BACKGROUND: An accurate and rapid method for specise identification of coagulase negative staphylococci(CNS) has been increasingly necessary for the clinical significance and planning the management of patients with staphylococcal infections. Recently, it has been reported that there is a highly conserved area on their 60KDa heat shock protein(HSP60) gene sequences between the interspecies of CNS and it can be amplified by a set of universal degenerate primer. This led us our attention to focus on whether the PCR-based RFLP method using Mse / restriction enzyme could be a useful tool for the species identification of CNS. METHODS: In the present study, we performed PCR-based RFLP analysis using a set of degenerate primers covering HSP60 and Mse / restriction enzyme on the reference strains and 25 clinical isolates(10 of S. epidermidis, 10 of S. haemolyticus, 4 of S. lugdunensis and 1 of S. warneri) which were previously identified by the API-STAPH, Vitek GPI card and/or with conventional biochemical test. RESULT: All the seven reference strains revealed that each strain has a distinct electrophoresed band patterns with combination of different number (up to 8) and size of fragments. And these distinct band patterns showed remarkable concordance with the seven reference strains and 25 clinical isolates. CONCLUSION: These result strongly suggest that the PCR-RFLP method using degenerate primers covering the HSP60 gene and Mse / digestion enzyme offer a convenient and accurate tool for species-specific identification of CNS.
Chaperonin 60* ; Coagulase* ; Digestion ; Heat-Shock Proteins* ; Hot Temperature* ; Humans ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Shock ; Staphylococcal Infections

Toxoplasma gondii can infect all the vertebrates including human, and leads to serious toxoplasmosis and considerable veterinary problems. T. gondii heat shock protein 60 (HSP60) is associated with the activation of antigen presenting cells by inducing initial immune responses and releasing inflammatory cytokines. It might be a potential DNA vaccine candidate for this parasite. A pVAX-HSP60 DNA vaccine was constructed and immune responses was evaluated in Kunming mice in this study. Our data indicated that the innate and adaptive immune responses was elicited by successive immunizations with pVAX-HSP60 DNA, showing apparent increases of CD3e+CD4+ and CD3e+CD8a+ T cells in spleen tissues of the HSP60 DNA-immunized mice (24.70±1.23% and 10.90±0.89%, P < 0.05) and higher levels of specific antibodies in sera. Furthermore, the survival period of the immunized mice (10.53±4.78 day) were significantly prolonged during the acute T. gondii infection. Decrease of brain cysts was significant in the experimental group during the chronic infection (P < 0.01). Taken together, TgHSP60 DNA can be as a vaccine candidate to prevent the acute and chronic T. gondii infections.
Animals ; Antibodies ; Antigen-Presenting Cells ; Brain ; Chaperonin 60 ; Cytokines ; DNA ; Humans ; Immunization ; Mice ; Parasites ; Spleen ; T-Lymphocytes ; Toxoplasma ; Toxoplasmosis ; Vertebrates

As thermostable enzymes and organisms are much more needed, researches on heat shock proteins(HSPs) of hyperthermophilic archaea have drawn more concerns. HSPs from hyperthermophilic archaea are concise only with HSP60, sHSP, prefoldin and AAA+proteins, but without HSP100s, HSP90s, HSP70 (DnaK), HSP40 (DnaJ) and GrpE which are common in mesophilic or thermophilic archaea. Accordingly, studies on the structure, function and operation mechanism of these four groups are much more important and meaningful. This review focuses on the recent progress in the researchs on the structure, function, operation mechanism and cooperation of the HSPs from hyperthermophilic archaea. The problems and obfuscations in these HSPs are analyzed, and farther research direction and key points are put out.
Archaea ; classification ; metabolism ; Archaeal Proteins ; metabolism ; Chaperonin 60 ; metabolism ; Heat-Shock Proteins ; genetics ; metabolism ; Molecular Chaperones ; metabolism

OBJECTIVETo identify the single nucleotide polymorphisms (SNPs) in the regulatory and coding regions of heat shock protein 60 gene and search for its genetic makers in Chinese Han people.

METHODSThe 5' flank region, parts of the exons and introns of hsp60 gene were resequenced to identify the SNPs in Chinese Han people, and then the sequenced results to the Japanese, European and African's data in National Center for Biotechnology Information (NCBI) and HapMap databases were compared.

RESULTSOne novel SNP was identified in exon 2 resulting in synonymous variant and the G allele frequency was 0.025. There were 11 reported SNPs in the sequenced region. The minor allele frequencies of rs1116734, rs3749095, rs1050347, rs8539 were 0.51, 0.30, 0.29, 0.49. The heterozygosity of the other 7 SNPs was 0. The distributions of rs1116734, rs1050347, rs8539, rs3749095 in Chinese Han people were similar to the Japanese's. The hsp60 rs3749095 which was not found in Japanese people was a high-frequency SNP in Chinese Han people; the distribution of rs8539 in Chinese Han people was quite different from European and African's (P < 0.01).

CONCLUSIONThe SNPs of hsp60 in Chinese Han people are different from the other peoples. The SNPs of hsp60 gene rs1116734, rs3749095, rs1050347, rs8539 are very common in Chinese Han people and might be used for candidate genetic markers of hsp60 gene.

Adolescent ; Adult ; Alleles ; Chaperonin 60 ; genetics ; China ; ethnology ; Exons ; genetics ; Female ; Gene Frequency ; Humans ; Male ; Polymorphism, Single Nucleotide

OBJECTIVE: To investigate the changes of creatine kinase-MB (CK-MB) and heat shock protein 60 (HSP 60) in rats without electric marks after electric injury, to identify the relationship of the CK-MB, HSP 60 and the time of electric injuries, and to evaluate the damage to cells after electric injury. METHODS: The animal model of electric injury without electric marks was established by alternating current (voltage 110 V). Automatic biochemistry analyzer was used to detect the serum CK-MB and immunohistochemical staining technology was used to analyze the tissues of myocardium and left lobe of liver. RESULTS: The amount of serum CK-MB was increased when the rats were injuried, and reached the peak at 30min. Then the amount of CK-MB began to decrease and showed a slight downward trend in 3-5 h after electric injury, and leveled off at 6 h. Immunohistochemistry staining also showed the changes of HSP 60 of rats' myocardial cells and hepatic cells regularly after electric injury. CONCLUSION The regular changes of serum CK-MB and tissular HSP 60 in rats can be used to diagnosis electric injury and assess the injury of internal organs after the electric injury without electric marks.
Animals ; Chaperonin 60/metabolism* ; Creatine Kinase, MB Form/metabolism* ; Electric Injuries/complications* ; Immunohistochemistry ; Liver/pathology* ; Myocardium/pathology* ; Rats

PURPOSE: Cervical cancer caused by the human papilloma virus (HPV) continues to be the cause of yearly death among women. However, it is a curable disease when diagnosed at an early stage. Recently, several researches have reported that heat shock protein (HSP) 60, a chaperone protein of molecular weight of 60 kDa, is involved in carcinogenesis and apoptosis. In order to evaluate the prognostic significance of HSP60 in cervical cancer, we examined differences in the HSP60 expression between cervical cancer and normal tissues in women. MATERIALS AND METHODS: Tissue samples were collected from 20 cervical cancer patients and 20 normal controls. HSP60 expression of cervical cancer and normal tissues were verified by the 2D gel proteomics, semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analyses. RESULTS: In 2D proteomic analysis, an increase of HSP60 expression was detected in cervical cancer tissues and confirmed by Western blot analysis (p < 0.05). However, messenger RNA (mRNA) levels of HSP60 did not display any significant differences between cervical cancer and normal tissues. CONCLUSION: These results suggest that HSP60 may be involved in the development of cervical cancer and have profound biological and prognostic significance.
Adult ; Aged ; Blotting, Western ; Chaperonin 60/*metabolism ; Electrophoresis, Gel, Two-Dimensional ; Female ; Humans ; Male ; Middle Aged ; Reverse Transcriptase Polymerase Chain Reaction ; Uterine Cervical Neoplasms/*metabolism

PURPOSE: The aim of this study was to define the immunoreactive specificity of Porphyromonas gingivalis (P. gingivalis) heat shock protein (HSP) 60 in periodontitis and atherosclerosis. METHODS: In an attempt to define the cross-reactive bacterial heat-shock protein with human self-antigen at molecular level, we have introduced a novel strategy for cloning hybridoma producing anti-P. gingivalis HSP 60 which is polyreactive to bacterial HSPs or to the human homolog. RESULTS: Five cross-reactive clones were obtained which recognized the #19 peptide (TLVVNRLRGSLKICAVKAPG) among 37 synthetic peptides (20-mer, 5 amino acids overlapping) spanning the whole molecule of P. gingivalis HSP 60. We have also established three anti-P. gingivalis HSP 60 monoclonal antibodies demonstrating mono-specificity. These clones recognized the #29 peptide (TVPGGGTTYIRAIAALEGLK). CONCLUSIONS: Peptide #19 and #29 of P. gingivalis HSP 60 might be important immunoreactive epitopes in the immunopathogenic mechanism of bacterial antigen-triggered autoimmune diseases.
Amino Acids ; Antibodies ; Antibodies, Monoclonal ; Autoimmune Diseases ; Chaperonin 60 ; Clone Cells ; Cloning, Organism ; Epitopes ; Heat-Shock Proteins ; Humans ; Hybridomas ; Peptides ; Periodontitis ; Porphyromonas ; Porphyromonas gingivalis ; Sensitivity and Specificity

Deer serve as reservoirs of tick-borne pathogens that impact on medical and veterinary health worldwide. In the Republic of Korea, the population of Korean water deer (KWD, Hydropotes inermis argyropus) has greatly increased from 1982 to 2011, in part, as a result of reforestation programs established following the Korean War when much of the land was barren of trees. Eighty seven Haemaphysalis flava, 228 Haemaphysalis longicornis, 8 Ixodes nipponensis, and 40 Ixodes persulcatus (21 larvae, 114 nymphs, and 228 adults) were collected from 27 out of 70 KWD. A total of 89/363 ticks (266 pools, 24.5% minimum infection rate) and 5 (1.4%) fed ticks were positive for Anaplasma phagocytophilum using nested PCR targeting the 16S rRNA and groEL genes, respectively. The 16S rRNA gene fragment sequences of 88/89 (98.9%) of positive samples for A. phagocytophilum corresponded to previously described gene sequences from KWD spleen tissues. The 16S rRNA gene fragment sequences of 20/363 (5.5%) of the ticks were positive for A. bovis and were identical to previously reported sequences. Using the ITS specific nested PCR, 11/363 (3.0%) of the ticks were positive for Bartonella spp. This is the first report of Anaplasma and Bartonella spp. detected in ticks collected from KWD, suggesting that ticks are vectors of Anaplasma and Bartonella spp. between reservoir hosts in natural surroundings.
Anaplasma/genetics/*physiology ; Animals ; Arachnid Vectors/microbiology ; Bartonella/genetics/*physiology ; Chaperonin 60/genetics ; Deer/parasitology ; Disease Reservoirs/veterinary ; RNA, Ribosomal, 16S/genetics ; Republic of Korea/epidemiology ; Ticks/*microbiology

OBJECTIVETo construct a prokaryotic expression system of groEL gene of Leptospira interrogans serogroup Icterohaemorrhagia serovar Lai strain Lai, and to determine the immunoprotective effect of recombinant GroEL protein (rGroEL) in LVG hamsters.

METHODSThe groEL gene was amplified by high fidelity PCR and the amplification products were then sequenced. A prokaryotic expression system of groEL gene was constructed using routine genetic engineering technique. SDS-PAGE plus Bio-Rad Gel Image Analyzer was applied to examine the expression and dissolubility of rGroEL protein while Ni-NTA affinity chromatography was used to extract the expressed rGroEL. The immunoprotective rate in rGroEL-immunized LVG hamsters was determined after challenge with L.interrogans strain Lai. The cross agglutination titers of sera from immunized hamsters with different L.interrogans serogroups were detected using MAT.

RESULTSThe nucleotide and amino acid sequences of the cloned groEL gene were the same as those reported in GenBank. The constructed prokaryotic expression system of groEL gene expressed soluble rGroEL. The immunoprotective rates of 100 and 200 μg rGroEL in LVG hamsters were 50.0 % and 75.0%, respectively. The sera from the rGroEL-immunized LVG hamsters agglutinated all the L.interrogans serogroups tested with different levels.

CONCLUSIONThe GroEL protein is a genus-specific immunoprotective antigen of L.interrogans and can be used to develop an universal genetically engineering vaccine of Leptospira.

Agglutination Tests ; Animals ; Antigens, Bacterial ; immunology ; Chaperonin 60 ; genetics ; immunology ; Cricetinae ; Gene Expression ; Leptospira interrogans ; genetics ; immunology ; Recombinant Proteins ; genetics ; immunology

OBJECTIVETo determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism.

METHODSOmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays.

RESULTSThe bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P <0.01), whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated (P<0.01). The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes (P <0.01). Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes (P <0.01).

CONCLUSIONExpression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Antigens, Bacterial ; genetics ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; metabolism ; Cell Line ; Chaperonin 60 ; genetics ; metabolism ; Humans ; Leptospira interrogans ; genetics ; immunology ; pathogenicity ; Lipoproteins ; genetics ; metabolism ; Macrophages ; microbiology