Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

Inhibition of lipid transfer by a unique high density lipoprotein subclass containing an inhibitor protein

We have isolated from human plasma a unique subclass of the high density lipoproteins (HDL) which contains a potent lipid transfer inhibitor protein (LTIP) that inhibited cholesteryl ester, triglyceride, and phospholipid transfer mediated by the lipid transfer protein, LTP-I, and phospholipid transfer mediated by the phospholipid transfer protein, LTP-II. This HDL subclass not only inhibited cholesteryl ester transfer from HDL to LDL or VLDL, but also inhibited cholesteryl ester transfer from HDL to HDL. The inhibitor protein was isolated by sequential chromatography of human whole plasma on dextran sulfate-cellulose, phenyl-Sepharose, and chromatofocusing chromatography. Isolated LTIP had the following characteristics: an apparent molecular weight of 29,000 +/- 1,000, (n = 10) by sodium dodecyl sulfate gel electrophoresis, and an isoelectric point of 4.6 as determined by chromatofocusing. LTIP remained functional following delipidation with organic solvents. Antibody to LTIP was produced, and an immunoaffinity column of the anti-LTIP was prepared. Passage of human, rat, or pig whole plasma over the anti-LTIP column enhanced cholesteryl ester transfer activity in human (17%), pig (200%), and rat plasma (125%). The HDL subclass containing LTIP was isolated from whole human HDL (d 1.063-1.21 g/ml) by immunoaffinity chromatography. The isolated LTIP-HDL complex was shown to: i) contain about 60% protein and 40% lipid, ii) have alpha and pre-beta electrophoretic mobility, iii) have particle size distribution somewhat smaller than whole HDL, about 100,000 daltons, as determined by gradient gel electrophoresis, and iv) contain only a small amount of apoA-I (less than 5%) and a trace amount of apoA-II. Assay of ultracentrifugally obtained lipoprotein fractions revealed that approximately 85% of the total functional LTIP activity was in the d 1.063-1.21 g/ml HDL fraction. Furthermore, immunoblot analysis of whole plasma by nondenaturing gradient gel electrophoresis revealed that LTIP was found predominantly in particles in the size range of HDL. This unique HDL subclass may play an important role in the regulation of plasma lipid transfer and metabolism.     

Cloning and functional analysis of molecular chaperone genes from Bacillus stearothermophilus SIC1

Genes homologous to groES and groEL, which are recognized as molecular chaperone genes, from Bacillus stearothermophilus SIC1 were cloned and sequenced. By addition of GroES, GroEL and ATP in vitro, remaning activity of the alcohol dehydrogenase from Saccharomyces cerevisiae after heat treatment at 50°C for 6 min was improved from 55% to 90%. Furthermore, even though inclusion bodies were formed when a single chain Fv(sFv) was expressed in E. coli cells, during in vivo coexpression with molecular chaperone, a significant amount of the antibody protein could be recovered from the soluble fraction.     

Alteration of Substrate Specificity and Optimum pH of Sarcosine Oxidase by Random and Site-Directed Mutagenesis

The sarcosine oxidase gene was randomly mutated, and F103V, which showed altered specificity, was screened with the plate assay. Five different mutants at position 103 were also constructed. Each mutant exhibited a rather striking change in substrate specificity, except for F103W. The optimum pH was also shifted, depending on the substrate.     

Molecular analysis of the dhp1 + gene of Schizosaccharomyces pombe: an essential gene that has homology to the DST 2 and RAT 1 genes of Saccharomyces cerevisiae

We report here the first cloning of a chalcone flavonone isomerase gene (CHI) from maize. Northern blot experiments indicate that the maize CHI gene (ZmCHI1) is regulated in the pericarp by the P gene, a myb homologue. The ZmCHI1 gene encodes a 24.3 kDa product 55% and 58% identical to CHI-A and CHI-B from Petunia, respectively. This maize CHI gene has four exons and an intron-exon structure identical to the CHI-B gene of Petunia hybrida. RFLP mapping data indicate that some inbred lines contain two additional CHI-homologous sequences, suggesting an organization more complex than that found in Petunia or bean. The possibility that the additional CHI-homologous sequences are responsible for the lack of CHI mutants in maize will be discussed.     

Effects of alginates on the ingestion and excretion of cholesterol in the rat

The effects of three forms of alginate (free acid, sodium and calcium salts) on ingestion and excretion of cholesterol in the rat were studied. For two weeks, Male Sprague-Dawley rats were fed a cholesterol-rich diet containing 3% alginate from the brown alga Ascophyllum nodosum. The food efficiency of the three types of alginate was: Na-alginate > Ca-alginate > alginic acid. A significant increase in the weight of cecum was also observed in alginate diets. Alginate was not effective in preventing the elevation of serum total cholesterol levels, although irregularly changing patterns were observed. The cholesterol level of liver showed a tendency to decrease with alginate feeding, while fecal excretion of cholesterol increased.     

Immunocytochemical localization of 5α-reductase type 1 in the prostate of normal and castrated rats

We immunohistochemically studied the localization of 5-reductase type 1 in combination with androgen receptor (AR) expression in individual lobes of the prostates of intact and castrated rats. In the normal rat prostate, 5-reductase was localized in the cytoplasm of most epithelial cells in the ventral, dorsal, and lateral type 1 (L1) lobes. Epithelial cells of lateral type 2 (L2) lobes were negative for 5-reductase. AR was present in the nuclei of all epithelial and stromal cells throughout the prostate. The number of 5-reductase-immunoreactive cells rapidly decreased in the ventral and L1 lobes after castration, whereas many positive cells remained in the dorsal lobe even at 4 weeks after castration. AR immunostaining was lost in the ventral, dorsal, and L1 lobes at 1 week after castration, but remained in the L2 lobe of 4-week-castrated rats. Electron microscopic immunocytochemistry showed that 5-reductase was exclusively localized in the rough endoplasmic reticulum membranes and that there were no distinct structural differences between the positively and negatively stained epithelial cells. These findings suggested that the expression of 5-reductase type 1 in the epithelial cell is heterogeneous within and among the individual lobes of the rat prostate, and does not correspond to AR expression.