Heteromeric complexes of heat shock protein 70 (HSP70) family members,including Hsp70B′, in differentiated human neuronal cells

Human neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis have been termed “protein misfolding disorders.” Upregulation of heat shock proteins that target misfolded aggregation-prone proteins has been proposed as a potential therapeutic strategy to counter neurodegenerative disorders. The heat shock protein 70 (HSP70) family is well characterized for its cytoprotective effects against cell death and has been implicated in neuroprotection by overexpression studies. HSP70 family members exhibit sequence and structural conservation. The significance of the multiplicity of HSP70 proteins is unknown. In this study, coimmunoprecipitation was employed to determine if association of HSP70 family members occurs, including Hsp70B′ which is present in the human genome but not in mouse and rat. Heteromeric complexes of Hsp70B′, Hsp70, and Hsc70 were detected in differentiated human SH-SY5Y neuronal cells. Hsp70B′ also formed complexes with Hsp40 suggesting a common co-chaperone for HSP70 family members.     

盐芥热激同源蛋白70基因(Thhsc70)的分子克隆及鉴定(英文)

热激蛋白70(hsp70s)具有分子伴侣的功能,其中在非胁迫条件下表达的hsp70s称为热激同源蛋白70(hsc70)。为更好地了解hsc70基因的特性,鉴定了盐芥(Thellungiella halophila(C. A. Mey. )O. E. Schulz)中编码胞质hsc70蛋白的基因Thhsc70。实验结果表明:在非胁迫条件下,Thhsc70基因具有组织特异性表达;Thhsc70基因受热胁迫和冷胁迫的诱导表达,但几乎不受盐诱导和干旱诱导。Thhsc70基因在拟南芥中过量表达后提高了转基因拟南芥的热耐受性和冷耐受性。     

HSP_(70)基因表达对高粱育性的影响(英文)

高粱细胞质雄性不育系３１９７Ａ（３Ａ）在常温条件下是不育的（Ｆｉｇｓ．１１＆２）,经热激（４５℃）诱导不同程度地恢复了育性（Ｆｉｇｓ．１３＆４）,为研究其不育机理提供了线索。热激２ｈ后,３Ａ中即可产生一类线粒体热激蛋白（ＨＳＰｓ）。其中,分子量为７０ｋＤ的ＨＳＰ７０含量最高,也最为稳定。不过,３Ａ中ＨＳＰｓ的稳定性弱于保持系３１９７Ｂ（３Ｂ）（Ｆｉｇ．２,Ｐａｎｅｌｓ１～４）。放线菌素Ｄ抑制ＨＳＰｓ的合成,而氯霉素无此作用（Ｆｉｇ．２,Ｐａｎｅｌｓ５＆６）,表明：ＨＳＰｓ是由核基因编码、在细胞质中合成、再跨膜转运到线粒体中的。３Ａ幼穗经热激后,线粒体的总蛋白量猛增了２．７倍（Ｆｉｇ．３）,达到３Ｂ的水平,育性亦变为可育的。Ｆｉｇ．４表明：ＨＳＰ７０反义链ｃＤＮＡ（Ｒ１）能进入到３Ｂ花药细胞中,并与靶ＲＮＡ（ＨＳＣ７０ｍＲＮＡ）结合,而对照、正义链ｃＤＮＡ（Ｄ）链无此反应。由此、再增加一个通用保守序列的反义链ｃＤＮＡ（Ｒ２）、共两个探针（Ｒ１、Ｒ２）,可以检测到：３Ａ在常温下没有能力合成ＨＳＣ７０ｍＲＮＡ（Ｆｉｇ．５）,而在热激条件下,转变为有能力（Ｆｉｇ．６）。启示：３Ａ在热激条件下由不育转变为可育     

Stress-induced localization of HSPA6 (HSP70B′) and HSPA1A (HSP70-1) proteins to centrioles in human neuronal cells

The localization of yellow fluorescent protein (YFP)-tagged HSP70 proteins was employed to identify stress-sensitive sites in human neurons following temperature elevation. Stable lines of human SH-SY5Y neuronal cells were established that expressed YFP-tagged protein products of the human inducible HSP70 genes HSPA6 (HSP70B′) and HSPA1A (HSP70-1). Following a brief period of thermal stress, YFP-tagged HSPA6 and HSPA1A rapidly appeared at centrioles in the cytoplasm of human neuronal cells, with HSPA6 demonstrating a more prolonged signal compared to HSPA1A. Each centriole is composed of a distal end and a proximal end, the latter linking the centriole doublet. The YFP-tagged HSP70 proteins targeted the proximal end of centrioles (identified by γ-tubulin marker) rather than the distal end (centrin marker). Centrioles play key roles in cellular polarity and migration during neuronal differentiation. The proximal end of the centriole, which is involved in centriole stabilization, may be stress-sensitive in post-mitotic, differentiating human neurons.     

Vegetation change in created emergent wetlands (1988–1996) in Connecticut (USA)

Changes in hydrology, water quality and vegetation were evaluated in four palustrine emergent wetland pairs, each including created and reference sites. Located along interstate highways, they were initially sampled in 1988 (Confer and Niering, 1992) and again in 1996. Overall, created sites showed significant decreases in open water and water depth between 1989 and 1996 compared to more stable conditions in reference sites. Total nitrogen was generally higher in created sites compared to reference sites, as was specific conductivity, with chloride levels exceeding 800 mg/L, apparently related to road salt. Emergent plant cover increased from 30 to 39% at three created sites, and decreased at a fourth, whereas reference sites remained relatively stable. Wetland species richness also increased from 31 to 39 species at created sites and 35 to 42 species at reference wetlands between the surveys. By 1996 there was an increase in invasive species, particularly Phragmites australis (common reed) and Lythrum salicaria (purple loosestrife). Phragmites increased from <1 to 15% at created sites, while Lythrum increased at one reference site from <1 to 16%. Typha latifolia (common cattail), dominant in the created wetlands in 1988, decreased from 16 to 5% while Typha angustifolia (narrow-leaved cattail) increased from 2 to 10%. At two created sites experiencing increased sedimentation, Phragmites is now dominant or co-dominant with Typha spp., whereas Carex stricta (tussock sedge) and T. latifolia continue to dominate at reference sites. At the one created, permanent flow-through-hydrology wetland, a three-fold decrease in T. latifolia and an eight-fold increase in Phragmites cover have occurred, the latter correlated with sedimentation from road culverts. Increases in alien or invasive species such as Phragmites and Lythrum can serve as indicators of wetland disturbance. Although these created wetlands provide the services of sediment retention, flood storage, and wildlife habitat, a greater range of wetland functions should be possible by constructing two-tiered systems that remove excess sediments and nutrients upstream of the wetland designed to compensate for wetland loss.     

重金属对HeLa细胞中热激蛋白70(HSP70)表达的影响

热激蛋白(HSPs)是受热等因素刺激后而诱导产生的蛋白质,是一类可以调节应激反应并且保护机体防止细胞损伤的蛋白质,在机体的应激反应中具有重要作用。它们作为一般标志物被广泛应用于环境监测中。CdCl2,Cu2+,Zn2+这三种重金属是普遍存在的环境污染物,对人体和动物的一些主要器官造成损伤。以HeLa细胞(子宫肿瘤细胞)为材料,采用不同浓度的CdCl2,Cu2+,Zn2+三种重金属物质诱导细胞,并利用免疫荧光染色(IFS),SDS-PAGE,Western blotting和RT-PCR四种手段分别从基因和蛋白质的水平来研究重金属对HSP70表达的影响。结果表明,三种金属对HSP70表达的影响程度为CdCl2>Zn2+>Cu2+,且HSP70的产生量与重金属的浓度呈正相关。通过研究,以建立一种对HSPs的表达更有效的检测手段用于以后的研究。     

IUBMB Symposia in 2000(英文)

ＦｒｏｎｔｉｅｒｓｏｆＰｒｏｔｅｉｎＳｔｒｕｃｔｕｒｅａｎｄＦｕｎｃｔｉｏｎＭａｒｃｈ２６３１,２０００ＳａｎＦｒａｎｃｉｓｃｏ,Ｃａｌｉｆｏｒｎｉａ,ＵＳＡＤｒ．ＲｏｂｅｒｔＧｅｎｎｉｓＤｅｐｔ．ｏｆＢｉｏｃｈｅｍｉｓｔｒｙ,ＵｎｉｖｅｒｓｉｔｙｏｆＩｌｌｉｎｏｉｓ６００ＳｏｕｔｈＭａｔｈｅｗｓＡｖｅｎｕｅ,Ｕｒｂａｎａ,ＩＬ６１８０１３３６４,ＵＳＡＴｅｌ：＋１２１７３３３９０７５;Ｆａｘ：＋１２１７２４４３１８６;Ｅｍａｉｌ：ｒｇｅｎｎｉｓ＠ｕｉｕｃ．ｅｄｕ１３ｔｈＩｎｔｅｒｎａ…     

Enhancement of Fermentative Hydrogen Production in an Extreme-Thermophilic (70°C) Mixed-Culture Environment by Repeated Batch Cultivation

Repeated batch cultivation was applied to enrich hydrogen fermentative microflora under extreme-thermophilic (70°C) environment. Initial inoculums received from a hydrogen producing reactor fed with organic fraction of household solid wastes. In total seven transfers was conducted and maximum hydrogen yield reached 296 ml H₂/g (2.38 mol/mol) glucose and 252 ml H₂/g (2.03 mol/mol) for 1 and 2 g/l glucose medium, respectively. It was found that hydrogen production was firstly decreased and got increased gradually from third generation. Acetate was found to be the main metabolic by-product in all batch cultivation. Furthermore, the diversity of bacterial community got decreased after repeated batch cultivation. It was proved that repeated batch cultivation was a good method to enhance the hydrogen production by enriching the mixed cultures of dominant species.     

盐芥热激同源蛋白70基因(Thhsc70)的分子克隆及鉴定

热激蛋白70(hsp70s)具有分子伴侣的功能,其中在非胁迫条件下表达的hsp70s称为热激同源蛋白70(hsc70).为更好地了解hsc70基因的特性,鉴定了盐芥(Thellungiella halophila(C.A.Mey.)O.E. Schulz)中编码胞质hsc70蛋白的基因Thhsc70.实验结果表明:在非胁迫条件下,Thhsc70基因具有组织特异性表达;Thhsc70基因受热胁迫和冷胁迫的诱导表达,但几乎不受盐诱导和干旱诱导.Thhsc70基因在拟南芥中过量表达后提高了转基因拟南芥的热耐受性和冷耐受性.     

A pentanucleotide tandem duplication polymorphism in the 3′ untranslated region of the HLA-linked heat-shock protein 70-2 (HSP70-2) gene

A novel two-allele pentanucleotide tandem duplication polymorphism is described within the 3 untranslated region of the HLA-linked HSP70-2 gene, for which a PstI polymorphism is known. All four haplotype combinations were found.     

Acidification of lakes in Šumava (Bohemia) and in the High Tatra Mountains (Slovakia)

Acidification of lakes takes place when pH of rainwater is less than 4.5 and the catchments lie on sensitive geology. Both conditions are met for most lakes in Bohemia and Slovakia. Since 1978 we have studied mountain lakes in the Sumava and in the High Tatra Mountains. In Šumava the three lakes under study are of glacial origin. The catchments are small, with steep sides covered by spruce. The bedrocks are biotite-rich paragneiss, together with gneiss, quartzite and granite. In summer 1936 surface pH was 5.7–6.9 in the Lake Čertovo and 6.9–7.0 in the Lake Černé. Now the pH values are 4.3–4.8 in the two lakes and in the Lake Prášilské as well. Old reports on zooplankton are from the years 1871, 1892–96, 1935–37, 1947 and 1960. Since 1979 we have not found any planktonic Crustacea in the lakes Černé and Čertovo. Lake Prášilské is inhabited by Daphnia longispina and Cyclops abyssorum. In July 1989 the pH values were 4.4, 4.7 and 4.7, concentrations of labile monomeric Al were 0.83, 0.68 and 0.24 mg l^-1 in the lakes Čertovo, Černé and Prašilské, respectively. High levels of toxic Al compounds might be responsible for the extinction of planktonic Crustacea in the lakes Čertovo and Černé. All the three lakes are void of fish at present. In the High Tatra Mts. we examined more than 40 lakes above timberline in altitudes 1612–2145 m. They are all clearwater, naturally fishless lakes. The bedrock is granite. Owing to different levels of calcium the lakes are now in different stages of acidification. According to recent changes in the zooplankton they can be divided into three groups: (1) Species composition of planktonic Crustacea has not changed. (2) Planktonic Crustacea were present until 1973 but are absent now. (3) From the original species of Crustacea only Chydorus sphaericus is present. The three groups are well separated along the gradients of calcium, ANC and pH. They can be identified with the Henriksen's bicarbonate (our group 1), intermediate (our group 2) and acid (our group 3) lakes. We suppose that in the process of acidification the lakes of the group (2) have been shifted from oligotrophy to ultraoligotrophy.     

Common micro RNAs (miRNAs) target complement factor H (CFH) regulation in Alzheimer’s disease (AD) and in age-related macular degeneration (AMD)

Alzheimer’s disease (AD) and age-related macular degeneration (AMD) are complex and progressive inflammatory degenerations of the human neocortex and retina. Recent molecular, genetic and epigenetic evidence indicate that at least 4 micro RNAs (miRNAs) - including the NF-кB-regulated miRNA-9, miRNA-125b, miRNA-146a and miRNA-155 - are progressively up-regulated in both AD and AMD. This quartet of up-regulated miRNAs in turn down-regulate a small brain- and retinal-cell-relevant family of target mRNAs, including that encoding complement factor H (CFH), a major negative regulator of the innate immune and inflammatory response. Together miRNA-146a and miRNA-155 recognize an overlapping miRNA regulatory control (MiRC) region in the CFH 3’-untranslated region (3’- UTR; 5’-TTTAGTATTAA-3’) to which either of these miRNAs may interact. Progressive, pathogenic increases in specific miRNA binding to the entire 232 nucleotide CFH 3’-UTR appears to be a major regulator of CFH expression down-regulation, and the inflammatory pathology that characterizes both AMD and AD. The data presented in this report provides evidence that up-regulation of brain- and retinal- abundant miRNAs, including miRNA-9, miRNA-125b, miRNA-146a and miRNA-155, are common to the pathogenetic mechanism of CFH deficiency that drives inflammatory neurodegeneration, and for the first time indicates multiple, independent miRNA-mediated regulation of the CFH mRNA 3’-UTR.     

日本三角涡虫热休克蛋白70(DjHSP70)C-端多肽表达及其抗血清制备

首先运用在线生物学软件对日本三角涡虫(Degusia japonica)热休克蛋白70(DjHSP70)氨基酸序列进行亲水区分析,发现该蛋白C-端含有较多亲水性氨基酸,然后以该段多肽序列为基础构建原核表达载体.采用PCR方法扩增450 bp cDNA片段,编码DjHSP70 C-端150个氨基酸多肽.将双酶切的cDNA...     

Dermatophytes in Portugal (1972–1981)

For the years 1972–1981, 7 333 isolates of dermatophytes belonging to 14 species were obtained from glabrous skin (32%), feet (28%), groin (19%), scalp (8%), toenails (7%), fingernails (3%) and beard (1%)., T. rubrum represented 50% of all the isolates and was the most frequent species on glabrous skin, groin and nails. T. mentagrophytes (24%) was mainly obtained from the feet, E. floccosum (9%) from the groin and T. megninii (4%) from uncovered areas of the skin, fingernail and beard. These 4 species predominated in men. M. canis was the commonest agent on the scalp and in children up to 11 years. T. violaceum, previously the main cause of tinea capitis, and T. tonsurans have been decreasing for the period of this study, just as T. schoenleinii for the years 1962–71.The rising prevalence of T. rubrum was observed since 1962. In the whole it seems stable after 1969, but the analysis of the main sites involved shows that in the glabrous skin this species increased from 1962 to 1974; in the groin it was gone up from 30% during 1962–1965, to 64% in the years 1969–1971; in the feet the evolution was slower and only in 1980 T. rubrum became more frequent than T. mentagrophytes.The increase in certain species, whereas others become rare, lacks a satisfactory explanation.

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Résumé Depuis 1972 jusqu'à 1981, 7 333 isolements de dermatophytes concernant 14 espèces ont été obtenus de la peau glabre (32%), des pieds (28%), des plis inguinaux (19%), des ongles des orteils (7%), des ongles des mains (3%) et de la barde (1%). T. rubrum a réprésenté 50% de tous les isolements, étant l'espèce la plus fréquente dans la peau glabre, aux plis inguinaux et dans les ongles. T. mentagrophytes (24%) a été isolé surtout des pieds; E.floccosum (9%) des plis inguinaux; et T. megninii (4%) des régions découvertes de la peau, des ongles de la main et de la barbe. Ces 4 espèces ont prédominé chez des adultes du sexe masculin. M. canis a été l'agent le plus commun dans le cuir chevelu et chez des enfants jusqu'à 11 ans. T. violaceum (auparavant la cause principale des teignes du cuir chevelu) et T. tonsurans sont devenus beaucoup moins fréquents pendant cette étude, comme d'ailleurs T. schoenleinii était devenu rare au cours d'une enquête anterieur (1962–1971).La prévalence de T. rubrum s'est beaucoup elevée dès 1962. Dans l'ensemble, elle parait stabilisée depuis 1969, mais l'analyse des principales localisations de la maladie démontre que dans la peau glabre cette espèce a augmenté depuis 1962 jusqu'à 1974; aux plis inguinaux elle a changé de 30% en 1962–1965 pour 64% en 1969–1971; aux pieds l'évolution a été moins rapide et seulement dès 1980 T. rubrum est devenu plus fréquent que T. mentagrophytes.L'augmentation de certaines espèces tandis que d'autres deviennent rares n'a pas trouvée une interpretation satisfaisante.

     

A Mn(II)–Mn(II) center in human prolidase

Human prolidase, the enzyme responsible for the hydrolysis of the Xaa-Pro/Hyp peptide bonds, is a key player in the recycling of imino acids during the final stage of protein catabolism and extracellular matrix remodeling. Its metal active site composition corresponding to the maximal catalytic activity is still unknown, although prolidase function is of increasing interest due to the link with carcinogenesis and mutations in prolidase gene cause a severe connective tissue disorder. Here, using EPR and ICP-MS on human recombinant prolidase produced in Escherichia coli (hRecProl), the Mn(II) ion organized in a dinuclear Mn(II)–Mn(II) center was identified as the protein cofactor. Furthermore, thermal denaturation, CD/fluorescence spectroscopy and limited proteolysis revealed that the Mn(II) is required for the proper protein folding and that a protein conformational modification is needed in the transition from apo- to Mn(II)loaded-enzyme. The collected data provided a better knowledge of the human holo-prolidase and, although limited to the recombinant enzyme, the exact identity and organization of the metal cofactor as well as the conformational change required for activity were proven.     

microRNA (miRNA) speciation in Alzheimer’s disease (AD) cerebrospinal fluid (CSF) and extracellular fluid (ECF)

Human cerebrospinal fluid (CSF), produced by the choroid plexus and secreted into the brain ventricles and subarachnoid space, plays critical roles in intra-cerebral transport and the biophysical and immune protection of the brain. CSF composition provides valuable insight into soluble pathogenic bio-markers that may be diagnostic for brain disease. In these experiments we analyzed amyloid beta (Aβ) peptide and micro RNA (miRNA) abundance in CSF and in short post-mortem interval (PMI <2.1 hr) brain tissue-derived extracellular fluid (ECF) from Alzheimer’s disease (AD) and age-matched control neocortex. There was a trend for decreased abundance of Aβ42 in the CSF and ECF in AD but it did not reach statistical significance (mean age ~72 yr; N=12; p~0.06, ANOVA). The most abundant nucleic acids in AD CSF and ECF were miRNAs, and their speciation and inducibility were studied further. Fluorescent miRNA-array-based analysis indicated significant increases in miRNA-9, miRNA-125b, miRNA-146a, miRNA-155 in AD CSF and ECF (N=12; p<0.01, ANOVA). Primary human neuronal-glial (HNG) cell co-cultures stressed with AD-derived ECF also displayed an up-regulation of these miRNAs, an effect that was quenched using the anti-NF-кB agents caffeic acid phenethyl ester (CAPE) or 1-fluoro-2-[2-(4-methoxy-phenyl)-ethenyl]-benzene (CAY10512). Increases in miRNAs were confirmed independently using a highly sensitive LED-Northern dot-blot assay. Several of these NF-кB-sensitive miRNAs are known to be up-regulated in AD brain, and associate with the progressive spreading of inflammatory neurodegeneration. The results indicate that miRNA-9, miRNA-125b, miRNA-146a and miRNA-155 are CSF- and ECF-abundant, NF-кB-sensitive pro-inflammatory miRNAs, and their enrichment in circulating CSF and ECF suggest that they may be involved in the modulation or proliferation of miRNA-triggered pathogenic signaling throughout the brain and central nervous system (CNS).