Involvement of luxS in Biofilm Formation by Capnocytophaga ochracea

Capnocytophaga ochracea is present in the dental plaque biofilm of patients with periodontitis. Biofilm cells change their phenotype through quorum sensing in response to fluctuations in cell-population density. Quorum sensing is mediated by auto-inducers (AIs). AI-2 is involved in intercellular signaling, and production of its distant precursor is catalyzed by LuxS, an enzyme involved in the activated methyl cycle. Our aim was to clarify the role of LuxS in biofilm formation by C. ochracea. Two luxS-deficient mutants, TmAI2 and LKT7, were constructed from C. ochracea ATCC 27872 by homologous recombination. The mutants produced significantly less AI-2 than the wild type. The growth rates of these mutants were similar to that of the wild-type in both undiluted Tryptic soy broth and 0.5 × Tryptic soy broth. However, according to crystal violet staining, they produced significantly less biofilm than the wild type. Confocal laser scanning microscopy and scanning electron microscopy showed that the biofilm of the TmAI2 strain had a rougher structure than that of the wild type. Complementation of TmAI-2 with extrinsic AI-2 from the culture supernatant of wild-type strain did not restore biofilm formation by the TmAI2 strain, but complementation of LKT7 strain with luxS partially restored biofilm formation. These results indicate that LuxS is involved in biofilm formation by C. ochracea, and that the attenuation of biofilm formation by the mutants is likely caused by a defect in the activated methyl cycle rather than by a loss of AI-2.     

Increase of pancreatic somatostatin concentration in early phases of streptozotocin diabetes in rats

A small yet significant increase of immunoassayable pancreatic somatostatin concentration (0.107 +/- 0.005 vs. 0.156 +/- 0.017 microgram/g at 24 hr, p less than 0.05) was found in rats, 24 hr as well as 7 days after treatment with a diabetogenic dose of streptozotocin (65 mg/kg BW). These animals were characterized by marked decreases of insulin in the pancreas without any significant changes in pancreatic glucagon concentration. These results suggest that an abrupt deprivation of insulin from islets results in an elevation of pancreatic somatostatin concentration, and that glucagon in the pancreas plays a minor role in determining pancreatic somatostatin concentration in rats with insulin-deprived diabetes of short duration.     

Use of 13C-labeled glucose for measuring exogenous glucose oxidation in mice

The author modified a respiratory gas analyzer to analyze the respiratory 13CO2 of 12 small laboratory animals all at once. To investigate the practical use of this system, mice were orally (OR) or intravenously (i.v.) given glucose solutions containing three different amounts of 13C-labeled glucose. Expired 13CO2 derived from exogenous glucose was detected within 10 minutes after administration in OR mice, but about 30 minutes in i.v. mice. The height of the peak of 13CO2 expiration was correlated with the administered 13C-glucose mass.     

Confirmation of the human thymidine kinase locus, 17q21 → 17qter,by means of a man-mouse somatic cell hybrid,D98/AH-2 X LMTK−Cl-1D

Summary A large metacentric marker chromosome, m20, in a line of human D98/AH-2 cells was identified by Q bands as being a translocation (1;17)(p36;q21). This was confirmed by means of somatic cell hybridization between D98/AH-2 and thymidine kinase (TK) deficient mouse cells. The hybrid clones by HAT selective system retained m20, indicating the presence of TK locus on this chromosome. The results also provide evidence that TK gene is located on the distal region of the breakpoint in 17q21 but not on 17q21 17pter.     

A novel type of myofibril-bound serine protease from white croaker (Argyrosomus argentatus)

Myofibril-bound serine protease (MBSP) was purified from the myofibril fraction of white croaker (Argyrosomus argentatus) muscle and its enzymatic properties were compared with other fish MBSPs. White croaker MBSP was extracted by the heat treatment of myofibrils and then purified by a series of column chromatographies on Q-Sepharose, Sephacryl S-300, hydroxyapatite and Benzamidine Sepharose. The purified MBSP migrated as a single protein band at 67 kDa in SDS-PAGE under both reducing and non-reducing conditions. It was inhibited by Pefabloc SC, soybean trypsin inhibitor (STI), aprotinin and benzamidine, and was not affected by E-64, pepstatin A and EDTA. The enzyme was most active against Boc-Phe-Ser-Arg-MCA at pH 7.0 and 50 degrees C, and preferentially hydrolyzed Boc-Val-Pro-Arg-MCA and Boc-Asp-Pro-Arg-MCA. Unlike other marine fish MBSPs, white croaker MBSP considerably hydrolyzed Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA. Some enzymatic characteristics including the molecular structure and the substrate specificity for a lysine residue at the P(1) position are quite different not only from other fish MBSPs but also from soluble serine protease obtained from white croaker muscle (MSSP). White croaker MBSP could be therefore classified into a novel type of fish muscle MBSP.     

A case of insulin autoimmune syndrome associated with small insulinomas and rheumatoid arthritis

Twenty five cases of insulin autoimmune syndrome including this case has been reported so far without having the pathogenesis clarified. This paper describes a case which suggests one aspect of pathogenesis. The patient, a housewife concurrently had insulinoma and severe rheumatoid arthritis, complaining of hypoglycemic syncope attacks. During the attacks her blood sugar levels ranged from 19 to 22 mg%. Her serum extractable immunoreactive insulin (IRI) and insulin binding antibody levels were 557 microunits/ml and 0.390 mU/ml, respectively. gamma-Globulin-bound insulin was also measured electrophoretically. Bio-Gel P 10 column chromatography eluted almost all IRI at the void volume at pH 7.4 and a smaller but significant IRI peak also at pH 3.0. Selective angiography revealed a tumor-like staining in the pancreas body. Pancreatectomy relieved her of hypoglycemic attacks. Histology disclosed two small insulinomas. Insulinoma, rheumatoid arthritis and insulin autoimmune syndrome coexisted in this case, suggesting some causal relationship among them.     

Promotion of Ccn2 expression and osteoblastic differentiation by actin polymerization, which is induced by laminar fluid flow stress

Fluid flow stress (FSS) is a major mechanical stress that induces bone remodeling upon orthodontic tooth movement, whereas CCN family protein 2 (CCN2) is a potent regenerator of bone defects. In this study, we initially evaluated the effect of laminar FSS on Ccn2 expression and investigated its mechanism in osteoblastic MC3T3-E1 cells. The Ccn2 expression was drastically induced by uniform FSS in an intensity dependent manner. Of note, the observed effect was inhibited by a Rho kinase inhibitor Y27632. Moreover, the inhibition of actin polymerization blocked the FSS-induced activation of Ccn2, whereas inducing F-actin formation using cytochalasin D and jasplakinolide enhanced Ccn2 expression in the same cells. Finally, F-actin formation was found to induce osteoblastic differentiation. In addition, activation of cyclic AMP-dependent kinase, which inhibits Rho signaling, abolished the effect of FSS. Collectively, these findings indicate the critical role of actin polymerization and Rho signaling in CCN2 induction and bone remodeling provoked by FSS.