Effect of low ambient temperature on protein turnover and heat production in chicks

1. Effect of low ambient temperature on protein turnover in the liver and whole body was investigated in chicks together with the contribution of protein synthesis to the total heat production. 2. Both protein synthesis and degradation in the whole body were increased, the latter to a larger extent, at low ambient temperature (LT, 22 degrees C) compared with adequate temperature (AT, 30 degrees C). Liver protein synthesis was not significantly altered by the temperature treatment. 3. The total heat production of LT group was as high as 160% of the AT group. 4. The increased heat production due to enhanced whole-body protein synthesis accounted for only 1.4% of the heat increment in thermogenesis at low ambient temperature, suggesting that protein synthesis would contribute little, if any, to cold-induced thermogenesis in chicks.     

Radioimmunoassay of chenodeoxycholic acid-3-sulfate in urine

A sensitive and specific radioimmunoassay (RIA) for the measurement of urinary total chenodeoxycholic acid-3-sulfate (SCDCA) was developed and the accuracy was confirmed. SCDCA bound to bovine serum albumin as the antigen and emulsified with Freund's complete adjuvant was injected into rabbits. The antiserum obtained was capable of binding 75% of [11,12-3H]SCDCA at 1:1000 dilution. The percentage of bound radioactivity decreased linearly with logarithmic increases in unlabeled SCDCA, from 8 to 200 pmol/ml. The antiserum showed an extremely high specificity for SCDCA (free and conjugated), and the values determined by RIA indicated a close correlation with those found by gas-liquid chromatography. The daily urinary SCDCA level was determined using SCDCA-RIA in 12 disease-free humans and 74 patients with chronic liver diseases. In the normal subjects the daily urinary SCDCA level was 0.74 +/- 0.83 mg/day and increased levels were evident in all groups with chronic liver diseases. The daily urinary SCDCA level corresponds closely with the extent of hepatic dysfunction.     

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.     

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.     

Influence of the gut microflora on protein synthesis in tissues and in the whole body of chicks.

1. The influence of the gut microflora on protein synthesis in individual tissues and in the whole body of young chicks was investigated by the large-dose injection of [3H]phenylalanine. 2. Growth of germ-free chicks was significantly better than that of conventional controls. Wet weights of liver, spleen, duodenum, jejunum + ileum and caeca were heavier in conventional birds than in germ-free counterparts. 3. Fractional rates of protein synthesis were higher in jejunum + ileum and whole body of conventional birds than in those of germ-free birds. Amounts of protein synthesized were larger in liver, jejunum + ileum and caeca in the presence of the gut microflora. 4. When tissues were classified into gut + liver and the remainder of the carcass, in the presence of the gut microflora an enhanced protein synthesis in fractional and absolute rate was found in the gut + liver, which is in direct contact or in close association with micro-organisms, whereas virtually no effect of the gut micro-organisms was detected in the remainder of the carcass. 5. The contribution of protein synthesis of gut + liver to that of the whole body was larger in conventional chicks than in germ-free birds, whereas the reverse was true for the remainder of the carcass.     

Molecular relationship between two types of phospholipase B from Penicillium notatum and reconstitution of active enzyme from its peptide fragments

Two types of phospholipase B of Penicillium notatum, the native type and the modified type that is thought to be generated by the introduction of some nicks into the native type of enzyme by the endogenous protease(s), were distinguished on a slab sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under a nonreducing condition. The native form migrated with a rate corresponding to 95K Da, whereas the modified form migrated more slowly, corresponding to 106K Da, presumably because of its more extended conformation. That the "106K" protein was indeed a nicked product of the "95K" protein was confirmed by amino acid analysis, peptide mapping, N- and C-terminal sequence analyses, and immunoblotting. The peptide fragments (70K and 37K + 32K) comprising the modified protein were isolated by gel filtration in the presence of SDS and 2-mercaptoethanol (the 32K peptide was suggested to be a partial proteolytic product of the 37K peptide). When the "95K" protein was subjected to the same treatment under denaturing condition, it retained a low, but significant, enzymatic activity; in contrast, the separated peptide fragments did not show any significant activity. By a coincubation of these fragments, however, a restoration of enzymatic activity was observed through a reformation of the active complex, corresponding to the original modified protein. The enzymatic activity of this complex was further increased by a treatment with guanidine X HCl, followed by dialysis. The association of peptide fragments appears to occur through the formation of interpeptidal disulfide bonds.