Developmental ontogeny of NAD+ kinase in the rat conceptus.

The ubiquitous NAD+ kinase (NADK) is the only known enzyme to catalyze formation of NADP+ from NAD+. The capacity to maintain an adequate supply of NADP(H) has important implications for development because of its requirement as a cofactor and electron donor in biosynthesis and detoxication reactions. Modulation of NADK may directly influence NADP(H) concentrations and cell sensitivity to embryotoxicants. Measurable activities of NADK were not detected in gestational day (GD) 10 rat conceptuses. By GD 11, specific activities of 1.8 and 7.0 pmol NADP+/min/microg protein were measured in embryos and visceral yolk sacs (VYSs), respectively. The VYS specific activities decreased thereafter to 0.5 pmol NADP+/min/microg protein by GD 18. Specific activities of NADK in placenta increased from 1.3 pmol NADP+/min/microg protein on GD 11 to 32.7 pmol NADP+/min/microg protein on GD 15. Specific activities in the liver increased from 1.7 pmol NADP+/min/microg protein on GD 15 to 51.1 pmol NADP+/min/microg protein on GD 21. NADK specific activities were also determined in other developmentally relevant tissues such as the heart and the brain. In the heart, NADK activity was at its lowest just before birth while in the brain it peaked at 5.4 pmol NADP+/min/microg protein just prior to birth. In the lung, activity increased from 0.9 pmol NADP+/min/microg protein on GD 17 to 5.9 pmol NADP+/min/microg protein on GD 21. However, activities dropped in the kidney from 2.0 pmol NADP+/min/microg protein on GD 17 to 1.1 pmol NADP+/min/microg protein on GD 21. These results demonstrate dramatic temporal and spatial variations in NADK activity. Tissue variations in NADK activities may reflect alterations in functional needs for cofactors during differentiation and a cooperation between tissues to optimize detoxification capacity. This is particularly important when chemical exposure during pregnancy disrupts pyridine nucleotide redox status and the conceptus must rely on NADK to provide additional NADP(H).

Comparison of in vitro and in utero ethanol exposure on indices of oxidative stress.

Prenatal ethanol exposure produces neural tube defects and growth retardation in experimental animals. Because ethanol's teratogenic effects may involve oxidative stress and effects may differ in vitro and in utero, glutathione, cysteine and ATP were evaluated in gestational day 10 rat conceptuses exposed to ethanol. Cultured embryos exposed to ethanol (1.5 or 3.0 mg/mL) maintained a concentration-dependent decrease in glutathione of 21 or 35%, respectively, at 6 h; visceral yolk sac (VYS) glutathione (GSH) decreased by 22 or 18%, respectively, at 3 h. Maternal ethanol exposure (4.5 g/kg) decreased glutathione by 30% in embryos and VYSs at 3 h, but values rebounded. Cultured embryonic cysteine decreased after 30 min by 42% with both doses and after 6 h by 32 or 38% with 1.5 or 3.0 mg/mL, respectively. Ethanol (1.5 mg/mL) increased VYS cysteine by 35% after 30 min. In utero ethanol exposure decreased embryonic cysteine by 58% at 3 h. Ethanol (1.5 mg/mL) decreased adenosine triphosphate (ATP) by 30-60% in embryos and VYSs at 30 min. After 6 h, embryonic ATP decreased by 41 and 30% with 1.5 and 3.0 mg/mL, respectively, while VYS ATP decreased by 38% with 1.5 mg/mL. In utero ethanol exposure decreased ATP by 31% at 3 h in VYSs. While decreases in GSH and cysteine were evident earlier in utero than in vitro, values returned to control suggesting embryos exposed in utero respond rapidly to chemical-induced oxidative stress due to maternal protective mechanisms. Differences between in vitro and in utero responses to ethanol have important implications for interpretation of in vitro developmental studies.

Pyridine nucleotide flux and glutathione oxidation in the cultured rat conceptus.

It is proposed that protection of the developing embryo from chemical and environmental insults that produces oxidative stress requires a proper glutathione (GSH) and pyridine nucleotide status in both the embryo and extra-embryonic membranes. Modulation of pyridine nucleotide flux [NAD(H) and NAD(P)H] in the visceral yolk sac (VYS) by the thiol oxidants diamide and tert-butyl hydroperoxide (tBH) was studied in real time using microfiberoptic sensors in GD 10 rat conceptuses. Consecutive 5-min exposures to 125- and 250-microM diamide resulted in a fluorescence decrease of 14 and 32 Arbitrary Fluorescence Units (AFU). An additional consecutive exposure to 500-microM diamide caused an attenuated decrease followed by a rebound increase of 22 AFU. Consecutive 5-min exposures to tBH at 250 and 500 microM produced fluorescence decreases similar to that of 500 microM diamide, but the decreases were attenuated at 1000 microM. However, there was variability in the rebound increase. A 5-min exposure to tBH (500 microM) alone caused a fluorescence decrease of 14 AFU followed by a rebound increase of 8 AFU. The rate of fluorescence decrease was attenuated by 50% with pretreatment with the glutathione reductase (GSSG-Rd) inhibitor, BCNU (1,3, bis(2 chloroethyl)-1-nitrosourea), indicating that the decrease in surface fluorescence was probably attributable to a decrease in NADPH. Decreases in fluorescence, observed from the surface of the VYS, correlated with decreases in GSH/GSSG ratios in the embryos and the VYS. After exposure to tBH, GSH levels in conceptuses decreased at the end of 5 and 15 min, with a corresponding increase in oxidized glutathione (GSSG) at the end of 3, 5, and 15 min. Our results demonstrate that the increased production of GSSG on exposure to thiol oxidants correlates with a decrease in the reduced pyridine nucleotide, implying the presence of an active GSSG-Rd pathway in the conceptus during organogenesis, and implicating an important role of the pyridine nucleotides in the restoration of GSH homeostasis in the developing rat conceptus during organogenesis.

Monoclonal antibodies to Escherichia coli ribosomal proteins L9 and L10. Effects on ribosome function and localization of L9 on the surface of the 50 S ribosomal subunit.

Monoclonal antibodies against Escherichia coli ribosomal proteins L9 and L10 were obtained and their specificity confirmed by Western blot analysis of total ribosomal protein. This was particularly important for the L9 antibody, since the immunizing antigen mixture contained predominantly L11. Each antibody recognized both 70 S ribosomes and 50 S subunits. Affinity-purified antibodies were tested for their effect on in vitro assays of ribosome function. Anti-L10 and anti-L9 inhibited poly(U)-directed polyphenylalanine synthesis almost completely. The antibodies had no effect on subunit association or dissociation and neither antibody inhibited peptidyltransferase activity. Both antibodies inhibited the binding of the ternary complex that consisted of aminoacyl-tRNA, guanylyl beta, gamma-methylenediphosphonate, and elongation factor Tu, and the binding of elongation factor G to the ribosome. The intact antibodies were more potent inhibitors than the Fab fragments. In contrast to the previously established location of L10 at the base of the L7/L12 stalk near the factor-binding site, the site of anti-L9 binding to 50 S subunits was shown by immune electron microscopy to be on the L1 lateral protuberance opposite the L7/L12 stalk as viewed in the quasisymmetric projection. The inhibition of factor binding by both antibodies, although consistent with established properties of L10 in the ribosome, suggests a long range effect on subunit structure that is triggered by the binding of anti-L9.