Expression level of enzymes related to in situ estrogen synthesis and clinicopathological parameters in breast cancer patients

In order to evaluate the importance of estrogen production in tumor and surrounding tissues, we measured mRNA expression levels of 5 enzymes participating to estrogen synthesis in situ and 4 breast cancer-related proteins in 27 pairs of tumor and non-malignant tissues. Steroid sulfatase (STS) mRNA was more frequently detected in tumor tissues rather than in their non-malignant counterparts. Estrogen sulfotransferase (EST) was constantly expressed with high level not only in tumor tissues but also in their surrounding non-malignant counterparts. In contrast, mRNA expression levels of aromatase, and 17β-hydroxysteroid dehydrogenase type I and II were relatively low and detected only in small proportion of the patients. We also measured the mRNA expression levels of the same nine genes in tumor tissues of 197 breast cancer patients, and analyzed relationship between the mRNA expression level and the clinicopathological parameters. The mRNA expression levels of STS, aromatase and erbB2 in tumor tissues increased as breast cancer progressed. The tumoral mRNA expression levels of STS, estrogen receptor β, and erbB2 in patients with recurrence were higher than those in patients without recurrence. Upregulation of STS expression plays an important role in tumor progression of human breast cancer and is considered to be responsible for estrogen production in tumor and surrounding tissues.     

The Schizosaccharomyces pombe cdc6 gene encodes the catalytic subunit of DNA polymerase δ

The cdc6 mutants of Schizosaccharomyces pombe have been classified as being defective in progression through the G2 phase of the cell cycle. We cloned an S. pombe gene that could complement the temperature-sensitive growth of the cdc6-23 mutant. Unexpectedly, the cloned gene was allelic to pol3, which encodes the catalytic subunit of DNA polymerase δ. Integration mapping confirmed that cdc6 and pol3 are identical. The cdc6-23 mutant carries one amino acid substitution in the conserved N3 region of Pol3. Received: 17 October 1996 / Accepted: 19 November 1996     

Purification and characterization of α-glucosidases produced by Saccharomyces in response to three distinct maltose genes

α-Glucosidases or maltases (EC 3.2.1.20) were purified to electrophoretic homogeneity from a respective strain of Sacchromyces cerevisiae which carries a single MAL gene, either MALα, MALβ or MALγ, using gluconate-Sepharose affinity chromography and isoelectrofocusing. Of these maltases, two types of maltase were obtained from the MALγ strain, the pI values of which were 5.6 and 5.9. From the MALα and MALβ strain was obtained only one type of maltase with the pI at 5.6 which was identical to one of the maltases from the MALγ strain. These four maltases possessed the same properties, except for pI. They were monomers with molecular weights of between 66 000 and 67 000. With regard to the substrate specificity, they hydrolyzed maltose and sucrose exclusively but not α-methulglucoside nor maltooligosaccharide. They did not differ in immunological properties.     

Gelation Phenomena Induced by Alkali-alcohol Treatment of 7S and 11S Components in Soybean Globulins

Transparent gels containing about 2% protein were obtained by mixing alkaline dope solution of 7S or 11S soybean proteins with alcohol. The 7S component showed the ability to form a stronger gel than the 11S. This phenomenon depended on pH and alcohol concentration. In 66 % ethanol, the viscosity of the 7S and 11S reached maxima at pH 11.4 and 11.2, respectively. Above these pH levels where further unfolding and dissociation into subunits of the protein molecules occur, the viscosity decreased rather. The effectiveness of alcohol to increase viscosity increased in the order; n-butanol < tert-butanol < n-propanol < iso-propanol < ethanol < methanol. Alcohols having minor hydrophobicity were more effective for increasing viscosity, but ethylene glycol was ineffective. The addition of NaCl or 2-mercaptoethanol to ethanol-mixed alkaline dope solutions resulted in the remarkable increment of the viscosity, especially for the 7S.     

The flaFIX gene product of Salmonella typhimurium is a flagellar basal body component with a signal peptide for export.

flaFIX, the structural gene for the periplasmic P ring of the flagellar basal body of Salmonella typhimurium, was cloned. Two gene products with apparent molecular weights of 38,000 and 40,000 were identified by minicell analysis. Data from pulse-chase and membrane fractionation experiments and data on the inhibitory effect of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone all indicated that the 40-kilodalton protein was a precursor form which, after export across the cytoplasmic membrane accompanied by cleavage of a signal peptide, gave rise to the mature protein in the periplasm. The N-terminal amino acid sequence of the FlaFIX protein, predicted from the DNA sequence, conformed well to known signal peptide sequences. The results indicate that the P-ring protein of the basal body (unlike flagellin and possible some other external flagellar components) crosses the cytoplasmic membrane in a conventional signal peptide-dependent manner.     

Identification of flagellar hook and basal body gene products (FlaFV, FlaFVI, FlaFVII and FlaFVIII) in Salmonella typhimurium.

The flagellar genes flaFV, flaFVII, and flaFVIII of Salmonella typhimurium were cloned, and their presence on a given plasmid was verified by complementation of Escherichia coli mutants defective in the homologous genes. The gene products were identified by radiolabeling in a minicell system as being proteins of the following molecular masses: FlaFV, 42 kilodaltons (kDa); FlaFVI, 32 kDa; FlaFVII, 30 kDA; and FlaFVIII, 27 kDa. These data, together with isoelectric focusing data, confirm gene product assignments of flagellar components made indirectly from mutant studies. Flagellar components are transported by either a signal peptide-dependent or a flagellar-specific pathway. Consistent with its location in the outer membrane ring of the basal body, protein FlaFVIII seems to use the signal peptide-dependent pathway, since it was synthesized in a precursor form and processed, presumably by peptide cleavage, to a mature form; the maturation process was inhibited by addition of a proton ionophore. Proteins synthesized in minicells were localized as follows: FlaFVI was localized to the soluble fraction (cytoplasm); pre-FlaFVIII and FlaFVIII were localized to the particulate fraction (membrane or high-molecular-weight aggregate); FlaFV and FlaFVII were localized to both fractions. The significance of these locations in terms of known or suspected roles in the flagellar apparatus is discussed.     

Synthesis of acetone glycerol acyl esters by immobilized lipase ofMucor miehei

Summary The applications of immobilized lipase ofMucor miehei for the synthesis of acetone glycerol acyl ester from acetone glycerol and fatty acid, which is the first step for monoglyceride production was investigated. With a high oleic acid to acetone glycerol ratio (O/A, mol/mol), a high catalytic activity was observed under low water content in the reaction mixture. By the combination of high O/A ratio (>3) and removal of water which was produced during the reaction, the conversion degree was increased to almost 100%. With the O/A ratio of 3, the approximate half-life of the immobilized lipase and productivity of ester was estimated to be 20 days and 869 g product/g immobilized enzyme per 2 half-lives, respectively.