Sex differences in the control of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Interaction of estrogen, testosterone and insulin in the regulation of enzyme levels in vivo and in cultured hepatocytes

Control of the activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malate dehydrogenase was investigated in intact rats and in hepatocyte cultures. 1) Adult females had 2-fold greater activities of hepatic glucose-6-phosphate- and 6-phosphogluconate dehydrogenases than adult males, but similar activities of malate dehydrogenase. Castrated males showed decreased activities of all three enzymes in comparison to age- and weight-matched intact controls. In starved animals the activities of all three enzymes decreased significantly. After refeeding with nonpurified diet the activities returned to the prestarved levels in females, but increased to clearly higher values in intact and castrated males. 2) Estrogen levels were in the same range in immature and adult male and female rats. Testosterone levels were highest in adult males, clearly lower in adult females (1/8) and immature males (1/8), still lower in immature females (1/15) and lowest in castrated males (1/40). A simple correlation of the sex differences in these hormone levels to sex differences in glucose-6-phosphate- and 6-phosphogluconate dehydrogenase activities was not apparent. 3) In serum-free, dexamethasone-supplemented 48-h cultures of hepatocytes from both male and female rats the basal activities of glucose-6-phosphate dehydrogenase were the same; they were increased 2-3 fold by insulin alone, 1.5 fold by estrogen alone and 4-5 fold by insulin plus estrogen. Apparently sex differences did not persist in 48-h cell cultures. 4) In 48-h cultures of male hepatocytes, then used as the experimental model, insulin alone increased the activity not only of glucose-6-phosphate dehydrogenase but also of 6-phosphogluconate and malate dehydrogenases.(ABSTRACT TRUNCATED AT 250 WORDS)     

HLA-DP and HLA-DO genes in presumptive HLA-identical siblings: structural and functional identification of allelic variation

We analyzed HLA class II genomic polymorphisms in three families in which bone marrow transplantation was performed between individuals presumed to be HLA identical, but in which unexplained mixed lymphocyte culture reactivity was observed. These families were characterized by classical HLA serology, MLC, and DP typing. In each family, a pair of "HLA-identical" siblings demonstrated a small proliferative response in bidirectional MLC. Southern blotting analysis performed with cDNA probes for DQ alpha, DP alpha, and DP beta identified DP genomic differences in each case. Hybridization of Bgl II-digested genomic DNA with a DP alpha cDNA probe revealed three prominent polymorphic fragments (7.7, 5.8, and 3.7 kb), which discriminated between presumptive identical siblings and indicated crossover events within HLA. Similarly, hybridization of SstI-digested genomic DNA with a DP beta cDNA probe, although resulting in a more complex pattern, identified DP genomic disparity between the presumed HLA identical siblings. Hybridization of SstI-digested DNA from two families with evidence of DP recombination was performed by using an oligonucleotide probe specific for the newly described HLA class II gene DO beta. Two major polymorphic fragments, at 6.2 and 3.3 kb, segregated in these families and localized the crossovers flanking the DO beta gene between the DQ and DP loci. The contribution of the antigenic differences marked by these HLA DP and DO DNA polymorphisms to allorecognition in MLR and in graft-vs-host disease are discussed.     

The interaction of the xid and me genes

The murine "motheaten" (me) mutation has been bred onto the NFS background and combined with the X-linked immunodeficiency (xid) mutation to investigate the effect of the xid-induced B cell maturational block on the widespread immune dysfunction, high levels of autoantibodies, and early mortality found in the motheaten mice. The xid markedly reduced spontaneous IgM secretion by spleen cells, serum IgM, anti-ssDNA antibodies, anti-bromelain-treated-erythrocyte antibodies, and T cell binding (but not thymocytotoxic) antibodies; however, neither phenotype nor mortality was affected, suggesting that other factors are responsible for early death. Marked expansion of the Ly-1+ B cell pool was prevented by xid in the motheaten mouse leaving only a very small population of sIgM-positive B cells. This failure of non-Ly-1+ B cell development in me/me X xid mice suggests that me/me leads to inhibition of non-Ly-1+ B cells and preferential expansion of Ly-1+ B cells in motheaten mice, perhaps as a result of their high levels of maturation and activation factors.     

Presence of the major progesterone-induced proteins of the sheep endometrium in fetal fluids

Allantoic and amniotic fluids were collected on Days 60 (n = 3), 100 (n = 4), and 140 (n = 3) of pregnancy. The presence of uterine milk proteins (UTM-proteins) in these samples was evaluated by Ouchterlony immunodiffusion and enzyme-linked immunoabsorbant assay (ELISA). Eight of ten samples of allantoic fluid and three of ten samples of amniotic fluid produced one or two immunoprecipitin bands against antiserum to UTM-proteins. Each band fused with immunoprecipitin bands from UTM-proteins purified from uterine fluid. Data from a semi-quantitative ELISA indicated that allantoic fluid from all ewes and amniotic fluid from six of ten ewes contained immunoreactive UTM-proteins. Concentrations of UTM-proteins in these fluids were not statistically affected by day of gestation (p greater than 0.10), but tended to decline as gestation advanced. Greater concentrations of UTM-proteins were detected in allantoic fluid than in amniotic fluid (p less than 0.05). The physical characteristics of the immunoreactive material in allantoic and amniotic fluids were examined by polyacrylamide gel electrophoresis and Western blotting. The immunoreactive material was found to possess pIs and molecular weights identical to UTM-proteins. These results indicate that fetal fluids contain material that reacts with antiserum to UTM-proteins and has physical properties similar to UTM-proteins. It is likely, therefore, that the UTM-proteins are transported across the placenta during gestation, perhaps to serve some function in the fetal compartment.     

In vivo genetic engineering: homologous recombination as a tool for plasmid construction

This paper describes a novel method for creating exact DNA fusions between any two points in a plasmid carried in Bacillus subtilis. It exploits the homologous in vivo recombination between directly repeated sequences that can be established by insertion of a synthetic oligodeoxyribonucleotide. The method was used to enhance the productivity in B. subtilis of a cloned alpha-amylase (Amy)-encoding gene originating from Bacillus stearothermophilus. Thus, an exact fusion between nucleotide sequences encoding the expression signals, including the signal peptide, of a Bacillus licheniformis Amy-encoding gene and the mature Amy of B. stearothermophilus, was created. The resulting hybrid translational product was processed correctly in B. subtilis during secretion, giving rise to an Amy identical to the mature Amy secreted by B. stearothermophilus.     

Production and immunohistochemical characterization of monoclonal antibodies directed against renal basement membranes of rats

Basement membranes were separated from rat glomeruli and purified by mild procedures, which led to a highly enriched basement membrane fraction. Here, the production and characterization of five monoclonal antibodies against tubular and glomerular basement membranes are described. These antibodies were analyzed immunohistochemically on frozen sections of rat, bovine, and human kidneys as well as on rat embryos. One monoclonal antibody (BM O II) exclusively recognized the glomerular basement membranes, another one (BM O VII) bound to tubular basement membranes and to Bowman's capsule. Three antibodies (BM O IV, BM M II, BM M III) recognized their antigens in both glomerular and tubular basement membranes as well as in mesangial cells. The BM O II antibody showed a stringent species specificity and bound only to glomerular basement membranes of the rat. The other four antibodies cross-reacted with human and bovine glomerular basement membrane and mesangial antigens; they also bound to other tissues in the developing rat embryo. Antibody binding to specific purified components of the basement membranes such as collagen type IV, laminin, heparan sulphate proteoglycan, and fibronectin was investigated by enzyme-linked immunosorbent assay (ELISA). None of these antibodies reacted with any of these known basement membrane components, indicating that the antibodies may serve as useful tools in future investigations of so far unidentified components of basement membranes.     

Initial reactions in the mineralization of 2-sulfobenzoate by Pseudomonas sp. RW611

Abstract Pseudomonas sp. strain RW611 utilized the ammonium salt of 2-sulfobenzoate as sole source of carbon, sulfur, nitrogen, and energy. The xenobiotic sulfo substituent was dioxygenolytically eliminated as sulfite, which was then slowly oxidized to sulfate. 2,3-Dihydroxybenzoate, which resulted from desulfonation underwent meta -cleavage, mediated by 2,3-dihydroxybenzoate 3,4-dioxygenase activity. This enzyme was inhibited by 3-chlorocatechol and 2,3,4-trihydroxybenzoate.