An enzyme‐linked immunosorbent assay for heat‐shock protein 70 in plants

Procedures are described for the production of antibodies against heat‐shock protein 70 (HSP70) from plants and for the development of an enzyme‐linked immunosorbent assay (ELISA) for the antigen in crude plant extracts. Competitive and two‐site sandwich variants of the ELISA are compared. Results are presented for a preliminary analysis of HSP70 levels in control and heat‐shocked mung bean shoots.     

Determination of intracellular heat shock protein 70 using a newly developed cell lysate immunometric assay

Heat shock proteins (Hsp) have been associated to several clinical relevant conditions. Currently used methods to determine Hsp 70 possess certain drawbacks. Therefore, we developed a cell lysate immunometric assay (CLIA) for the quantification of intracellular Hsp 70. This CLIA uses a combination of two distinct monoclonal antibodies that recognize different epitopes on the Hsp 70 molecule. A recombinant human Hsp 70 was used as the standard material. The detection range of the CLIA was 4-4000 ng/ml. The intra- and interassay coefficients of variation were, on average, 5% and 12%, respectively. The recovery varied between 81% and 116%. The Hsp 70 levels assayed after serial dilution of cell lysates varied linearly with dilution (between 97% and 120%).The reliability of the CLIA was assessed by comparison with the values determined by flow cytometric procedure; these two sets of values showed a highly significant correlation (r=0.896, p<0.0001), indicating that the two methods are comparable. We conclude that this assay represents a low-cost alternative of the flow cytometric technique.     

The heat shock protein 90 of Leishmania donovani

The 90-kDa heat shock protein (Hsp90) of Leishmania donovani is a highly abundant cytoplasmic protein and is involved in a variety of cellular processes. Pharmacological deactivation of Hsp90 leads to growth arrest and induces the synthesis of heat shock proteins. Moreover, treatment of promastigote parasites with Hsp90 inhibitors induces the synthesis of amastigote-specific marker proteins and a morphological alteration similar to axenic amastigote differentiation. We propose a role for Hsp90 in the feedback control of the cellular stress response and in the control of the parasite's life cycle.     

The polymorphism of heat shock protein 70 genes in Chinese Han population in Fujian province

Heat shock protein 70 (HSP70) genes are themost important and conserve gene members intheheat shock protein family,and locate in an areaadjacent tothe TNFgenesinthe classⅢregionofmajor histocompatibilitycomplex(MHC) .Its geneproductsHSP70proteins are encoded by 3 differ-ent genes ,HSP70-1,HSP70-2andHSP70-hom.Previous studies showed that three kinds ofpolymorphisms existin3loci of thesethree genes ,i .e .+190 G/CBsrBⅠrestrictionsite onHSP70-1, +1267 A/GPstⅠrestriction site onHSP…     

Usefulness of a PCR-based method in the detection and species identification of Leishmania from clinical samples

The aim of this study is to assess the usefulness of a simple, low-cost method for the detection and species identification of Leishmania isolated by in vitro culture or detected directly from clinical samples. A total of 110 samples were used in this study. Among these, 21 were human and canine peripheral bloods, 63 skin lesion material samples, eight reference strains and 18 Leishmania culture. Detection of Leishmania DNA with PCR using primers designed to amplify the internal transcribed spacer 1 (ITS1) region of the rRNA gene proved sufficiently sensitive at the level of 0.1 parasites per PCR reaction. Furthermore, followed by single-strand conformational polymorphism (SSCP), the PCR-ITS1 allowed the species identification of Leishmania. The inter-specific polymorphism of Leishmania was first validated on reference strains, and then this method was applied on clinical samples and culture. Typing identified all human and canine visceral leishmaniasis samples (21 samples) as L. infantum, 95.23% of the cutaneous leishmaniasis samples as L. major and 3.17% as L. killicki and 1.58% as L. infantum. A scheme of the PCR diagnosis procedure for the detection and identification of Leishmania parasites is proposed in this study.     

Facets of heat shock protein 70 show immunotherapeutic potential

Amongst the families of intracellular molecules that chaperone and assist with the trafficking of other proteins, notably during conditions of cellular stress, heat shock protein (hsp) 70 is one of the most studied. Although its name suggests that expression is exclusively induced during cellular hyperthermia, members of the hsp70 family of proteins can be constitutively expressed and/or induced by a range of other cellular insults. The ubiquitous presence of hsp70 in eukaryotic and prokaryotic cells, combined with its high degree of sequence homology and intrinsic immunogenicity, have prompted the suggestion that inappropriate immune reactivity to hsp70 might lead to pro-inflammatory responses and the development of autoimmune disease. Indeed, hsp70 has been shown to be a potent activator of innate immunity and aberrant expression of hsp70 in certain organs promotes immunopathology. However, studies also suggest that hsp70 might have immunotherapeutic potential, as hsp70 purified from malignant and virally infected cells can transfer and deliver antigenic peptides to antigen-presenting cells to elicit peptide-specific immunity and, in contrast to its reported pro-inflammatory effects, the administration of recombinant hsp70 can attenuate experimental autoimmune disease. This review focuses on the immunoregulatory capacity of hsp70 and its potential therapeutic value.     

Immunohistochemical expression of heat shock protein 70 in vitiligo

Heat shock proteins (HSPs) are proteins that are expressed under variety of stresses including pathologic conditions. How stresses affect vitiligo is not fully understood and little is known about the role of HSPs generally and Hsp70 specifically in vitiligo. The current study investigated the expression of Hsp70 in vitiliginous (32) and normal skin (10) by immunohistochemistry together with correlating this expression with the clinicopathologic parameters in the studied vitiligo group. Hsp70 was expressed in the cytoplasm of epidermis in all normal skin compared with its localization to the cytoplasm in 35.5% and to the nuclei in 64.5% of epidermis in vitiligo lesions. Intense (P < .001) and diffuse (P < .001) expression of Hsp70 was in favor of vitiligo skin compared with normal skin. Nuclear form of Hsp70 tended to be expressed in progressive forms of the disease. The percentage of Hsp70 expression tended to be decreased with the duration of the disease. From the present study, up-regulation of HSP 70, in the form of its intense and diffuse expression, may be blamed in pathogenesis of vitiligo. Nuclear localization of HSP 70 may be more important than its presence or absence, beside it may be related to progression of the disease.     

Serum heat shock protein 70 level as a biomarker of exceptional longevity

Heat shock proteins are highly conserved proteins that, when produced intracellularly, protect stress exposed cells. In contrast, extracellular heat shock protein 70 (Hsp70) has been shown to have both protective and deleterious effects. In this study, we assessed heat shock protein 70 for its potential role in human longevity. Because of the importance of HSP to disease processes, cellular protection, and inflammation, we hypothesized that: (1) Hsp70 levels in centenarians and centenarian offspring are different from controls and (2) alleles in genes associated with Hsp70 explain these differences. In this cross-sectional study, we assessed serum Hsp70 levels from participants enrolled in either the New England Centenarian Study (NECS) or the Longevity Genes Project (LGP): 87 centenarians (from LGP), 93 centenarian offspring (from NECS), and 126 controls (43 from NECS, 83 from LGP). We also examined genotypic and allelic frequencies of polymorphisms in HSP70-A1A and HSP70-A1B in 347 centenarians (266 from the NECS, 81 from the LGP), 260 NECS centenarian offspring, and 238 controls (NECS: 53 spousal controls and 106 septuagenarian offspring controls; LGP: 79 spousal controls). The adjusted mean serum Hsp70 levels (ng/mL) for the NECS centenarian offspring, LGP centenarians, LGP spousal controls, and NECS controls were 1.05, 1.13, 3.07, 6.93, respectively, suggesting that a low serum Hsp70 level is associated with longevity; however, no genetic associations were found with two SNPs within two hsp70 genes.     

Pepino mosaic virus capsid protein interacts with a tomato heat shock protein cognate 70

Plant viral capsid proteins (CP) can be involved in virus movement, replication and symptom development as a result of their interaction with host factors. The identification of such interactions may thus provide information about viral pathogenesis. In this study, Pepino mosaic virus (PepMV) CP was used as bait to screen a tomato (Solanum lycopersicum) cDNA library for potential interactors in yeast. Of seven independent interacting clones, six were predicted to encode the C-termini of the heat shock cognate 70 (Hsc70) proteins. Three full length tomato Hsc70s (named Hsc70.1, .2, .3) were used to confirm the interaction in the yeast two hybrid assay and bimolecular fluorescent complementation (BiFC) in planta. The PepMV CP-Hsc70 interaction was confirmed only in the case of Hsc70.3 for both assays. In BiFC, the interaction was visualized in the cytoplasm and nucleus of agroinfiltrated Nicotiana benthamiana epidermal cells. During PepMV infection, Hsc70.3 mRNA levels were induced and protein accumulation increased at 48 and 72 h post inoculation. In transmission electron microscopy using immunogold labelling techniques, Hsc70 was detected to co-localize with virions in the phloem of PepMV-infected tomato leaves. These observations, together with the co-purification of Hsc70 with PepMV virions further support the notion of a PepMV CP/Hsc70 interaction during virus infection.     

Immunostimulatory properties of the Leishmania infantum heat shock proteins HSP70 and HSP83

Emerging evidence indicates that the heat shock proteins (HSPs), a set of highly evolutionary conserved proteins, are playing essential roles in both normal processes of the immune system and specific immune responses. In a previous work, we demonstrated that the Leishmania infantum HSP70 possesses remarkable immunostimulatory properties. In the present work, we have extended the study to another HSP from this parasite, the HSP83. We show that this protein also has an adjuvant effect to an accompanying protein by stimulation of the humoral response when both proteins are fused and co-administered to BALBjc mice. The analysis of the IgG isotypes, IgG1 and IgG2a, indicated that the immunisations with the Leishmania HSPs, mainly the HSP70, potentiate a Thl-type response. It was found that the amino-terminal domain of the HSP70, the most evolutionary conserved region of the molecule, maintains the ability to stimulate the humoral response, whereas the carboxyl-terminal domain does not have a similar effect. Unexpectedly, we found that the L. infantum HSP70 and HSP83 recombinant proteins stimulated the proliferation of spleen cells from unprimed BALB/c mice. Remarkably, this proliferation was abolished either by thermal denaturing of the proteins or by using specific antibodies. The use of the T-cell inhibitor cyclosporin A in the splenocytes proliferation assays suggested that both T- and non-T-cells are stimulated by the Leishmania HSPs. These findings may be relevant for therapeutic and prophylactic applications.     

Effects of heat shock protein 70 (Hsp70) on arsenite-induced genotoxicity

Arsenic, a human carcinogen, is genotoxic, although its mechanism(s) of action for tumorigenesis is not well understood. Among the toxicity-related properties of this chemical are its clastogenic and aneugenic activities, as well as its capacity for inducing stress-response in the form of elevated heat shock protein (HSP) expression. In the present study, we evaluated the effects of Hsp70 expression on arsenite (As)-induced structural and numerical chromosome anomalies in human cells. Human MCF-7 Tet-off cells stably transfected with a pTRE/Hsp70-1 transgene construct were used to regulate Hsp70 levels prior to in vitro As exposures. Separate cultures of relatively high vs. low Hsp70-expressing cells were established. A cytokinesis block micronucleus assay with kinetochore immunostaining was used to detect micronuclei (MN) derived from chromosome breakage (K-MN) or loss (K+MN). These studies demonstrated significant increases in micronucleus frequencies in response to As following either a long exposure (5 or 10 microM for 46 hr), or short exposure (10 or 40 microM for 8 hr) protocol. Overall, the long protocol was more efficient in producing K+MN and cells with multiple MN. Overexpressing Hsp70 resulted in significant reductions in the percent of cells positive for MN for both the long and short As exposure protocols. Both K+ and K- types of As-induced MN were lower in cells with elevated Hsp70 as compared to cells without overexpression of Hsp70. We conclude that the dose and duration of As exposure influence the type as well as amount of chromosomal alteration produced and that inducible Hsp70 protects against both the clastogenic and aneugenic effects of this chemical.     

人热休克蛋白70基因的原核表达

目的 表达人热休克蛋白70（HSP70）并进行鉴定。方法 用PCR方法扩增人HSP70基因片段，经T-A克隆法克隆到载体pUCm-T中，并进行DNA测序，将人HSP70基因片段从载体pUCm-T中酶切后，构建重组表达载体pGEX-4T-1-HSP70，并转化大肠杆菌JM109，用IPTG诱导，收集细菌，菌体裂解后进行SDS-PAGE及Western blot检测。结果 人HSP70基因的PCR产物约为1.9kb。序列测定结果证实，所获目的序列与文献[3]报道的相一致，经EcoRⅠ和XhoⅠ酶切鉴定证实。人HSP70基因已成功地克隆到表达载体pGEX-4T-1中，构建的表达载体pGEX-4T-1-HSP70，能很好地在大肠杆菌中表达相对分子质量（Mr)为96000并具有抗原特性的融合蛋白，结论 成功地克隆并表达了HSP70基因，为研究HSP70的结构。功能与临床应用提供了必要条件。     

Expression of 65- and 67-kilodalton heat-regulated proteins and a 70-kilodalton heat shock cognate protein of Leishmania donovani in macrophages.

Heat shock protein (HSP) expression was examined in murine bone marrow-derived macrophages infected with stationary-phase promastigotes of Leishmania donovani. Immunoblotting performed with a rabbit polyclonal antiserum raised against HSP60 from Heliothis virescens (moth) revealed the de novo appearance of 65- and 67-kDa proteins in leishmania-infected macrophages. A third protein of 60 kDa, which represented murine HSP60, was also detected, and its expression did not change in response to infection. In contrast, expression of the novel 65- and 67-kDa proteins in infected cells was coordinately regulated and, at 24 h of infection, reached maximal levels of 52 to 100% increases above initial levels determined at 3 h. Proteins which had identical electrophoretic mobilities and were similarly regulated in response to heat were also detected in promastigotes. The appearance of these proteins in macrophages was specific to leishmania infection in that neither protein was detected in noninfected cells either in the basal state or following several treatments, including (i) infection with Yersinia pseudotuberculosis, (ii) phagocytosis of Staphylococcus aureus, (iii) NaAsO2 treatment, and (iv) heat shock. Expression of the 65- and 67-kDa heat-regulated Leishmania proteins was also observed to be selective, in that as their concentration was increasing, the abundance of the Leishmania surface protease gp63 in infected cells was noted to decrease. Murine HSP60 but not the Leishmania heat-regulated proteins was also recognized by a distinct rabbit antiserum raised against human HSP60, suggesting the presence of specific determinants within these Leishmania proteins. A monoclonal antibody that recognizes both mammalian HSP70 and HSP70 from plasmodia detected single isoforms of both Leishmania and murine HSP70 in infected cells, and the level of neither protein changed during infection. Moreover, although a murine HSP of 73 kDa was induced in response to both heat shock and NaAsO2 treatment, it was not induced to detectable levels by infection. The rapid and relatively high level of expression of inducible HSP60-related proteins of L. donovani and Leishmania HSP70 in infected macrophages suggests that these proteins are involved in pathogenesis and may be important targets of the immune response.     

Three new sensitive and specific heat-shock protein 70 PCRs for global Leishmania species identification

The heat-shock protein 70 gene (hsp70) has been exploited for Leishmania species identification in the New and Old World, using PCR followed by restriction fragment length polymorphism (RFLP) analysis. Current PCR presents limitations in terms of sensitivity, which hampers its use for analyzing clinical and biological samples, and specificity, which makes it inappropriate to discriminate between Leishmania and other trypanosomatids. The aim of the study was to improve the sensitivity and specificity of a previously reported hsp70 PCR using alternative PCR primers and RFLPs. Following in silico analysis of available sequences, three new PCR primer sets and restriction digest schemes were tested on a globally representative panel of 114 Leishmania strains, various other infectious agents, and clinical samples. The largest new PCR fragment retained the discriminatory power from RFLP, while two smaller fragments discriminated less species. The detection limit of the new PCRs was between 0.05 and 0.5 parasite genomes, they amplified clinical samples more efficiently, and were Leishmania specific. We succeeded in significantly improving the specificity and sensitivity of the PCRs for hsp70 Leishmania species typing. The improved PCR-RFLP assays can impact diagnosis, treatment, and epidemiological studies of leishmaniasis in any setting worldwide.     

Identification of heat shock protein 70 in the rabies virion.

We investigated a 73-kDa polypeptide (p73), a minor component of the rabies virion (HEP-Flury and ERA strains), accounting for as much as 1% of total virion proteins. Two-dimensional gel electrophoresis and immunoblotting with the antiserum against the heat shock protein 70 (hsp70) demonstrated that p73 was identical to a constitutive type of cellular hsp70. The antiserum also detected p73/hsp70 in the purified virions of other negative-stranded RNA viruses, such as vesicular stomatitis virus (New Jersey serotype), Newcastle disease virus (Miyadera strain), and influenza A virus (PR8 strain), among which, however, the contents were variable.     

血清热休克蛋白70与妊娠的相关性研究

目的探讨妊娠期间母体与血清热休克蛋白之间的关系。方法用双抗体夹心ELISA法检测114例孕妇血清热休克蛋白含量,按孕3、5、7、8个月和足月共分五组,与非孕对照组比较。结果孕3-8个月组血清HSP70蛋白的含量与非孕对照组比较无差异（P〉0.05）,足月孕妇组血清HSP70含量明显升高,与非孕对照组比较有非常显著性差异（P〈0.001）;重度妊高征患者血清热休克蛋白70含量明显高于同孕龄正常孕妇组（P〈0.001）。结论了解不同孕月健康孕妇血清HSP70含量,可为妊娠期间某些疾病引起的血清HSP70升高,提供参考依据。     

Distribution of 70kDa heat shock protein in rabbit brains after heat stress and heat stroke

OBJECTIVE: Evaluation of the relationship between the induction of 70kDa heat shock protein in rabbit brains and heat stress. METHODS: HSP70 was detected using monoclonal antibody by ABC method in rabbit hypothalamus, hippocampus and cerberal cortex. RESULTS: Intense HSP70 staining was displayed in rabbit brains of the heat stroke group (rectal temperature 43 degrees C to death). Positive cells were distributed mainly in the CA1, CA2 regions of the hippocampus; granular cell layer I and pyramidal layer (II) of the cerebral cortex; and the periventricular area of hypothalamus. HSP70-psoitive substances were localized in the cytoplasm and neuronal processes, a few neurons exhibited dark staining nucle. Hosever, the rabbit brains of the general heat stress group (rectal temperature 42.0 degrees C, 30 minutes) had much weaker staining. CONCLUSION: Hyperthermia causes neuronal expression of HSP70, particularly under strong heat stress, and may be sustained till death.     

Development of a PCR assay for typing and subtyping of Brucella species

In the course of this study, examinations were carried out to develop a PCR-based test which allows discrimination of Brucella species and biovars not targeted by the currently established gel-based PCR assays. Appropriate primers were designed based on specific deletions and insertions in the different Brucella genomes as determined by RAPD-PCR and whole-genome comparisons. After testing the specificity of the primers with a set of 22 Brucella reference strains of all species and biovars, they were used to supplement the existing PCR assays resulting in a 19-primer multiplex PCR. In addition to the commonly used PCR assays, the developed assay specifically identified B. neotomae, B. pinnipedialis, B. ceti, and B. microti. Furthermore, it differentiated B. abortus biovars 1, 2, 4 from biovars 3, 5, 6, 9, as well as between B. suis biovar 1, biovars 3, 4, and biovars 2 and 5. When tested in the multiplex assay, all Brucella type and reference strains and the majority of 118 field strains examined could be accurately identified by their respective banding patterns according to their previous typing. B. canis strains were subdivided into 2 groups, one exhibiting a unique pattern and the other one a banding pattern shared with B. suis biovars 3 and 4. Species of the closely related genus Ochrobactrum and several other clinically relevant bacteria showed no amplification product. Hence, the developed PCR assay is useful for rapid identification of Brucella at the species and at the biovar level.