Androgenic regulation of hepatic gene expression

Androgen-dependent synthesis of alpha 2u globulin in the rat liver has been used in our laboratory as a model for studying the effect of sex hormones on hepatic gene expression. alpha 2u Globulin is a group of low molecular weight (Mr approximately 18,000) male specific urinary proteins synthesized and secreted by hepatocytes. In the male rat hepatic synthesis of alpha 2u globulin begins at puberty (approximately 40 days), reaches a peak level (approximately 20 mg/day) at about 75 days and declines during old age. Androgens can induce alpha 2u globulin in ovariectomized female rats in vivo and in the liver perfusion system in vitro. However, both prepubertal and senescent (greater than 800 days) male rats not only do not produce alpha 2u globulin but are also refractory to androgen administration. alpha 2u Globulin is coded by a multigene family comprising about 20-30 gene copies per haploid genome. All of these gene copies seem to express translationally active mRNAs giving rise to individual isoforms of alpha 2u globulin. Appearance and disappearance of the cytoplasmic androgen-binding protein (CAB) correlates with the androgen responsiveness of hepatocytes. Photoaffinity labeling of the hepatic cytosol shows that the biologically active binding protein, found in the cytosol of the mature male rat liver, has a molecular weight of 31 kDa. A molecular transition of the 31-kDa CAB to a biologically inactive 29-kDa form may be the basis of hepatic androgen insensitivity during prepuberty and senescence.     

Distribution, purification and characterization of rat intestinal UDPgalactose: N-acetylglucosaminyl(beta 1----4)galactosyltransferase

Rat intestinal UDPgalactose: N-acetylglucosaminyl(beta 1----4)galactosyltransferase activity was studied as to its intestinal and villus-to-crypt distribution, and then purified and characterized. Rapid UDPgalactose hydrolysis was noted in the duodenum and jejunum; little to no breakdown was detected in the distal ileum, cecum and proximal colon. Product analysis suggested that UDPgalactose hydrolysis was due to nucleotide-sugar pyrophosphatase and galactose-1-phosphate phosphatase activities; ileum appeared to have little of the first activity and none of the latter. An aboral gradient of galactosyltransferase activity was noted, activity being 3-4-fold higher in the ileum, cecum and proximal colon. Total homogenate exogenous acceptor galactosyltransferase activities showed no villus-crypt differences but activity measured with intact isolated cells demonstrated higher activity with crypt cells; this was particularly evident in the ileum. Galactosyltransferase activity was purified from ileal-colonic mucosa. An over 4000-fold purification with 75 percent yield was achieved. Only one band of approx. 70-75 kDa was noted on sodium dodecyl sulfate polyacrylamide electrophoresis. As with other eukaryotic galactosyltransferase activities, there was an absolute requirement for Mn2+; the concentration required for half maximal activity was only 2.5 microM and higher concentrations did not inhibit. The Km for UDPgalactose was 30 microM.     

Biochemical changes in the gastric mucosa after injury in young and aged rats

Changes in gastric mucosal thymidine kinase (TK) activity (an indicator of proliferative activity) were examined in young (4 month) and aged (24 month) Fischer-344 male rats 6 h after intragastric administration of either 2 M NaCl (1 ml/130 g b.w.) or an equivalent volume of water (control). These changes were related to the expression of c-myc gene, tyrosine kinase (Tyr-K) activity and tyrosine-specific phosphorylation of proteins in the gastric mucosa. Basal gastric mucosal TK activity (data from the controls) in the aged rats was found to be 75% (P less than 0.001) above the young animals. This was accompanied by increased expression of c-myc gene and a 67% (P less than 0.001) enhancement in Tyr-K activity. Intragastric administration of 2 M NaCl resulted in gastric mucosal damage (as evidenced by lesions index) in both age groups. However, in aged rats, the lesions index was found to be about 75% higher than in their younger counterparts. In young rats, mucosal injury resulted in a 95% rise in TK activity, whereas in aged rats it was increased by only 38%, when compared with corresponding controls. This 2-fold rise in TK activity in young rats was also associated with increased expression of the c-myc gene. In young rats, administration of hypertonic saline caused a 90% (P less than 0.001) increment in Tyr-K activity and significantly stimulated tyrosine-specific phosphorylation of five mucosal proteins with an apparent molecular mass of 170, 120, 100, 55 and 43 kDa. On the other hand, administration of hypertonic saline to the aged rats caused only a small 16% (P less than 0.025) increase in Tyr-K activity, and produced no apparent change in either expression of c-myc gene or tyrosine-specific phosphorylation of any of the proteins in the gastric mucosa, when compared with the corresponding controls. We conclude that aging increases the susceptibility of the gastric mucosa to damaging agents and diminishes its regenerative capacity. We also suggest that Tyr-K may play a role in determining these events.     

Ca2(+)-dependent glucose transport in chicken intestine

Transport of glucose was measured in the intestine of white leghorn layers in vivo using ligated upper small intestinal segment in the presence of Ca2+ and other ions either singly or in combination. Transport of glucose across the intestine was very significantly increased with Ca2+ than Na+, K+ and Po4(3-) individually, but when Ca2+ was combined with Na+, K+ and PO4(3-), the glucose absorption increased significantly over that achieved by Na+ ions alone. These data revealed that Ca2+ ions might be exerting the major influence on glucose transport processes of the chicken intestine.     

Melting characteristics of highly supercoiled DNA

The effect of high supercoil densities on the melting characteristics of a supercoiled DNA has been studied. It is found that although the melting temperature increases abruptly on converting a linear DNA merely into the relaxed circular form, it falls back substantially at high supercoil densities. It is further predicted, in such cases, that the number of melted base pairs should be significantly enhanced even at the physiological temperature, which may facilitate the binding of other molecules to the highly supercoiled DNA.     

Structure and expression of the Chlorobium vibrioforme hemA gene

The green sulfur bacterium, Chlorobium vibrioforme, synthesizes the tetrapyrrole precursor, -aminolevulinic acid (ALA), from glutamate via the RNA-dependent five-carbon pathway. A 1.9-kb clone of genomic DNA from C. vibrioforme that is capable of transforming a glutamyl-tRNA reductase-deficient, ALA-dependent, hemA mutant of Escherichia coli to prototrophy was sequenced. The transforming C. vibrioforme DNA has significant sequence similarity to the E. coli, Salmonella typhimurium, and Bacillus subtilis hemA genes and contains a 1245 base open reading frame that encodes a 415 amino acid polypeptide with a calculated molecular weight of 46174. This polypeptide has over 28% amino acid identity with the polypeptides deduced from the nucleic acid sequences of the E. coli, S. typhimurium, and B. subtilis hemA genes. No sequence similarity was detected, at either the nucleic acid or the peptide level, with the Rhodobacter capsulatus or Bradyrhizobium japonicum hemA genes, which encode ALA synthase, or with the S. typhimurium hemL gene, which encodes glutamate-1-semialdehyde aminotransferase. These results establish that hemA encodes glutamyl-tRNA reductase in species that use the five-carbon ALA biosynthetic pathway. A second region of the cloned DNA, located downstream from the hemA gene, has significant sequence similarity to the E. coli and B. subtilis hemC genes. This region contains a potential open reading frame that encodes a polypeptide that has high sequence identity to the deduced E. coli and B. subtilis HemC peptides. hemC encodes the tetrapyrrole biosynthetic enzyme, porphobilinogen deaminase, in these species. Preliminary evidence was obtained for the existence of a 3.0-kb polycistronic meassge that includes the hemA sequence, in exponentially growing C. vibrioforme cells. Results of condon usage analysis for the C. vibrioforme hemA gene indicate that green sulfur bacteria are more closely related to purple nonsulfur bacteria than to enteric bacteria. Sequences corresponding to a polyadenylation signal and a poly(A) attachment site were found immediately downstream from the 3 end of the hemA open reading frame.     

Effect of changes in the osmolarity of the growth medium on Vibrio cholerae cells.

The rate and extent of lysis of Vibrio cholerae cells under nongrowing conditions were dependent on the osmolarity of the growth medium. Gross alterations in cellular morphology were observed when V. cholerae cells were grown in media of high and low osmolarity. The rate of lysis of V. cholerae cells under nongrowing conditions increased after treatment with chloramphenicol. Chloramphenicol-treated V. cholerae 569B cells showed formation of sphaeroplast-like bodies in medium of high osmolarity, but not in low osmolarity. Changes in the osmolarity of the growth medium also regulated the expression of the outer membrane proteins. This regulation was abolished if V. cholerae cells were grown in Pi-depleted medium. Analysis of the lytic behavior and composition of outer membrane proteins of an osmotically fragile mutant strain revealed a similar dependence on the osmolarity of the growth medium.