Involvement of geranylgeranylation of Rho and Rac GTPases in adipogenic and RANKL expression, which was inhibited by simvastatin.

Simvastatin suppresses myoblast differentiation via inhibition of Rac GTPase, which is involved in the mevalonic acid pathway that produces cholesterol. Statins also inhibit adipogenic differentiation and receptor activator of NFκB ligand (RANKL) expression, possibly through the mevalonic acid pathway, although the involvement of that pathway and effector proteins in these cellular events has not been fully clarified. In the present study, we aimed to elucidate the mechanism of the effects of simvastatin on adipogenic differentiation and calcitriol-induced RANKL expression in bone marrow stromal ST2 cells. Adipogenesis and mRNA up-regulation of peroxisome proliferator-activated receptor γ and adipocyte fatty acid-binding protein were induced by troglitazone, and those events were efficiently inhibited by simvastatin. In addition, RANKL expression induced by calcitriol was abrogated by simvastatin in ST2 cells. The inhibitory effects of simvastatin were adequately compensated by the addition of either mevalonic acid or an intermediate of the mevalonic acid pathway, geranylgeranyl pyrophosphate, but not by another intermediate, farnesyl pyrophosphate. These findings suggest that protein geranylgeranylation is related to cellular differentiation in those two directions. Furthermore, inhibitor analysis demonstrated that Rac GTPase is involved in adipogenic differentiation, whereas Rho GTPase was found to be involved in RANKL expression. Taken together, the present findings suggest that geranylgeranylation of Rho family GTPase is involved in both adipogenesis and RANKL expression of stromal cells, while Rac GTPase is involved in adipogenesis and Rho GTPase in RANKL expression.

Propeptide processing and proteolytic activity of proenzymes of the staphylococcal and enterococcal GluV8-family protease.

Proenzymes with various lengths of propeptides have been observed in GluV8 from Staphylococcus aureus and GluSE from S. epidermidis. However, the production mechanism of these proenzymes and roles of truncated propeptides have yet to be elucidated. Here we demonstrate that shortening of propeptide commonly occurs in an auto-catalytic manner in GluV8-family members, including those from coagulase negative Staphylococci and Enterococcus faecalis. Accompanied with propeptide shortening, the pro-mature junction (Asn/Ser_1-Val1) becomes more susceptible towards the hetero-catalytic maturation enzymes. The auto-catalytic propeptide truncation is not observed in Ser169Ala inert molecules of GluV8-family members. A faint proteolytic activity of proenzymes from Staphylococcus caprae and E. faecalis is detected. In addition, proteolytic activity of proenzyme of GluV8 carrying Arg-3AlaAsn.1 is demonstrated with synthetic peptide substrates LLE/Q-MCA. These results suggest that GluV8-family proenzymes with shortened propeptides intrinsically possess proteolytic activity and are involved in the propeptide shortening that facilitates the final hetero-catalytic maturation.

Relationship of quantitative salivary levels of Streptococcus mutans and S. sobrinus in mothers to caries status and colonization of mutans streptococci in plaque in their 2.5-year-old children.

OBJECTIVES: The aim of this study was to assess the relationships of quantitative salivary levels of Streptococcus mutans and S. sobrinus in mothers with the colonization of mutans streptococci (MS) in plaque and caries status in their 2.5-year-old children. Furthermore, the dynamics of caries status in the children was evaluated in a 2-year follow-up survey. METHODS: After oral examination of 54 mother-and-child pairs, the saliva samples from the mothers and the plaque samples from the children were collected. The levels (log DNA copies/ml saliva) of S. mutans and S. sobrinus were quantified using real-time polymerase chain reaction (PCR) assays, while MS in the plaque samples were detected using a cultivation method. In addition, 50 of the 54 children participated in a 2-year follow-up survey of caries prevalence. RESULTS: In the 2.5-year-old children, the percentage of dft-positive subjects and mean number of dft were significantly higher in the MS(+) group when compared with the MS(-) group. Findings from the 2-year follow-up survey indicated that MS(+) subjects had a persistently higher mean number of dft at 4.5 years. The 2.5-year-old children were divided into three groups based on the quantitative levels of salivary S. mutans and S. sobrinus in their mothers: those whose mothers had low levels of S. mutans (<4 log DNA copies/ml) and S. sobrinus (<2) (group 1); those whose mothers had a high level of S. mutans (> or = 4) and low level of S. sobrinus (<2) (group 2); and those whose mothers had high levels of both (> or = 4 and > or = 2, respectively) (group 3). Among the three groups, the percentages of MS(+) and dft-positive children were highest in group 3 and lowest in group 1. Furthermore, multiple logistic regression analyses revealed that grouping the mothers based on salivary level of S. mutans and S. sobrinus was an efficient means to predict both MS colonization (OR = 2.96) and prevalence of dental caries (OR = 9.39) in children at 2.5 years of age. CONCLUSIONS: In the 54 mother-and-child pairs tested, the maternal salivary levels of S. mutans and S. sobrinus determined by real-time PCR were significantly related to MS colonization in plaque as well as dental caries in their children at 2.5 years of age. Thus, determination of maternal levels of both organisms using the present cut-off values is proposed as an efficient method to indicate the risks of maternal transmission of MS and childhood dental caries.

Occurrence of staphylococci in the oral cavities of healthy adults and nasal oral trafficking of the bacteria.

To investigate a possible peroral route of infective endocarditis (IE), the occurrence of staphylococci in the oral cavity was examined using saliva and supragingival plaque specimens from 56 systemically and periodontally healthy adults aged 22-43 years old (27.1+/-5.3). Nine Staphylococcus species and 334 isolates were identified. In saliva, the total occurrence rate was 83.9 % and the total number of bacteria was 10(2)-10(4) c.f.u. ml(-1). Staphylococcus aureus was the most frequent species (46.4 %), followed by Staphylococcus epidermidis (41.1 %) and others (Staphylococcus hominis, Staphylococcus warneri, Staphylococcus intermedius, Staphylococcus capitis, Staphylococcus haemolyticus, Staphylococcus lugdunensis and Staphylococcus gallinarum, isolation frequencies ranging in order from 12.5 to 1.8 %). A similar isolation tendency was observed in supragingival plaque, with a total occurrence rate of 73.2 % and amounts of bacteria ranging from 10(2) to 10(5) c.f.u. g(-1). Four common Staphylococcus species (S. aureus, S. epidermidis, S. lugdunensis and S. hominis) were isolated from nasal swab samples taken from the oral staphylococci-positive subjects. Genotyping of all 18 combinations of oral- and nasal-derived isolates by PFGE indicated that identical clones or close relatives were commonly distributed in these two cavities. Since the provision of micro-organisms from the nasal cavity was shown and occurrence rates in the oral cavity were adequate, these results suggest a possible peroral route of staphylococcal IE, as in cases of viridans streptococcal IE.

Streptococcus anginosus infection in oral cancer and its infection route.

OBJECTIVE: To elucidate a possible involvement of Streptococcus anginosus in oral cancer, we assessed the frequency of S. anginosus infection in oral cancer tissues, and investigated its infection route. MATERIALS AND METHOD: The tissue specimens were obtained from 46 oral cancer and three precancerous leukoplakia subjects. Frequency of S. anginosus infection was assessed by a species-specific polymerase chain reaction (PCR) assay. The genotype of the clinical isolates taken from cancer tissue and dental plaque samples was analyzed using pulsed-field gel electrophoresis (PFGE). RESULTS: S. anginosus DNA was frequently detected in squamous cell carcinoma (19/42), but not in other types of cancer (lymphoma and rhabdomyosarcoma) or leukoplakia samples. A subject-based analysis revealed that S. anginosus was solely detected in dental plaque and not in saliva from all 19 S. anginosus-positive squamous cell carcinoma cases. Further, the genotype of S. anginosus isolated from cancer tissue was identical to that from dental plaque of the same patients. CONCLUSION: Infection of S. anginosus could occur frequently in oral squamous cell carcinoma and that dental plaque could be a dominant reservoir of the S. anginosus.

PCR-based identification of Staphylococcus epidermidis targeting gseA encoding the glutamic-acid-specific protease.

The frequency of the gseA gene encoding a glutamic acid-specific serine protease, GluSE, of Staphylococcus epidermidis was investigated. DNA hybridization analysis demonstrated that gseA existed exclusively in S. epidermidis but not in other bacteria examined. A single step PCR assay with a set of designed primers yielded amplification of gseA from all 69 clinical isolates of S. epidermidis taken from patients and healthy adults, whereas production of GluSE was observed in 74% (51/69) of the isolates. Furthermore, none of the 46 clinical isolates of other species of coagulase-negative staphylococci and 45 clinical isolates of Staphylococcus aureus showed amplification, except a Staphylococcus capitis strain. However, this strain was positive for a S. epidermidis-specific DNA region and the DNA sequence of the 16S rRNA gene showed 99% identity with that of S. epidermidis. Therefore, these results indicated that the present PCR assay for gseA was ubiquitous and highly specific for detection of S. epidermidis.

Identification of a new variant of fimA gene of Porphyromonas gingivalis and its distribution in adults and disabled populations with periodontitis.

Porphyromonas gingivalis fimbriae are critical for the promotion of bacterial infection. The fimA gene encoding fimbrillin, a subunit of fimbriae, has been classified into five genotypes (types I to V) based on their nucleotide sequences. Using a fimA type-specific PCR assay, our previous study demonstrated a close relationship between P. gingivalis possessing type II and type IV fimA genes and adult periodontitis. In that study, some clinical specimens were found to be positive for both types I- and II- fimA specific primers, likely due to the coexistence of two clonal types or a single clone of an unknown genotype in the samples. In the present study, we cloned a new variant of the fimA gene, designated as type Ib fimA, from P. gingivalis HG1691. The nucleotide sequence of the cloned fimA gene showed a 97.1% homology with that of type I fimA, indicating it as a clonal variant of type I fimA. Organisms with type Ib fimA were detected in 13.5% of periodontitis patients and in 2.9% of periodontal healthy adults. Statistical analysis revealed a strong relationship between periodontitis and specific fimA types such as type Ib [odds ratio (OR) 6.51], type II (OR 77.8), and type IV (OR 7.54). Moreover, type Ib fimA-organisms were also found to be related to periodontitis in Down's syndrome (OR 1.91) and mentally disabled populations (OR 4.00). These findings suggest that P. gingivalis with type Ib fimA is closely associated with the progression of periodontitis, similar to organisms with type II and IV fimA.

Antigenic characterisation of a novel Streptococcus anginosus antigen that induces nitric oxide synthesis by murine peritoneal exudate cells.

A novel antigen that induces nitric oxide (NO) synthesis by murine peritoneal exudate cells (PEC) was prepared from a culture supernate of Streptococcus anginosus NCTC 10713 in dialysed medium by column chromatography with DEAE-Sephacel followed by size-exclusion high performance liquid chromatography (HPLC). A chemical analysis of the S. anginosus antigen (SAA) revealed that it mainly consisted of carbohydrates (rhamnose, N-acetylglucosamine, glucose and galactose), smaller quantities of protein and a trace amount of phosphorus. The SAA stimulated PEC from C57BL/6N mice to produce NO and accumulate induced NO synthetase (iNOS) mRNA in a dose-dependent manner, reaching a plateau with 10-30 microg/ml. Furthermore, a reverse transcription-PCR assay revealed that SAA 10 microg/ml could induce mRNA accumulation of tumour necrosis factor-alpha, interleukin (IL)-1beta and IL-6 as well as iNOS. In contrast, Rantz-Randall antigen (RRA), a carbohydrate antigen prepared from the organisms, could not induce NO synthesis or cause the accumulation of iNOS mRNA, although cytokine production was observed after stimulation. The SAA-induced NO synthesis, but not the cytokine production, was sensitive to heat. Furthermore, an immunoblot analysis of SAA indicated that the 43-kDa protein band reacted with anti-SAA but not anti-RRA antibodies. In immunodiffusion, SAA reacted with both anti-SAA and anti-RRA antibodies, and the precipitin bands formed crossing lines, suggesting that SAA could possess two different antigenic components--one that reacts specificially with anti-SAA antibodies and another that has an identity similar to that of RRA. Taken together, SAA, a novel antigen of S. anginosus, was found to induce NO synthesis as well as produce inflammatory cytokines in murine PEC. It is suggested that the protein molecule of SAA may exclusively induce NO synthesis, and its carbohydrate component(s) could have a relationship to cytokine production.

Establishment of gingival epithelial cell lines from transgenic mice harboring temperature sensitive simian virus 40 large T-antigen gene.

We established two gingival epithelial cell lines (GE1 and GE6), originating from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene. GE1 and GE6 grew at a permissive temperature (33 degrees C) in a pavement arrangement and solely formed multilayers that exhibited morphological features similar to those of the stratified oral epithelium, with neither the use of stromal equivalents nor feeder layers. Both GE cells underwent apoptosis at a non-permissive temperature (39 degrees C). Characteristic keratin peptides, keratin 4 and 13, for mucosal epithelium were obviously expressed in the suprabasal cells, and keratohyalin granules and involucrin were present in the surface flat cells in the multilayered culture. Keratin 10 (one of the markers for higher keratinized gingival epithelium) was rarely found in some uppermost cells, and filaggrin (a component of keratohyalin granules) appeared sparsely in uppermost desquamating cells in the older cultures. These observations indicated that GE1 and GE6 cells exhibited the phenotype characterizing nonkeratinized sulcular epithelium, which possessed the potency undergoing keratinization in such highly stratified cultures as oral gingival epithelium. GE cells increased the expression levels of mRNA of interleukin-1beta and tumor necrosis factor alpha by the stimulation of lipopolysaccharide and extracellular substances of oral streptococci. The GE cell lines thus could serve as an excellent experimental system for further studies on the physiology of gingival epithelium and corresponding diseases, such as periodontal disease, epithelial hyperplasia, and gingival tumors.

The immunohistochemical localization of Fas and Fas ligand in jaw bone and tooth germ of human fetuses.

The cellular localization and roles of bone morphogenetic protein (BMP)-2 and apoptosis-associating factors in human orofacial development remain unclear. In this study, BMP-2, osteocalcin, and TGF-beta, which are bone-differentiating markers, apoptosis-associating factors (i.e., Bcl-2, Bax, Fas, and Fas ligand), apoptotic cells detected by the in situ 3'-end labeling method (TUNEL), and proliferating cell nuclear antigen (PCNA) were immunohistochemically examined in the heads (in particular, the jaw bone and tooth germs) of human fetuses of 11-week pregnancy. BMP-2 was positive in osteoblasts and newly formed osteoid of the incisive and palatal bone of the maxilla and the mandible, which indicated that BMP-2 was exclusively involved in intramembranous ossification in the human fetal head. Fas was positive in the cytoplasm of osteocytes and a few osteoblasts. In contrast, Fas ligand was positive in the cytoplasm of osteoblasts and abundant in the stroma of the osteoblastic layer, periosteum, and perichondrium. The Fas ligand in the stroma was recognized as the soluble form, which was possibly produced by osteoblasts. TUNEL-positive apoptotic cells were found in a few osteocytes and a few osteoblastic cells in new bone, and in monocytes of degenerate Meckel's cartilage. The induction of apoptosis observed in monocytes seems to be caused via a Fas-Fas ligand cell death system, because some of these monocytes were Fas-positive, and most of them were Fas ligand-positive. Interestingly, the abundant soluble Fas ligand observed in the periosteum probably protects the bone-formative zone from the invasion of the activated lymphocytes by binding to Fas expressing in these lymphocytes and killing these cells. Fas and Fas ligand were focally positive in the dental lamina and inner enamel epithelium and cusps of the enamel organ, nevertheless, the presence of TUNEL-positive cells was very rare. Bcl-2 was clearly and Bax was weakly positive in the cells throughout the dental lamina and enamel organ. These findings indicated that Fas-mediated apoptosis was inhibited by the Bcl-2 family in the development of teeth.

Potential of Escherichia coli GTP cyclohydrolase II for hydrolyzing 8-oxo-dGTP, a mutagenic substrate for DNA synthesis.

MutT protein of Escherichia coli prevents the occurrence of A:T --> C:G transversion by hydrolyzing an oxidized form of dGTP, 8-oxo-7, 8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), which is produced by active oxygen species. In a search for mutT-related genes, we found that the ribA gene, encoding GTP cyclohydrolase II, is able to reduce the increased level of mutation frequency of the mutT strain to almost the normal level, provided that the gene product is overproduced. Purified preparations of Escherichia coli GTP cyclohydrolase II protein as well as the histidine hexamer-tagged recombinant GTP cyclohydrolase II protein efficiently hydrolyze 8-oxo-dGTP and 8-oxo-GTP, producing 8-oxo-dGMP and 8-oxo-GMP, respectively. dGTP was not hydrolyzed by these preparations. GTP cyclohydrolase II catalyzes conversion of GTP to 2, 5-diamino-6-hydroxy-4-(5-phosphoribosylamino)-pyrimidine, which constitutes the first step for riboflavin synthesis. The Km values for the three types of guanine nucleotides, GTP, 8-oxo-GTP, and 8-oxo-dGTP, were almost the same. In the mutT- background, ribA- cells showed higher spontaneous mutation frequencies as compared with that of ribA+ cells. Thus, GTP cyclohydrolase II, the ribA gene product, has a potential to protect genetic material from the untoward effects of endogenous oxygen radicals.

Identification of Abiotrophia adiacens and Abiotrophia defectiva by 16S rRNA gene PCR and restriction fragment length polymorphism analysis.

Abiotrophia adiacens and Abiotrophia defectiva, previously referred to as nutritionally variant streptococci, Streptococcus adjacens and Streptococcus defectivus, respectively, are causes of infective endocarditis. We describe a method of identifying these two species and also of distinguishing them from 15 other major etiological pathogens of infective endocarditis by means of 16S rRNA gene PCR amplification followed by restriction fragment length polymorphism analysis (PCR-RFLP). The 16S rRNA genes were successfully amplified with a set of universal primers from all 17 species of bacteria examined, including viridans group streptococci. The RFLP patterns of A. adiacens and A. defectiva obtained by HaeIII or MspI digestion were readily distinguished from each other and from those of other bacteria. When PCR analysis was performed with the supernatant of a suspension of a boiled colony, the 16S rRNA genes of 80 of 82 isolates (97%) of A. adiacens and all isolates (11 of 11) of A. defectiva were amplified. The HaeIII RFLP patterns of the isolates were the same as those of the corresponding type strains, although 28% of A. adiacens isolates revealed intraspecies polymorphism. The detection limit of this method was 0.1 pg of genomic DNA, as assessed by using the digoxigenin-labeling DNA detection system. Thus, the PCR-RFLP analysis that we developed is applicable for the routine detection of Abiotrophia from clinical specimens.

Expression of bone morphogenetic proteins of human neoplastic epithelial cells.

Bone morphogenetic proteins (BMPs) are crucial factors of osteogenesis. We investigated the expressions of BMP subtypes in human salivary adenocarcinoma cell line (HSG-S8), tongue squamous cell (HSC-4) and gingival squamous cell (Ca9-22) carcinoma cell lines, gastric poorly differentiated adenocarcinoma cell (MNK45) and signet ring cell (KATOIII) carcinoma cell lines, rectal adenocarcinoma (RCM-1, RCM-2, and RCM-3), and thyroid (8505C) and bladder (T24) carcinoma cell lines by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR disclosed that BMP-1 was expressed in all cell lines examined, and BMP-2 was amplified in almost all cells except MKN45. Two squamous cell carcinomas, HSC-4 and Ca9-22, and KATOIII expressed only BMP-1 and BMP-2. MKN45 did not express BMP-2, but expressed BMP-7 and weakly BMP-4 and BMP-5. In addition to the expression BMP-7, and HSG-S8 expressed BMP-6. These findings indicated that the neoplastic epithelial cells possessed a rather great potency to express BMP mRNAs. On the other hand, among these carcinoma cells, HSG-S8 solely induced bone in nude mouse tumors, and HSC-4 and KATOIII contained many calcified masses in tumors while the rest did not induce either.

[Genotyping of clinical isolates of MRSA by PFGE and AP-PCR].

Genotyping of 67 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) separated from patients in a hospital in Mizusawa City in 1994 and 1995 was studied by pulsed field gel electrophoresis (PFGE) and arbitrarily primed-polymerase chain reaction (AP-PCR). Two main genotypes were observed by PFGE, and more than 70% of the 67 MRSA isolates produced coagulase type II. One group diverged well and gained higher tolerance in 1994, but was not isolated in 1995. The other group was continually isolated during the two-year period and showed moderate tolerance in 1994, and higher tolerance in 1995. AP-PCR was able to classify the genotypes of MRSA into 6 subgenotypes under the present conditions, which supported the results obtained by PFGE. These results suggest that AP-PCR could become a convenient and useful typing method by improving both sequence and length of a primer.

[Genotype analysis of enterohemorrhagic Escherichia coli O157:H7 strains isolated in Iwate Prefecture from 1996 to February 1997].

Genotypes of 38 isolates of enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolated from 11 sporadic cases and one outbreak in Iwate Prefecture from 1996 to February 1997 were studied by pulsed-field gel electrophoresis (PFGE), in comparison with a strain of EHEC O157:H7 isolated in 1992 in Ohazama-Cho, Iwate Prefecture, and two isolates of EHEC O157:NM. In order to substantiate the genotypes classified by PFGE, Southern blotting was performed to investigate integration sites of the Shiga toxin genes (stx) in the XbaI-digested genome DNA fragments. The stx1 gene existed on an approximately 130 kb fragment in all isolates except two ones. On the other hand, the stx2 gene was observed on 11 DNA fragments in different length from 600 kb to 155 kb, indicating that the stx2 gene integrates into more heterogeneous sites of genome DNA than stx1 does. From these analyses, EHEC O157:H7 isolates examined were classified into 7 genotypes. Since half of the isolates were the same genotype as that of the isolate in 1992, it is suggested that this type of EHEC O157:H7 strain is expanding from Ohazama-Cho and Morioka City in Iwate Prefecture.

Expression of T-cell receptor V beta 2 and type 1 helper T-cell-related cytokine mRNA in streptococcal pyrogenic exotoxin-C-activated human peripheral blood mononuclear cells.

Streptococcal pyrogenic exotoxin type C (SPE C) is a member of the bacterial superantigens that are potent stimulants of T cells. We expressed SPE C in Escherichia coli and characterized its selective stimulation properties on human T cells bearing specific V beta chains of T-cell receptors (TCRs). Cytokine profiles induced by SPE C were also examined. Recombinant SPE C significantly enhanced proliferation of human peripheral blood mononuclear cells (PBMCs) at concentrations as low as 10(-12)-10(-14)M. Reverse transcription of RNA, from SPE-C-stimulated PBMCs followed by polymerase chain reaction, revealed selective induction of TCR V beta 2 chain expression. SPE C raised the mRNA level of type 1 helper T cell (TH1) related cytokines, such as interferon gamma (IFN-gamma), interleukin 2 (IL-2), and tumor necrosis factor beta (TNF beta). The expression of TNF alpha was also increased. In contrast, the increase in mRNA levels of the p35 small fragment of IL-12 and type 2 helper T cell (TH2) related cytokines (i.e., IL-4 and IL-10) was not significantly affected by SPE C. The mRNA level of proinflammatory cytokine IL-6 was increased marginally. Consistent with the mRNA accumulation, protein concentrations of IFN gamma, IL-2, and TNF were increased in SPE-C-stimulated PBMCs, but IL-4 was not. From these results, we conclude that the stimuli of SPE C preferentially causes the TH1 responses in human T cells bearing TCR V beta 2.

Retinoic acid enhances expression of bone morphogenetic protein-2 in human adenocarcinoma cell line (HSG-S8).

Expression of bone morphogenetic protein (BMP)-2 mRNA was stimulated by retinoic acid in human adenocarcinoma cell line, HSG-S8, in a dose-dependent manner. Northern blot analysis demonstrated that retinoic acid most strongly increased the level of BMP-2 mRNA 6 h after the treatment and the stimulatory effect was maintained at 48 h. The mature peptides of 16 and 18 kDa molecular masses of BMP-2 were also increased in the conditioned medium by the treatment of retinoic acid on western blotting. The proliferation of HSG-S8 cells was inhibited by retinoic acid, however, retinoic acid did not cause morphological change showing cellular differentiation. 1 alpha, 25(OH)2D3, like retinoic acid, clearly increased the mRNA level of BMP-2, whereas dibuthryl cyclic AMP remarkably diminished it, and bromodeoxyuridine had no effect on the expression of BMP-2 mRNA.

Mechanism of dimer formation of the 90-kDa heat-shock protein.

This study describes the mechanism of homodimer formation of the 90-kDa heat-shock protein (HSP90). In eukaryotic cells, there are two HSP90 isoforms, alpha and beta, encoded by two separate genes. HSP90 alpha exists predominantly as a homodimer, HSP90 beta mainly as a monomer. Analysis by native PAGE revealed that bacterially expressed HSP90 alpha fused to glutathione S-transferase (GST) existed as a high-molecular-mass oligomer, and was converted to a homodimer following removal of the fusion enzyme by thrombin cleavage. A deletion mutant, HSP90 alpha D44-603, formed a monomer and an N-terminal truncated mutant, HSP90 alpha 533-732, existed as a dimer, indicating that the dimer-forming ability resides somewhere in the C-terminal 200 amino acids. Limited proteolysis of the C-terminal 200 amino acids of HSP90 alpha with chymotrypsin produced the C-terminal 16-kDa fragment (Met628/Ala629-Asp732) and its adjacent more N-terminal 13-kDa fragment (Val542-Tyr627/Met628). Size-exclusion HPLC and two-dimensional PAGE analyses demonstrated that these two chymotryptic fragments bound each other. The C-terminal 198 amino acids as well as the full-length form of HSP90 beta revealed a lower dimer-forming activity than HSP90 alpha. Expression of the chimeric proteins at the C-terminal 198 amino acids of the alpha and beta isoforms further indicated that the 16 amino acid substitutions locating between amino acids 561 and 685 account for the impeded dimerization of HSP90 beta. A leucine zipper motif (Met402-Leu423) was unlikely to be involved in the dimer formation. Taken together, these results indicate that the dimeric structure of HSP90 alpha is mediated by the C-terminal 191 amino acids and consists of duplicate interactions of the C-terminal region (Met628/Ala629-Asp732) of one subunit and the adjacent more N-terminal region (Val542-Try627/Met628) of the other subunit.

Cysteine protease activity of streptococcal pyrogenic exotoxin B.

Streptococcal pyrogenic exotoxin B (SPE B) was purified and its protease and mitogenic activities were investigated. The apparent molecular mass of SPE B purified in the presence of iodoacetic acid was 42 kDa, whereas 29-kDa SPE B was predominant without the reagent. A polyclonal antibody raised against the 29-kDa species reacted with both species, indicating that the 42-kDa species was a precursor of the 29-kDa entity. Both the 42- and 29-kDa species enhanced [3H]thymidine incorporation into human peripheral blood mononuclear cells, whereas neither had any effect on T cell depleted mononuclear cells. The 29-kDa SPE B possessed caseinolytic activity, with an optimal pH of 8, and the activity was specifically suppressed by the antibody. A group of cysteine protease inhibitors, but no serine-, metallo-, or acidic-protease inhibitors, limited the protease activity, whereas dithiothreitol increased the activity. The DNA sequence around a putative active cysteine residue was identical among the speB genes from Streptococcus pyogenes R70, NY-5, and T19. Taken together, these results indicate that SPE B is identical to a cysteine protease, streptopain (EC 3.4.22.10).