectively. Expression analysis reveals that the chicken Aven gene is expressed in the adult brain, heart, intestine, kidney, lung, stomach and spleen, as well as in the whole embryos of 4- and 6-days old. Phylogenetic analysis of the Aven ortholog proteins from various organisms clusters the chicken Aven in the same group with other bird Avens.