Primary structure of non-histone protein HMG1 revealed by the nucleotide sequence

The isolation and sequencing of a cDNA clone coding for the entire sequence of pig thymus non-histone protein HMG1 are described. The sequence analysis reveals a complete 2192-nucleotide sequence with a 5'-terminal untranslated region of 11 nucleotides, 642 nucleotides of an open reading frame that encoded 214 amino acids, and a 3'-terminal untranslated region of 1539 nucleotides. The HMG1 protein, deduced from the nucleotide sequence, has a molecular weight of 24,785 and a C-terminal of a continuous run of 30 acidic amino acids, encoded by a simple repeating sequence of (GAN)30. The predicted amino acid sequence is homologous to HMG1, HMG2, and HMG-T sequences from several sources, suggesting that the protein conformation is under evolutionary constraints. Northern blot analysis reveals that another hybridizable RNA species of smaller size is present. Southern blot analyses suggest that pig genome contains several HMG1 gene equivalents.     

Increase in peripheral large granular lymphocytes in postpartum autoimmune thyroiditis

In order to elucidate the mechanism of postpartum aggravation of autoimmune thyroid disease (AITD), we serially examined the change in the proportion of peripheral large granular lymphocytes (LGL), which have activities of NK, K and/or cytotoxic T cells, in their postpartum period. Within 6 months postpartum, the percentage of LGL increased transiently in patients with AITD who remained euthyroid, or developed destructive thyrotoxicosis and/or hypothyroidism due to thyroiditis and even in normal controls. These changes in the LGL percentage were more obvious in the patients who had marked postpartum thyroid dysfunction. In contrast, we did not find a definite increase in the LGL percentage within 6 months postpartum in patients with Graves' disease who relapsed into Graves' thyrotoxicosis. These deta suggest that the increase in LGL in the postpartum period may be related to the induction of postpartum destructive thyrotoxicosis and/or hypothyroidism in AITD.     

A noncleavable signal for mitochondrial import of 3-oxoacyl-CoA thiolase

Rat 3-oxoacyl-CoA thiolase, an enzyme of the fatty acid beta-oxidation cycle, is located in the mitochondrial matrix. Unlike most mitochondrial matrix proteins, the thiolase is synthesized with no transient presequence and possesses information for mitochondrial targeting and import in the mature protein of 397 amino acid residues. cDNA sequences encoding various portions of the thiolase were fused in frame to the cDNA encoding the mature portion of rat ornithine transcarbamylase (lacking its own presequence). The fusion genes were transfected into COS cells, and subcellular localization of the fusion proteins was analyzed by cell fractionation with digitonin. When the mature portion of ornithine transcarbamylase was expressed, it was recovered in the soluble fraction. On the other hand, the fusion proteins containing the NH2-terminal 392, 161, or 61 amino acid residues of the thiolase were recovered in the particulate fraction, whereas the fusion protein containing the COOH-terminal 331 residues (residues 62-392) was recovered in the soluble fraction. Enzyme immunocytochemical and immunoelectron microscopic analyses using an anti-ornithine transcarbamylase antibody showed mitochondrial localization of the fusion proteins containing the NH2-terminal portions of the thiolase. These results indicate that the NH2-terminal 61 amino acids of rat 3-oxoacyl-CoA thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. Pulse-chase experiments showed that the ornithine transcarbamylase precursor and the thiolase traveled from the cytosol to the mitochondria with half-lives of less than 5 min, whereas the three fusion proteins traveled with half-lives of 10-15 min. Interestingly, in the cells expressing the fusion proteins, the mitochondria showed abnormal shapes and were filled with immunogold-positive crystalloid structures.     

Formation of 5 alpha-products as major C19-steroids in estrogen-responsive mouse Leydig cell tumor lines

Homogenates of estrogen-responsive mouse Leydig cell tumors (T 124958-R and T 22137) or 28- and 120-day-old mouse testes were incubated with [3H]progesterone or [14C]4-androstene-3,17-dione in the presence of NADPH, and progesterone metabolism and enzyme activities were estimated. The growth of T 124958-R tumor transplanted in BALB/c mice was markedly stimulated by estrogenization of host mice, but the growth of T 22137 tumor was evidently suppressed by the estrogenization. The major C21-17-OH-steroids and C19-steroids formed from progesterone by both tumors and the testes of immature mice were 5 alpha-steroids, such as 3 alpha,17-dihydroxy-5 alpha-pregnan-20-one, 5 alpha-androstane-3,17-dione, androsterone, 3 beta-hydroxy-5 alpha-androstan-17-one and 5 alpha-androstane-3 alpha,17 beta-diol. In contrast, the major steroids formed by the testes of adult mice were testosterone and 4-androstene-3,17-dione, and no or little 5 alpha-steroids were produced. 5 alpha-Reductase activities in both tumor cells (40-50 nmol/l X 10(8) cells per h) were also found to be approx. 5-6 times higher than that in Leydig cells of adult mouse testes (8 nmol/l X 10(8) Leydig cells per h), though 17-hydroxylase activity was much higher in the Leydig cells of adult testes (730 nmol/l X 10(8) Leydig cells per h) than in both tumor cells (1-7 nmol/l X 10(8) cells per h). Furthermore, the presence of significant amounts of endogenous androsterone and/or 5 alpha-androstane-3 alpha,17 beta-diol was demonstrated in both tumors by radioimmunoassay. The present results demonstrate for the first time that C19-5 alpha-steroids are major C19-steroid products (immature type of testicular androgen production) in Leydig cell tumor lines.     

Cloning of Candida pelliculosa beta-glucosidase gene and its expression in Saccharomyces cerevisiae

Summary Candida pelliculosa var. acetaetherius is a strain of yeast which can utilize cellobiose as the carbon source. From a gene library prepared from this yeast, the -glucosidase gene has been cloned in a S. cerevisiae host using a chromogenic substrate, 5-bromo-4-chloro-3-indolyl--glucoside as an indicator. It was proved by Southern analysis that the DNA fragment carrying the -glucosidase gene originated from C. pelliculosa. -Glucosidase produced by S. cerevisiae transformants was secreted into the periplasmic space. In Candida, -glucosidase was not induced by cellobiose but was derepressed by lowering the concentration of glucose. The regulation of -glucosidase synthesis in S. cerevisiae carrying the cloned -glucosidase was not clear compared with that in Candida, however, the enzyme activity in low glucose medium (0.05%) was reproducibly higher than in high glucose medium (2%). We have found the sequence that controls the expression of the -glucosidase gene negatively in S. cerevisiae.     

Comparative study of different methods for detection and enumeration of Salmonella spp. in natural waters

Seven enrichment media (two proposed by the authors) for detecting salmonellas from polluted freshwater were compared. The Most Probable Number technique for enumeration of salmonellas in water samples was used, directly adding filtered water to buffered peptone water as the pre-enrichment medium. The results indicate that Rappaport-Vassiliadis/43 and Rappaport-Vassiliadis/43 supplemented with 10 micrograms of sodium novobiocin per ml are the best media for the recovery and enumeration of salmonellas from water samples.     

Isolation and characterization of the human ornithine transcarbamylase gene: structure of the 5'-end region

We isolated over 20 phage clones carrying the ornithine transcarbamylase (OTC) [EC 2.1.3.3] gene, from two independently constructed human genomic DNA libraries, using as probes either a rat OTC cDNA or several nuclear DNA fragments derived from some of these isolated clones. These clones, classified into 10 different groups, overlapped and spanned a region of more than 85 kilobase pairs of the human genomic DNA. Restriction mapping and Southern blot analyses demonstrated that one of the clones covers the 5'-end region of the OTC gene. We sequenced the 5'-end region of the OTC gene and found that it covered 665 base pairs of the 5'-flanking region, the complete first exon and a part of the first intron (150 base pairs). In the 5'-flanking region, there were two pairs of putative CAAT and TATA boxes and one enhancer core-like sequence, GTGGAAAG. The first exon contained a coding region for most of the OTC presequence, i.e. 26 out of 32 amino acid residues of the presequence, including the initiation methionine.     

4-Hydroxyaminoquinoline 1-oxide metabolism and DNA adducts in the early stage of tumorigenesis in rats: comparison of target organ pancreas with non-target organ liver

In order to probe key early molecular events which might be responsible for the initiation of rat pancreatic tumorigenesis by 4-hydroxyaminoquinoline 1-oxide (4-HAQO), the uptake and metabolism of carcinogen and the formation and subsequent repair of DNA adducts were monitored under conditions of high and low tumorigenicity, respectively in partially pancreatectomized and non-operated animals, and in the liver, a non-target organ for this carcinogen. Although uptake of radioactively labelled 4-HAQO was higher in the liver than in the pancreas, generation of DNA adducts was 20 times greater in the latter organ. This discrepancy was probably due to a difference in the metabolic profile of 4-HAQO. The spectrum of the adducts was qualitatively similar in both organs. No qualitative or quantitative differences could be established under the high and low tumorigenicity conditions with regard to DNA adduct formation or persistence. The major difference was the presence of a relatively large extent of pancreatic DNA replication under the high tumorigenic condition. The results indicated that metabolic profile of 4-HAQO, quantity of DNA adducts and levels of DNA replication are key factors involved in initiation of tumorigenesis.     

Influences of sulfated glycosaminoglycans on biosynthesis of hyaluronic acid in rabbit knee synovial membrane

The effect of various sulfated glycosaminoglycans on glycoconjugates syntheses in synovial membranes of rabbit knee joints in culture was investigated by two different approaches. In the first approach, synovial membranes isolated from rabbit knee joints were cultured in the presence of sulfated glycosaminoglycans and [14C]glucosamine. In the second approach, solutions of sulfated glycosaminoglycans were injected into rabbit knee joints and synovial membranes isolated from the joints were cultured in the presence of [14C]glucosamine. The major part of [14C]glucosamine-labeled glycoconjugates associated with the synovial membranes and secreted into culture medium was hyaluronic acid. Of the natural glycosaminoglycans tested, dermatan sulfate gave the maximum stimulation of hyaluronic acid synthesis followed by chondroitin 4- and 6-sulfate. Heparin, heparan sulfate, keratan sulfate, keratan polysulfate, and hyaluronic acid had no significant effect. Of the chemically polysulfated glycosaminoglycans, GAGPS (a persulfated derivative of chondroitin sulfate) gave high stimulation but N-acetylchitosan 3,6-disulfate had no effect. The effect of sulfated glycosaminoglycans on hyaluronic acid synthesis was the same in both experimental approaches. The increase in the amount of secreted hyaluronic acid in culture medium paralleled that in synovial membranes. The results indicate that the galactosamine-containing sulfated glycosaminoglycans have a specific stimulatory effect on hyaluronic acid synthesis. A high degree of sulfation of the molecules appeared to potentiate the stimulatory effect.