Hydrogen peroxide induces expression and activation of AMP-activated protein kinase in a dental pulp cell line.

AIM: To investigate the effects of hydrogen peroxide on cell viability and expression and activation of AMP-activated protein kinase (AMPK) in rat dental pulp cell line RPC-C2A. METHODOLOGY: RPC-C2A cells derived from rat dental pulp were maintained in MEM supplemented with 10% FBS at 37 degrees C, in a humidified atmosphere at 5% CO(2). Cells were cultured in the presence or absence of H(2)O(2) for up to 60 min at concentrations of from 0.1 to 3.0 mmol L(-1). Cell viability was analysed by WST-1 reduction assay. Expression of AMPK subunit isoforms was analysed by Western blotting using antibodies to the catalytic alpha1 and regulatory beta1 and gamma1 subunit isoforms. The effect of silencing AMPKalpha1 on cell viability was determined using siRNA. RESULTS: Exposure to H(2)O(2) decreased cell viability in a time- and dose-dependent manner. The catalytic AMPKalpha1 subunit and its activated form, phospho-AMPKalpha, increased with exposure to H(2)O(2) in a time- and dose-dependent manner, whereas the regulatory beta1 and gamma1 subunits showed no change. Downregulation of AMPKalpha1 resulted in a reduction in cell viability in H(2)O(2)-treated cells at a concentration of 0.1 mmol L(-1) for 30 min incubation, indicating an increased sensitivity to H(2)O(2). CONCLUSIONS: Reactive oxygen induced energy fuel gauge enzyme AMPKalpha expression and its activation by phosphorylation in RPC-C2A cells, suggesting that AMPK is essential for protection against H(2)O(2)-induced nonapoptotic cell death. Therefore, AMPK may be a therapeutic modulation target for treatment of the dentine-pulp complex injured by reactive oxygen.

Hypoxia induces expression and activation of AMPK in rat dental pulp cells.

AMP-activated protein kinase (AMPK) is a stress-responsive enzyme involved in cell adaptation to an energy crisis. We hypothesized that hypoxia suppresses oxidative phosphorylation and ATP production, resulting in AMPK activation to protect cells. We investigated the effects of hypoxia on cell proliferation, the expression of AMPK and hypoxia-inducible factor 1alpha (HIF-1alpha), the activation of AMPK, and the relationship between AMPK and HIF-1alpha expression in rat dental pulp RPC-C2A cells. AMPK in the cells was composed of catalytic alpha1, and regulatory beta1 and gamma1 subunit isoforms. Cell proliferation was initially suppressed under hypoxia, but it increased thereafter, together with an increase in the expression of AMPK and HIF-1alpha, and the activation of AMPK. Down-regulation of AMPKalpha1 by siRNA inhibited cell proliferation under both normoxia and hypoxia, revealing that AMPK induction and activation were required for cell proliferation, although HIF-1alpha expression under hypoxia was not affected.

One of two gbpC gene homologues is involved in dextran-dependent aggregation of Streptococcus sobrinus.

INTRODUCTION: Streptococcus sobrinus exhibits marked dextran-dependent aggregation mediated by glucan-binding proteins (GBPs). In contrast to Streptococcus mutans, in which the gbpC gene responsible for dextran-dependent aggregation of this organism has been characterized, genes encoding the S. sobrinus GBPs have not yet been identified. METHODS: Recently, we identified the gbpC gene homologue from Streptococcus macacae using polymerase chain reaction primers based on the conserved regions of the gbpC sequence exhibiting intraspecies variations. This method was applied to amplify a S. sobrinus homologue. RESULTS: Unexpectedly, two gbpC gene homologues were identified in S. sobrinus strain 100-4. One homologue, named gbpC, was more similar to the S. mutans gbpC gene than the other and was approximately half the molecular size of its homologue with similar regions interrupted by several non-similar stretches. However, the dextran-binding activity of the protein expressed from gbpC in Escherichia coli was not detected in contrast to the other homologue, a protein designated as Dbl, expressing this activity. The gbpC gene was shown to be intact on the chromosome of strain OMZ176, which does not exhibit dextran-dependent aggregation, while the dbl gene of this strain contained a single adenine nucleotide insertion at approximately one-third the distance from the 5'-end. The insertion mutation in the dbl gene resulted in translation of a premature protein missing its LPXTG sequence signature sequence of the wall-anchored proteins. CONCLUSION: These results suggest that the dbl gene is very likely responsible for S. sobrinus dextran-dependent aggregation.

Identification of a glucan-binding protein C gene homologue in Streptococcus macacae.

BACKGROUND/AIMS: The past few decades have seen the isolation of certain glucosyltransferases and a number of proteins from mutans streptococci. Some of these proteins have been shown to possess glucan-binding capabilities which confer an important virulence property on mutans streptococci for the role of these bacteria play in dental caries. Among these proteins is glucan-binding protein C, which is encoded by the gbpC gene, and which we have identified as being involved in the dextran-dependent aggregation of Streptococcus mutans. However, gbpC homologues have yet to be identified in other mutans streptococci. METHODS: We carried out polymerase chain reaction amplification of Streptococcus macacae using primers that were designed based on conserved sequences of S. mutans gbpC and identified a gbpC gene homologue. The gene of that homologue was then characterized. RESULTS: Nucleotide sequencing of the S. macacae gbpC homologue revealed a 1854 bp open reading frame encoding a protein with an N-terminal signal peptide. The molecular mass of the processed protein was calculated to be 67 kDa. We also found an LPxTG motif, the consensus sequence for gram-positive cocci cell wall-anchored surface proteins, which was followed by a characteristic sequence at the carboxal terminal region of the putative protein. This suggests that the S. macacae GbpC homologue protein was tethered to the cell wall. CONCLUSION: Based on these results, together with the demonstrated glucan-binding ability of the S. macacae GbpC homologue protein, we suggest that S. macacae cells are capable of binding dextran via the GbpC homologue protein, which is similar to the S. mutans GbpC protein. In addition, Southern hybridization analysis using the S. macacae gbpC homologue as a probe showed a distribution of gbpC homologues throughout the mutans streptococci.

Streptococcus mutans strains harboring collagen-binding adhesin.

A previously unidentified 120-kDa protein was detected in Streptococcus mutans strain Z1 and was involved in the cold-agglutination of the strain. We have identified the gene, designated cnm, as being involved in the agglutination of strain Z1 following random mutagenesis. The amino acid sequence of the deduced Cnm protein exhibited high similarity to those of collagen-binding adhesins from staphylococci and other organisms. To confirm whether the protein is involved in collagen-binding, we cloned a cnm gene fragment, overexpressed it in E.coli, and prepared crude extracts. The extracts containing recombinant protein exhibited binding to immobilized collagen and laminin but not to fibronectin. Compared with the parental strain Z1, the cold-agglutination-negative mutant 05A02 exhibited reduced binding to collagen and laminin but retained that to fibronectin. This gene was detected in some strains of S. mutans. Therefore, the cnm gene encoded a new strain-specific member of the collagen-binding adhesin family.

Streptococcus mutans binding to solid phase dextran mediated by the glucan-binding protein C.

Streptococcus mutans GbpC is a wall-anchored surface protein which is involved in dextran-dependent aggregation. The GbpC phenotype is observed only in cells grown under stress conditions. In order to detect the GbpC protein of S. mutans, we isolated the wall fraction following digestion of the cell wall of this organism by N-acetylmuramidase, and detected the GbpC protein from S. mutans cells by western analysis with anti-GbpC serum. Interestingly, S. mutans cells exhibiting the negative dextran(alpha-1,6 glucan)-dependent aggregation (ddag) phenotype expressed the protein and could bind to immobilized dextran.

gbpC and pac gene mutations detected in Streptococcus mutans strain GS-5.

The Streptococcus mutans gbpC gene encoding cell wall-anchoring glucan-binding protein C is involved in the dextran(alpha-1,6 glucan)-dependent aggregation (ddag) of this organism. Unlike cells of other strains of S. mutans, strain GS-5 cells did not exhibit dextran(alpha-1,6 glucan)-dependent aggregation under any conditions. We therefore hypothesized that the gbpC gene may be mutated in strain GS-5. Sequencing analysis of the 1752-nucleotide GS-5 gbpC gene revealed a point mutation that switched codon 65 to a TAA termination codon. Strain GS-5 was previously reported also to have a mutation in the pac gene encoding the cell wall-anchored major protein antigen. The laboratory-maintained strain GS-5 is regarded as having lower cariogenicity than the original isolate. The decreased cariogenicity developed during the laboratory culture of strain GS-5 may have been caused by mutations in an environment lacking appropriate selective pressures.

Novel protein kinase C delta isoform insensitive to caspase-3.

Protein kinase C delta (PKC delta) plays a key regulatory role in a variety of cellular functions, including apoptosis, as well as cell growth and differentiation. We previously reported that apoptosis was induced by pretreatment with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), an inhibitor of PKC, in mouse thymocytes. In the present study, we showed that a novel PKC delta isoform (PKC deltaII) was transiently expressed when thymocytes were pretreated with H-7. The analysis of the cDNA encoding PKC deltaII indicated that a 78 bp fragment was inserted into the caspase-3 sensitive site of the original PKC delta (PKC deltaI), presumably by alternative splicing. The PKC deltaII expressed in COS-1 cells was one product with a molecular mass of 81 kDa and with kinase activity similar to that of PKC deltaI. The expressed PKC deltaI protein (78 kDa) was in part cleaved into a 38 kDa fragment in vivo and in vitro, but the PKC deltaII protein was not. Cleavage of the PKC deltaI protein was inhibited by a specific inhibitor of caspase-3, indicating that PKC deltaII is insensitive to caspase-3. The PKC deltaII was highly expressed in the testis and ovary, and at a lower level in the thymocytes, brain and kidney, whereas PKC deltaI was detected in most tissues, suggesting that the function of PKC deltaII is different from that of PKC deltaI.

Characterization of the Streptococcus mutans pyruvate formate-lyase (PFL)-activating enzyme gene by complementary reconstitution of the In vitro PFL-reactivating system.

The act gene was identified and an act mutant as well as the pfl mutant was constructed in Streptococcus mutans. Pyruvate formate-lyase (PFL) activity was regenerated with the mixture of the respective cell extracts from these mutants by complementary reconstitution of the in vitro reactivating system. The S. mutans act gene encoded the sole enzyme able to activate the PFL protein in this organism.

Construction of region-specific partial duplication mutants (merodiploid mutants) to identify the regulatory gene for the glucan-binding protein C gene in vivo in Streptococcus mutans.

The gbpC gene encoding the glucan-binding protein C which is involved in dextran (glucan)-dependent aggregation (ddag) of Streptococcus mutans has been identified by random mutagenesis. We analyzed ddag(-) mutants containing the intact gbpC gene and found that these mutants possessed a large and characteristic duplication of a region of the chromosome which was responsible for the phenotype. Based upon characterization of these duplications, we developed a strategy to introduce a duplication into any specific region of the chromosome of these organisms. The 690-bp gene responsible for the ddag(-) phenotype was identified within a 60-kb region by observing ddag (positive or negative) phenotypes of successively constructed specific duplication mutants.

Xylitol-induced elevated expression of the gbpC gene in a population of Streptococcus mutans cells.

Xylitol possesses a unique property distinct from the other caries-preventive sweeteners. This sugar alcohol cannot be metabolized to acids but is taken up by Streptococcus mutans and accumulated as a toxic sugar-phosphate in the cells, resulting in growth inhibition. Due to the accumulation, xylitol induces biological responses including the emergence of xylitol-insensitive populations. Therefore, we expected another response induced by xylitol and found a new phenomenon, that cells repeatedly cultured in the presence of xylitol evolved into those exhibiting an elevated dextran-dependent aggregation phenotype. This phenotype was found to result from expression of the gbpC gene, which was previously reported to be expressed only under certain stress conditions. Construction of a Strep. mutans isogenic mutant carrying the gbpC::lacZ fusion gene indicated that gbpC expression of cells repeatedly cultured in the presence of xylitol was elevated 20-fold. DNA transfer experiments indicated that this phenotypic change did not appear to be due to a mutation. These cells also exhibited decreased adhesion to glass surfaces when grown in the presence of sucrose. This may be one of the ways by which some populations of Strep. mutans are removed from dental plaques.

Benzodiazepine receptor agonists modulate thymocyte apoptosis through reduction of the mitochondrial transmembrane potential.

Peripheral-type benzodiazepines have been shown to exert immunological effects. In this study, we examined the effects of the peripheral-type benzodiazepines on murine thymocytes. Murine thymocytes that were incubated with the peripheral-type benzodiazepines underwent apoptosis associated with the collapse of mitochondrial transmembrane potential (delta psi(m)). The drugs stimulated dexamethasone- and etoposide-induced apoptosis with the enhanced collapse of delta psi(m). The central-type benzodiazepines had no effect on either the delta psi(m) or apoptosis. The reduction of delta psi(m) depended on protein synthesis and protein phosphorylation. These results suggest that the immunomodulating effect of benzodiazepines is in part due to the modulation of thymocyte apoptosis associated with the collapse of delta psi(m).

Cloning of the TIS gene suppressed by topoisomerase inhibitors.

We recently cloned the cDNA TIS (topoisomerase inhibitor-suppressed) in RVC lymphoma cells exposed to the topoisomerase inhibitors. To elucidate the suppression mechanism of the TIS mRNA by camptothecin, we characterized the structures of the TIS gene. The gene spanned about 21 kb including 11 exons and was present as a single copy. The putative transcription site was present 192 bp upstream from the ATG codon. The typical TATA sequence and CCAAT promoter element were located at positions -21 and -81, respectively. The unidirectional deletion analysis of the 5'-flanking region revealed that [-132/+160] is the promoter region, which participates in the responsiveness to camptothecin. A Northern blot analysis showed that the TIS was expressed in most mouse tissues; at the highest level in the liver and to less extent in the heart and skeletal muscle. The present study showed that the expression of the TIS is suppressed at the transcriptional level by camptothecin. Considering that topoisomerase I is an essential enzyme in mammalian cells, the TIS protein may have an important role in camptothecin toxicity.

4-Piperidinopiperidine-resistant lymphoma cells were resistant to dexamethasone- and A23187-induced apoptosis.

4-Piperidinopiperidine is a side residue of CPT-11, a derivative of camptothecin. We have previously established a 4-piperidinopiperidine-resistant lymphoma cell line, 4-pp-R, which was co-resistant to CPT-11. We report here that this cell line is cross-resistant to dexamethasone and A23187 which induce apoptosis in parent RVC cells. Examination of apoptosis-related gene expression by RT-PCR showed that bcl-2 expression was greater in 4-pp-R than in RVC. p53, bax and bcl-xL were expressed at the same level in 4-pp-R and RVC cells. These results suggest that upregulation of bcl-2 in 4-pp-R cells is related to the resistance to CPT-11 as well as to A23187 or dexamethasone.

Preincubation of thymocytes with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) induces apoptosis in non-stimulated thymocytes.

1-(5-Isoquinolinesulfonyl1)-2-methylpiperazine hydrochloride (H-7), an inhibitor of protein kinases, has been shown to inhibit the thymocyte apoptosis induced by various apoptogenic agents. In the present study, when mouse thymocytes were pretreated with H-7, washed, and cultured for an additional time, apoptosis was induced depending on the preincubation time and the dose of H-7. The protein kinase C activity in the H-7-pretreated and -washed cells was not altered, suggesting that an alteration of a certain PKC isoform is related to both the triggering and the progression of apoptosis.

The refined crystal structure of Bacillus cereus oligo-1,6-glucosidase at 2.0 A resolution: structural characterization of proline-substitution sites for protein thermostabilization.

The crystal structure of oligo-1,6-glucosidase (dextrin 6-alpha-glucanohydrolase, EC 3.2.1.10) from Bacillus cereus ATCC7064 has been refined to 2.0 A resolution with an R-factor of 19.6% for 43,328 reflections. The final model contains 4646 protein atoms and 221 ordered water molecules with respective root-mean-square deviations of 0.015 A for bond lengths and of 3.166 degrees for bond angles from the ideal values. The structure consists of three domains: the N-terminal domain (residues 1 to 104 and 175 to 480), the subdomain (residues 105 to 174) and the C-terminal domain (residues 481 to 558). The N-terminal domain takes a (beta/alpha)8-barrel structure with additions of an alpha-helix (N alpha6') between the sixth strand Nbeta6 and the sixth helix N alpha6, an alpha-helix (N alpha7') between the seventh strand Nbeta7 and the seventh helix N alpha7 and three alpha-helices (N alpha8', N alpha8" and N alpha8'" between the eighth strand Nbeta8 and the eighth helix N alpha8. The subdomain is composed of an alpha-helix, a three-stranded antiparallel beta-sheet, and long intervening loops. The C-terminal domain has a beta-barrel structure of eight antiparallel beta-strands folded in double Greek key motifs, which is distorted in the sixth strand Cbeta6. Three catalytic residues, Asp199, Glu255 and Asp329, are located at the bottom of a deep cleft formed by the subdomain and a cluster of the two additional alpha-helices N alpha8' and N alpha8" in the (beta/alpha)8-barrel. The refined structure reveals the locations of 21 proline-substitution sites that are expected to be critical to protein thermostabilization from a sequence comparison among three Bacillus oligo-1,6-glucosidases with different thermostability. These sites lie in loops, beta-turns and alpha-helices, in order of frequency, except for Cys515 in the fourth beta-strand Cbeta4 of the C-terminal domain. The residues in beta-turns (Lys121, Glu208, Pro257, Glu290, Pro443, Lys457 and Glu487) are all found at their second positions, and those in alpha-helices (Asn109, Glu175, Thr261 and Ile403) are present at their N1 positions of the first helical turns. Those residues in both secondary structures adopt phi and phi values favorable for proline substitution. Residues preceding the 21 sites are mostly conserved upon proline occurrence at these 21 sites in more thermostable Bacillus oligo-1,6-glucosidases. These structural features with respect to the 21 sites indicate that the sites in beta-turns and alpha-helices have more essential prerequisites for proline substitution to thermostabilize the protein than those in loops. This well supports the previous finding that the replacement at the appropriate positions in beta-turns or alpha-helices is the most effective for protein thermostabilization by proline substitution.

Inflammation and apoptosis.

Apoptosis is a highly regulated form of programmed cell death defined by distinct morphological and biochemical features; it plays an important role in embryogenesis and the maintenance of cellular and tissue homeostasis in multicellular organisms. Its perturbation has been implicated in a wide range of patho-physiological stages, including autoimmune, bacterial, and viral disease, and degenerative disorders. The critical role of apoptosis in eliminating harmful or injured cells from tissues suggests its participation in inflammatory processes and in the resolution of inflammatory reactions. In this article, we briefly review the molecular mechanism of apoptosis and the role of apoptosis in inflammation.

Protein synthesis dependent and independent apoptosis of murine splenic T cells.

To elucidate the mechanism of apoptosis in matured T cells, we examined the effect of phorbol esters capable of activating protein kinase C, cAMP agonists, calcium ionophores, and dexamethasone on apoptosis in mouse splenic T cells. All of these agents induced apoptosis in thymocytes. Neither phorbol esters nor calcium ionophores induced apoptosis in the splenic T cells. Dibutyrylcyclic AMP induced apoptosis in splenic T cells but to a lesser extent than that in thymocytes. The results suggest that matured T cells are more resistant to various apoptogenic stimuli than thymocytes. Dexamethasone induced apoptotic cell death in splenic T cells as well as in thymocytes. Cycloheximide, an inhibitor of protein synthesis which completely inhibited thymocyte apoptosis, induced slight apoptosis in splenic T cells, but it inhibited the dexamethasone-induced apoptosis to a level at which cycloheximide induced apoptosis, indicating that at least two pathways of apoptosis exist in peripheral T cells; one protein synthesis dependent and the other independent. Taken together, the results suggest that the apoptosis of peripheral T cells is regulated by a mechanism different from that of thymocytes.

Peptidyl aldehyde inhibitors of proteasome induce apoptosis rapidly in mouse lymphoma RVC cells.

Proteases play an important role in regulation of apoptosis. To elucidate the role of proteasome in apoptosis, we examined the effects of the proteasome inhibitors, carbobenzoxy-L-isoleucyl-gamma-t-butyl-L-glutamyl-L-alanyl-L-leucinal and carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal on RVC lymphoma cells. Cells exposed to the proteasome inhibitors arrested at G2/M phase followed by internucleosomal DNA cleavage, chromatin condensation, and formation of apoptotic bodies dose- and time-dependently. Ubiquitinated histone H2A levels decreased in the exposed cells, suggesting a relationship between deubiquitination of histone H2A and the chromatin disarray seen in apoptosis. Northern blots showed an increase in expression of polyubiquitin genes early in the incubation. These findings suggest that the ubiquitin-mediated proteasome-proteolytic system is involved in regulating the cell cycle and apoptosis in RVC cells.