Role of hydrogen generation by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in the oral cavity

Some gastrointestinal bacteria synthesize hydrogen (H₂) by fermentation. Despite the presence of bactericidal factors in human saliva, a large number of bacteria also live in the oral cavity. It has never been shown that oral bacteria also produce H₂ or what role H₂ might play in the oral cavity. It was found that a significant amount of H₂ is synthesized in the oral cavity of healthy human subjects, and that its generation is enhanced by the presence of glucose but inhibited by either teeth brushing or sterilization with povidone iodine. These observations suggest the presence of H₂-generating bacteria in the oral cavity. The screening of commensal bacteria in the oral cavity revealed that a variety of anaerobic bacteria generate H₂. Among them, Klebsiella pneumoniae (K. pneumoniae) generated significantly large amounts of H₂ in the presence of glucose. Biochemical analysis revealed that various proteins in K. pneumoniae are carbonylated under standard culture conditions, and that oxidative stress induced by the presence of Fe⁺⁺ and H₂O₂ increases the number of carbonylated proteins, particularly when their hydrogenase activity is inhibited by KCN. Inhibition of H₂ generation markedly suppresses the growth of K. pneumoniae. These observations suggest that H₂ generation and/or the reduction of oxidative stress is important for the survival and growth of K. pneumoniae in the oral cavity.     

An evolutionally conserved Lys122 is essential for function in Rhodospirillum rubrum bona fide RuBisCO and Bacillus subtilis RuBisCO-like protein

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and RuBisCO-like protein (RLP) catalyze similar enolase-type reactions. Both enzymes have a conserved non-catalytic Lys122 or Arg122 on the β-strand E lying in the interface between the N- and C-terminal domains. We used site-directed mutagenesis to analyze the function of Lys122 in the form II Rhodospirillum rubrum RuBisCO (RrRuBisCO) and Bacillus subtilis RLP (BsRLP). The K122R mutant of RrRuBisCO had a 40% decrease in k_cat for carboxylase activity, a 2-fold increase in K_m for CO₂, and a 1.9-fold increase in K_m for ribulose-1,5-bisphosphate. K122M and K122E mutants of RrRuBisCO were almost inactive. None of the substitutions affected the thermal stability of RrRuBisCO. The K122R mutant of BsRLP had a 32% decrease in k_cat and lower thermal stability than the wild-type enzyme. The K122M and K122E mutants of BsRLP failed to form a catalytic dimer. Our results suggest that the lysine residue is essential for function in both enzymes, although in each case, its role is likely distinct.     

Identification of genes related to heart failure using global gene expression profiling of human failing myocardium

Although various management methods have been developed for heart failure, it is necessary to investigate the diagnostic or therapeutic targets of heart failure. Accordingly, we have developed different approaches for managing heart failure by using conventional microarray analyses. We analyzed gene expression profiles of myocardial samples from 12 patients with heart failure and constructed datasets of heart failure-associated genes using clinical parameters such as pulmonary artery pressure (PAP) and ejection fraction (EF). From these 12 genes, we selected four genes with high expression levels in the heart, and examined their novelty by performing a literature-based search. In addition, we included four G-protein-coupled receptor (GPCR)-encoding genes, three enzyme-encoding genes, and one ion-channel protein-encoding gene to identify a drug target for heart failure using in silico microarray database. After the in vitro functional screening using adenovirus transfections of 12 genes into rat cardiomyocytes, we generated gene-targeting mice of five candidate genes, namely, MYLK3, GPR37L1, GPR35, MMP23, and NBC1. The results revealed that systolic blood pressure differed significantly between GPR35-KO and GPR35-WT mice as well as between GPR37L1-Tg and GPR37L1-KO mice. Further, the heart weight/body weight ratio between MYLK3-Tg and MYLK3-WT mice and between GPR37L1-Tg and GPR37L1-KO mice differed significantly. Hence, microarray analysis combined with clinical parameters can be an effective method to identify novel therapeutic targets for the prevention or management of heart failure.     

Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.     

Electroencephalogram abnormalities in panic disorder patients: a study of symptom characteristics and pathology

Background  

Since the 1980s, a high EEG abnormality rate has been reported for patients with panic disorder. However, how the EEG abnormalities of panic disorder patients are related to the clinical features and pathology of these patients has yet to be clarified. In this study we investigated whether or not EEG abnormalities are related to the 13 symptoms in the DSM-IV criteria for a diagnosis of panic attacks.     

Identification of a novel type of CA19-9 carrier in human bile and sera of cancer patients: an implication of the involvement in nonsecretory exocytosis

Carbohydrate antigen 19-9 (CA19-9) is a well-known tumor marker for pancreatic cancer. Although the CA19-9 level is measured using anti-sialyl Lewis A antibodies, it remains unknown which molecules carry CA19-9 other than mucins. Here we report the identification and characterization of a novel type of CA19-9 carrier, BGM (bile globular membrane), which is thought to exist in normal bile and to be secreted into sera of patients with pancreatic cancer. We purified the BGM from bile juice using a β-casein column because surface plasmon resonance analysis could detect such carrier vesicles binding to β-casein in sera of patients with pancreatic cancer. We identified characteristic molecules for BGM such as AHNAK (desmoykoin) and a novel golgin family member, CABIN (CAsein Binding domain integral protein with golgIN motif) by mass spectrometry analysis. BGM was detected in the sera of patients with pancreatic cancer as well as athymic mice with transplanted pancreatic cancer cells. Down regulation of CABIN inhibited the secretion of CA19-9 on BGM in pancreatic cancer cell lines. We measured and visualized BGM in sera of patients with cancer. Thus, BGM might be another CA19-9 carrier (glyco-lipids on membrane vesicles) other than mucins and could be applied to the diagnosis of pancreatic cancer.     

ESR studies on the active intermediate in the enzymatic reduction of the Fe-bleomycin complex

The spin trapping method was applied to elucidate the active intermediate during the enzymatic reduction of Fe(III)-bleomycin in the presence of NADPH-cytochrome P-450 reductase and O2. Although the hydroxyl adducts to spin traps were observed, the adduct formation was not inhibited by catalase nor by SOD. Furthermore, in Tris-HCl buffer, no Tris adduct to the spin trap was observed. The results lead to the conclusion that there is no participation of free OH radical in the reactive intermediate in this reduction system. Effect of phosphate buffer on the reactivity of Fe(II)-bleomycin and spin state of Fe(III)-bleomycin were discussed.     

Identification of the reactive cysteinyl residue and ATP binding site in Bacillus cereus glutamine synthetase by chemical modification

Bacillus cereus glutamine synthetase was modified by reaction with a fluorescent SH reagent, N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS), or an ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The locations of the specific binding sites of these reagents were identified. IAEDANS inactivated Mg2(+)-dependent activity and activated Mn2(+)-dependent activity. FSBA inactivated only Mn2(+)-dependent activity. Mg2+ plus Mn2(+)-dependent activity was inactivated by IAEDANS or FSBA. Amino acid sequence analysis of the single AEDANS-labeled proteolytic fragment showed the cysteinyl residue at position 306 to be the site of modification. Cys 306 is one of three cysteines that are unique to Bacillus glutamine synthetase. The result suggested that the cysteine has a role in the active site of the enzyme. We also report that the amino acid residue modified by FSBA was the lysyl residue at position 43.