Tadao Hasegawa

The function of GADD34 is a recovery from a shutoff of protein synthesis induced by ER stress: elucidation by GADD34-deficient mice.

GADD34 is a protein that is induced by stresses such as DNA damage. The function of mammalian GADD34 has been proposed by in vitro transfection, but its function in vivo has not yet been elucidated. Here we generated and analyzed GADD34 knockout mice. Despite their embryonic stage‐ and tissue‐specific expressions, GADD34 knockout mice showed no abnormalities at fetal development and in early adult life. However, in GADD34−/− mouse embryonic fibroblasts (MEFs), recovery from a shutoff of protein synthesis was delayed when MEFs were exposed to endoplasmic reticulum (ER) stress. The phosphorylation of eukaryotic translation initiation factor 2 α (eIF2α) at Ser51 induced by thapsigargin or DTT was prolonged in GADD34−/− MEF, although following treatment with tunicamycin, the eIF2α phosphorylation level did not change in either GADD34+/+ or GADD34−/− cells. ER stress stimuli induced expressions of Bip (binding Ig protein) and CHOP (C/EBP homologous protein) in MEF of wild‐type mice. These expressions were strongly reduced in GADD34−/− MEF, which suggests that GADD34 up‐regulates Bip and CHOP. These results indicate that GADD34 works as a sensor of ER stress stimuli and recovers cells from shutoff of protein synthesis.

Similarity of Tetracycline Resistance Genes Isolated from Fish Farm Bacteria to Those from Clinical Isolates

ABSTRACT Tetracycline-resistant (Tetr) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000. Of the 66 Tetr gram-negative strains, 29 were identified as carrying tetB only. Four carried tetY, and another four carried tetD. Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG. Sequence analyses indicated the identity in Tetr genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY. Eleven of the Tetr strains transferred Tetr genes by conjugation to Escherichia coli HB-101. All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline. The donors included strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients. Because NaCl enhanced their growth, these Tetr strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria. Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains.

Characterization of Group C and G Streptococcal Strains That Cause Streptococcal Toxic Shock Syndrome

ABSTRACT Twelve strains (the largest number ever reported) of group C and G1 streptococci (GCS and GGS, respectively) that caused streptococcal toxic shock syndrome (STSS) were collected and characterized. Eleven strains were identified as Streptococcus dysgalactiae subsp. equisimilis, and one strain was identified as Streptococcus equi subsp. zooepidemicus. We found that it was the first reported case of STSS caused by S. equi subsp. zooepidemicus. Cluster analysis according to the 16S rRNA gene (rDNA) sequences revealed that the S. dysgalactiae strains belonged to clusters I and II, both of which were closely related. The emm types and the restriction patterns of chromosomal DNA measured by pulsed-field gel electrophoresis were highly variable in these strains except BL2719 and N1434. The 16S rDNA sequences and other characteristics of these two strains were indistinguishable, suggesting the clonal dissemination of this particular S. dysgalactiae strain in Japan. As the involvement of superantigens in the pathogenesis of group A streptococcus-related STSS has been suggested, we tried to detect known streptococcal superantigens in GCS and GGS strains. However, only the spegg gene was detected in seven S. dysgalactiae strains, with none of the other superantigen genes being detected in any of the strains. However, the sagA gene was detected in all of the strains except Tokyo1291. In the present study no apparent factor(s) responsible for the pathogenesis of STSS was identified, although close genetic relationships of GCS and GGS strains involved in this disease were suggested.

Both Sp1 and Sp3 are responsible for p21waf1 promoter activity induced by histone deacetylase inhibitor in NIH3T3 cells

Histone deacetylase inhibitor‐induced expression of p21WAF1 is p53 independent. In the present study, we provide evidence that trichostatin A (TSA), a specific inhibitor of histone deacetylase, can elevate H3 and H4 acetylation and p21WAF1 expression in NIH3T3 cells at first. To identify the transcription factor which is responsible for histone deacetylase inhibitor‐induced expression of p21WAF1 and understand the potential events occurred during this process, we analyze the response of the mouse p21WAF1 promoter to TSA in detail. The region responsive to TSA treatment in the p21 promoter is located −100 bp upstream from transcription initiation site and contains a GC‐box. The mutation introduced into this GC‐box decreases most of the basal and TSA‐induced promoter activity. The results from gel‐shift assay show that Sp1 and Sp3 bind to this GC‐rich region. Cotransfection with Sp1 and/or Sp3 expression constructs elevate both basal and induced promoter activity, and this elevation is dependent on the present of the GC‐box. By contrast, cotransfection with reverse oriented Sp1 or Sp3 cDNA decreased basal and induced‐promoter activity, as well as GC‐box dependency. These findings provide physical and functional evidence which strongly indicated that both Sp1 and Sp3 are responsible for TSA‐induced transactivation of the murine p21WAF1 promoter in NIH3T3 cells. J. Cell. Biochem. 73:291–302, 1999.

Heterozygosity with respect to Zfp148 causes complete loss of fetal germ cells during mouse embryogenesis

Zfp148 belongs to a large family of C2H2-type zinc-finger transcription factors. Zfp148 is expressed in fetal germ cells in 13.5-d-old (E13.5) mouse embryos. Germ-line transmission of mutations were not observed in chimeric Zfp148+/− mice, and some of these mice completely lacked spermatogonia. The number of primordial germ cells in Zfp148+/− tetraploid embryos was normal until E11.5, but declined from E11.5 to E13.5 and continued to decline until few germ cells were present at E18.5. This phenotype was not rescued by wild-type Sertoli or stromal cells, and is therefore a cell-autonomous phenotype. These results indicate that two functional alleles of Zfp148 are required for the normal development of fetal germ cells. Recent studies have shown that Zfp148 activates p53, which has an important role in cell-cycle regulation. Primordial germ cells stop proliferating at approximately E13.5, which correlates with induction of phosphorylation of p53 and its translocation to the nucleus. Phosphorylation of p53 is impaired in Zfp148+/− embryonic stem cells and in fetal germ cells from chimeric Zfp148+/− embryos. Thus, Zfp148 may be required for regulating p53 in the development of germ cells.

Effect of Subinhibitory Concentrations of Macrolides on Expression of Flagellin in Pseudomonas aeruginosa and Proteus mirabilis

ABSTRACT In the present study we showed by molecular analysis that the inhibition of motility by macrolides in Proteus mirabilisand Pseudomonas aeruginosa was well correlated with the loss of the expression of flagellin. Erythromycin, clarithromycin, and azithromycin at subinhibitory concentrations (sub-MICs) suppressed the expression of flagellin dose dependently. Azithromycin had the strongest inhibitory effect on the expression of P. aeruginosa flagellin, whereas 16-membered rokitamycin had only a weak inhibitory effect. These results indicate the potential effectiveness of sub-MICs of erythromycin, clarithromycin, and azithromycin for the treatment of patients with P. mirabilis and P. aeruginosa infections.

Involvement of surface polysaccharides in the organic acid resistance of Shiga Toxin-producing Escherichia coli O157:H7

In general, wild Escherichia coli strains can grow effectively under moderately acidic organic acid‐rich conditions. We found that the Shiga Toxin‐producing E. coli (STEC) O157:H7 NGY9 grows more quickly than a K‐12 strain in Luria–Bertani (LB)‐2‐morpholinoethanesulphonic acid (MES) broth supplemented with acetic acid (pH 5.4). Hypothesizing that the resistance of STEC O157:H7 to acetic acid is as a result of a mechanism(s) other than those known, we screened for STEC mutants sensitive to acetic acid. NGY9 was subjected to mini‐Tn5 mutagenesis and, from 50 000 colonies, five mutants that showed a clear acetic acid‐sensitive phenotype were isolated. The insertion of mini‐Tn5 in three mutants occurred at the fcl, wecA (rfe) and wecB (rffE) genes and caused loss of surface O‐polysaccharide, loss of both O‐polysaccharide and enterobacterial common antigen (ECA) and loss of ECA respectively. The other two mutants showed inactivation of the waaG (rfaG) gene but at different positions that caused a deep rough mutant with loss of the outer core oligosaccharide of lipopolysaccharide (LPS) as well as phenotypic loss of O‐polysaccharide and ECA. With the introduction of plasmids carrying the fcl, wecA, wecB and waaG genes, respectively, all mutants were complemented in their production of O‐polysaccharide and ECA, and normal growth was restored in organic acid‐rich culture conditions. We also found that the growth of Salmonella LPS mutants Ra, Rb1, Rc, Rd1, Rd2 and Re was suppressed in the presence of acetic acid compared with that of the parents. These results suggest that the full expression of LPS (including O‐polysaccharide) and ECA is indispensable to the resistance against acetic acid and other short chain fatty acids in STEC O157:H7 and Salmonella. To the best of our knowledge, this is a newly identified physiological role for O‐polysaccharide and ECA as well as an acid resistance mechanism.

A NF-?B p65 subunit is indispensable for activating manganese superoxide: Dismutase gene transcription mediated by tumor necrosis factor-?

Expression of the manganese superoxide dismutase (Mn‐SOD) is induced by tumor necrosis factor‐α (TNF‐α), interleukin‐1 (IL‐1), and lipopolysaccharide (LPS). Recently, a TNF‐responsive element (TNFRE) was identified within the second intron of the murine Mn‐SOD gene. The 5′ CCAAT/enhancer binding protein (C/EBP)‐related region within the TNFRE was responsive to TNF, whereas the 3′ NF‐κB‐related region alone was not. This report describes the minimal promoter region of the Mn‐SOD gene and investigates the cis‐acting elements and trans‐acting factors responsible for TNF‐α‐induced Mn‐SOD gene expression. Reporter plasmid transfection studies demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn‐SOD gene through the intronic enhancer region. Electrophoretic mobility shift assays demonstrated that after TNF‐α stimulation, p50 and p65 NF‐κB subunits bound specifically to the newly identified NF‐κB transcription factor‐binding site, distinct from the previously described NF‐κB site, within the intronic enhancer region. In addition, site‐directed mutagenesis and cotransfection studies demonstrated that the NF‐κB p65 subunit enhanced the transcriptional activity of the Mn‐SOD gene through the newly identified NF‐κB site. These results show that a NF‐κB p65 subunit is mainly involved in the molecular mechanisms controlling TNF‐α‐mediated Mn‐SOD gene transcription. J. Cell. Biochem. 77:474–486, 2000.

Effect of Antibiotics on Group A Streptococcus Exoprotein Production Analyzed by Two-Dimensional Gel Electrophoresis

ABSTRACT High-dose clindamycin (CLDM) and benzylpenicillin (PCG) are the recommended chemotherapeutic remedies for toxic shock-like syndrome caused by group A streptococci. One reason for this is that it has been shown that CLDM suppresses the expression of some exoproteins, e.g., SpeB, SpeA, and streptolysin O (Slo). We analyzed the effects of antibiotics on the production of whole exoproteins by two-dimensional gel electrophoresis. Unexpectedly, we found that the levels of several exoproteins, Slo, NAD+-glycohydrolase (Nga), M protein, and Sic, were increased by CLDM treatment, although we also confirmed previous findings that the levels of various exoproteins, including SpeB, were decreased. The increases in exoprotein levels were also detected by using other protein synthesis inhibitor antibiotics: erythromycin, kanamycin, tetracycline, chloramphenicol, and linezolid. Peptidoglycan synthesis inhibitors (such as PCG, cefazolin, and imipenem), DNA replication inhibitors (such as gatifloxacin), and an RNA polymerase inhibitor (rifampin) did not have significant effects on exoprotein production. The combination of CLDM and PCG had no advantageous effects with regard to exoprotein production compared to the effect achieved with CLDM alone. We also analyzed the transcriptional levels of slo and nga by reverse transcription-PCR and found that this change was also detected at the transcriptional level. Furthermore, the phenomenon was seen not only in strains of the M1 serotype but also in strains of the other M serotypes. Our study suggests that the clinical effectiveness of CLDM might be due to the inhibition of the production of a limited number of exoproteins.

Cooperative interaction of NF-κB and C/EBP binding sites is necessary for manganese superoxide dismutase gene transcription mediated by lipopolysaccharide and interferon-γ

Expression of the manganese superoxide dismutase (Mn‐SOD) is induced by pro‐inflammatory cytokines. We investigated the cis‐acting elements within a tumor necrosis factor‐responsive element (TNFRE) which was identified in the second intron of the murine Mn‐SOD gene. Site‐directed mutagenesis, reporter plasmid transfection studies and electrophoretic mobility shift assays demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn‐SOD gene through the TNFRE. The cooperation between proteins binding to the newly identified NF‐κB and C/EBP sites led to synergistic gene transcription. This report provides the first evidence that cooperation between two distinct cis‐acting elements may be required for induction of Mn‐SOD gene expression mediated by lipopolysaccharide and interferon‐γ.