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.     

Calcitonin-induced phosphorylation of rat liver cytosolic proteins

Calcitonin (CT) stimulated phosphorylation of two liver cytosolic proteins whose molecular weights are 67,000 and 93,000. Stimulation of 67,000-Mr protein phosphorylation began shortly after subcutaneous injection of CT, reaching a maximum at 5 min and decreasing to below the control level at 30 min. The reaction was independent of cyclic AMP or Ca2+, and was not influenced by a calmodulin antagonist, W7. Stimulation of 93,000-Mr protein phosphorylation became evident by 30 min. This reaction was also stimulated by administration of vasopressin or epinephrine, which is known to cause increased phosphorylation of glycogen phosphorylase having the same molecular weight. The phosphorylation of 93,000-Mr protein, stimulated by CT, was dependent on Ca2+ but not on cyclic AMP, and appeared to be inhibited by W7. In addition, CT did not influence the phosphorylation of 61,000-Mr protein, a major protein phosphorylated in a cyclic AMP-dependent manner. These results suggest that CT may exert its effect on liver cells through protein phosphorylation, most probably in a cyclic AMP-independent manner.     

Biochemical and clinical analysis of accumulated glycolipids in symptomatic heterozygotes of angiokeratoma corporis diffusum (Fabry's disease) in comparison with hemizygotes

Angiokeratoma corporis diffusum (Fabry's disease) is an X-linked disorder of glycosphingolipid catabolism. Heterozygous females, although usually asymptomatic, are occasionally as severely afflicted as hemizygous males; recently we identified a heterozygous patient with cardiomyopathy and severe pain in the extremities. In order to elucidate the difference of the clinical features, we analyzed the glycolipid composition of the heart, liver, and kidney obtained from the patient and from a hemizygote. Gas-liquid chromatography revealed that globotriaosylceramide (Gb3) was markedly increased in the heart (32.4 times higher than control) and increased to a lesser extent in the liver and kidney (3.74 and 6.79 times, respectively). The pattern of Gb3 accumulation in the heterozygote, where the highest increases were seen in the heart, was distinct from that in the hemizygote, where elevated levels of Gb3 and Ga2 were found in the kidney. Furthermore, the alpha-galactosidase activity in the heart, liver, and kidney of the heterozygote was 17%, 26%, and 36%, respectively, of normal controls, which correlated well with the accumulation of glycosphingolipid in the heart and with the disease's clinical manifestations. Two other hemizygotic patients, who were identified by low alpha-galactosidase activities, demonstrated the cardiac involvement.     

Production of a herbicide, 5-aminolevulinic acid,by Rhodobacter sphaeroides using the effluent of swine waste from an anaerobic digestor

Summary For the production of a herbicide, 5-amino-levulinic acid (ALA), from anaerobic digestion liquor, the utilization of the photosynthetic bacterium, Rhodobacter sphaeroides was examined. This bacterium could produce ALA extracelularly from this liquor with the addition of levulinic acid (LA), an inhibitor of ALA dehydratase (ALAD), and glycine, a precursor of ALA biosynthesis in the Shemin pathway. Succinate (another precursor) addition was unnecessary for ALA production. When repeated additions of LA were made together with glycine ALA production was significantly enhanced. However, above three additions of LA, ALA production was not further enhanced. The maximum value of ALA production attained was 4.2 mM (0.63 g/ 1), which was over double that of other ALA producers such as Chlorella vulgaris. Propionic acid was predominantly utilized compared with other lower fatty acids, suggesting that this might be converted to ALA via succinyl-coenzyme A (CoA) in the methylmalonyl-CoA pathway.Offprint requests to: Y. Nishizawa     

Nucleotide sequence and presumed secondary structure of the 28S rRNA of pea aphid implication for diversification of insect rRNA

Determination of the entire nucleotide sequence of the aphid 28S ribosomal RNA gene (28S rDNA) revealed that it is 4,147 by in length with a G + C content of 60.3%. Based on the nucleotide sequence, we constructed a presumed secondary-structure model of the aphid 28S rRNA which indicated that the aphid 28S rRNA is characterized by the length and high G + C content of its variable regions. The G + C content of the aphid's variable regions was much higher than that of the entire sequence of the 28S rRNA, which formed a striking contrast to those ofDrosophila with the G + C content much lower than the entire 28S molecule. In this respect, the aphid 28S rRNA somewhat resembled those of vertebrates. This is the third report of a complete large-subunit rRNA sequence from an arthropod, and the first 28S rRNA sequence for a nondipterous insect. Correspondence to: H. Ishikawa     

Promoter regions of cysteine endopeptidase genes from legumes confer germination-specific expression in transgenic tobacco seeds

Cysteine endopeptidases, SH-EP from Vigna mungo and EP-C1 from Phaseolus vulgaris, act to degrade seed storage protein during seed germination. Using transgenic tobacco plants, expression of SH-EP and promoter activity of the EP-C1 gene were analyzed in transgenic tobacco plants. The promoters of the two genes in tobacco seeds showed germination-specific activation, although post-translational processing of SH-EP and regulatory regions of promoter of the gene for EP-C1 were found to differ between leguminous seeds and transgenic tobacco seeds.     

Soybean Lipoxygenase L-4, a Component of the 94-kilodalton Storage Protein in Vegetative Tissues: Expression and Accumulation in Leaves Induced by Pod Removal and by Methyl Jasmonate

A lipoxygenase L-4 gene was isolated from a soybean genomiclibrary. The amino acid sequence of lipoxygenase L-4 is highlyhomologous with the partial amino acid sequence of the 94-kDavegetative storage protein, vsp94, found in paraveinal mesophyllcells in the leaves of depodded soybean plants. No L-4 expressionwas observed in maturing seeds. The L-4 gene is highly expressedin the vegetative tissues of young seedlings, including cotyledons,hypocotyls, roots and primary leaves. L-4 expression followedthe same pattern as lipoxygenase activity in cotyledons peaking3 to 5 days after germination, and returning to a basal levelby 9 days after germination. L-4 gene expression was low inthe roots, stems and leaves of 10-week-old plants. Exposureof 4-week-old plants to atmospheric methyl jasmonate increasedL-4 mRNA in leaves. Continuous pod removal from 7-week-old plantsover a 2 week period resulted in dramatic accumulation of L-4mRNA in leaves. Accumulation of the L-4 protein and three otherlipoxygenase fractions in the leaves of depodded plants wasdemonstrated by ion exchange chromatography. These results indicatethat lipoxygenase L-4 is a component of vsp94. (Received May 31, 1993; Accepted August 9, 1993)