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.     

Schizosaccharomyces pombe Pmr1p is essential for cell wall integrity and is required for polarized cell growth and cytokinesis

The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28 degrees C in minimal medium and a lethal thermosensitive phenotype at 37 degrees C. Cell growth is completely inhibited at 28 degrees C in a Ca2+-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear. The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions. The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca2+-ATPase homologue Pmr1p. The gene is not essential under optimal growth conditions but is required under conditions of low Ca2+ (<0.1 mM) or high temperature (>35 degrees C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Delta strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-beta-D-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca2+-free medium. The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells. These results indicate that S. pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally. Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca2+-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth.