Studies on Bacteriolytic Enzymes

About 100 soil samples were subjected to screening for microorganisms which were capable of producing lytic enzyme toward Staphylococcus aureus. A strain belonging to Streptomyces was isolated and found to produce lytic enzyme(s) noninduciblly, when grown aerobically at 37°C for 25 hr in a medium containing 7.5% soybean cake extract, 2% dextrin, 0.6% K₂HPO₄, 0.02% each of MgSO₄·7H₂O and KCl, pH 7.0. The crude enzyme preparation was active at pH values of 8.5 and 5.8 toward S. aureus, B. subtilis, L. bulgaricus and Str. faecalis but was completely inert against M. lysodeikticus, indicating the enzyme(s) to be distinguished from other bacteriolytic enzymes of Streptomyces so far reported.     

Studies of Hemicellulolytic Enzymes of Bacillus subtilis

Many strains of Bacillus subtilis were found to secrete several hemicellulolytic enzymes such as arabinoxylanase, galactomannanase, arabinogalactanase, etc. Chemical and enzymatic properties of certain strains of the bacterium were comparatively investigated. An arabinogalactanase was purified and obtained in a crystalline state. Its molecular weight and isoelectric point were estimated to be 3.7×10⁴ and 8.39, respectively. The enzyme showed an optimal pH for reactions at 6.0, and was stable in a pH range of 5.0 to 9.5 at 30°C. It required no metallic ions for its activity and hydrolyzed soybean arabinogalactan, forming galactobiose as the main product. No liberation of arabinose was observed in the hydrolysate. Also, the enzyme did not attack coffee bean arabinogalactan. Some implications of the experimental results are discussed.     

Studies on Bacterial Protease

Some physicochemical properties and amino acid composition of the alkaline protease of B. amylosacchariticus were determined. The molecular weight and sedimentation coefficient were estimated to be 22,700 and 2.89 s, respectively, and the amino terminal amino acid was identified to be alanine. The enzyme contained 15.9% of nitrogen and was composed of 220 residues of amino acid: lys₆, his₅, arg₃, asp₂₀, thr₁₄, ser₃₇, glu₁₂, pro₁₀, gly₂₅ ala₂₇ val₂₀, met₃, isoleu₁₂, leu₁₂, tyr₉, phe₂, try₃ and amide ammonia₁₆ The results indicate that protein nature and chemical properties of the alkaline protease presented here are distinct from those of alkaline proteases obtained from the other strains of B. subtilis, such as subtilopeptidase A, B and BPN’     

α-Amylase Formation and Nucleic Acid Metabolism of Bacillus subtilis

The nucleic acid metabolism in washed cells of Bacillus subtilis was investigated with special reference to amylase formation of the bacterium. On incubation of the suspension of the washed cells, purines, pyrimidines and their related compounds were observed in the medium. However, in the medium of the cells incubated with a calcium chelater, where no amylase formation occurred, were detected adenosine- and guanosine-monophosphate in addition to those described above. The addition of a calcium chelater was also found to decrease the quantity of the nucleic acids being involved in the lysozyme-sensitive fraction of the bacterial cells, suggesting the possibility that the metabolism of nucleic acids in this fraction is closely related to amylase formation of the cells.     

Molecular pathways of cyclic nucleotide-induced inhibition of arterial smooth muscle cell proliferation

Cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) are second messengers involved in the intracellular signal transduction of a wide variety of extracellular stimuli. These signals regulate many biological processes including cell proliferation, differentiation, migration, and apoptosis. Recently, significant progress has been achieved in the molecular basis underlying cyclic nucleotide regulation of cell proliferation. This review summarizes our knowledge of the signaling pathways regulated by cyclic nucleotides in arterial smooth muscle cells.     

Allelotypic characteristics of thorotrast-induced intrahepatic cholangiocarcinoma: comparison to liver cancers not associated with thorotrast

To elucidate the genetic alterations that are specific to Thorotrast-induced liver cancers and their possible roles in tumorigenesis, we analyzed loss of heterozygosity (LOH) at 37 loci. Our previous study of liver cancers that were not associated with Thorotrast found LOH at 9 of these loci to be characteristic of intrahepatic cholangiocarcinoma (ICC), at 19 to be characteristic of hepatocellular carcinoma (HCC), and at 9 to be common to both ICC and HCC. LOH analysis was also performed in tissues of cholangiolocellular carcinoma, which is thought to originate from a common stem cell progenitor of hepatocytes and bile duct epithelial cells. We found frequent LOH at D4S1538, D16S2624 and D17S1303 to be common to all the subtypes of liver cancers, independent of the specific carcinogenic agent. In contrast, LOH at D4S1652 generally was not observed in Thorotrast-induced ICC. LOH analysis revealed that Thorotrast-induced ICC shares some LOH features with both ICC and HCC that were not induced by Thorotrast; however, it is more similar to ICC than to HCC in terms of genetic changes. This study could narrow down the crucial chromosomal loci whose deletions are relevant to hepatobiliary carcinogenesis irrespective of the carcinogenic agent. The study of LOH at loci other the those crucial ones may help us understand how the phenotype of liver cancers is determined.     

The affinity of hemoglobin for oxygen affects ventilatory responses in mutant mice with Presbyterian hemoglobinopathy

The purpose of this study was to test whether chronically enhanced O2 delivery to tissues, without arterial hyperoxia, can change acute ventilatory responses to hypercapnia and hypoxia. The effects of decreased hemoglobin (Hb)-O2 affinity on ventilatory responses during hypercapnia (0, 5, 7, and 9% CO2 in O2) and hypoxia (10 and 15% O2 in N2) were assessed in mutant mice expressing Hb Presbyterian (mutation in the beta-globin gene, beta108 Asn --> Lys). O2 consumption during normoxia, measured via open-circuit methods, was significantly higher in the mutant mice than in wild-type mice. Respiratory measurements were conducted with a whole body, unrestrained, single-chamber plethysmograph under conscious conditions. During hypercapnia, there was no difference between the slopes of the hypercapnic ventilatory responses, whereas minute ventilation at the same levels of arterial PCO2 was lower in the Presbyterian mice than in the wild-type mice. During both hypoxic exposures, ventilatory responses were blunted in the mutant mice compared with responses in the wild-type mice. The effects of brief hyperoxia exposure (100% O2) after 10% hypoxia on ventilation were examined in anesthetized, spontaneously breathing mice with a double-chamber plethysmograph. No significant difference was found in ventilatory responses to brief hypoxia between both groups of mice, indicating possible involvement of central mechanisms in blunted ventilatory responses to hypoxia in Presbyterian mice. We conclude that chronically enhanced O2 delivery to peripheral tissues can reduce ventilation during acute hypercapnic and hypoxic exposures.