Glucose utilization by enzymatically-formed trophoblastic vesicles and Day-14 porcine blastocysts

The total glucose metabolism of 48-h spherical trophoblastic vesicles, Day-60 trophoblastic vesicles sections and Day-14 porcine blastocyst sections was measured by the method of O'Fallon and Wright (1). Trophoblastic vesicles were formed by enzyme dispersal in Day-14 porcine blastocysts. Glucose was based on DNA content of the tissue measured by diamino benzoic acid reaction with DNA (2). Slope of the lines (PMoles glucose utilized/4 h x DNA content) was different between Day-14 blastocyst sections and 48 h trophoblastic vesicles (P /= 0.05). Slopes of the lines were identical between 48-h trophoblastic vesicles and Day-60 trophoblastic vesicles sections (P >/= 0.87). Average glucose utilization on a per ng DNA basis was calculated. Day-14 blastocyst sections utilized 0.67 Pmoles glucose/4 h per ng DNA, Day-60 trophoblastic vesicles sections; 0.57; and 48-h sperical trophoblastic vesicles used 0.29. It is hypothesized that the change in glucose utilization between the Day-14 porcine blastocyst and enzymatically formed trophoblastic vesicles may be due to a decrease in metabolism as a consequence of in vitro culture. Further, it is theorized that Day-60 trophoblastic vesicles sections used higher quantities of glucose than 48-h sperical trophoblastic vesicles on a per ng DNA basis due to the increased availability of glucose to the cells of the inner layers, caused by the sectioning of the tissue. The results of this study identify changes in glucose metabolism of enzymatically formed porcine trophoblastic vesicles during culture. It is proposed that enzymatically-formed trophoblastic vesicles be used as a model system for the study of embyro metabolism.     

Effect of early postpartum treatment with prostaglandin F2alpha on subsequent fertility in the dairy cow

Eighty Holstein dairy cows were treated with 25 mg of prostaglandin F2alpha (PGF2alpha) on Days 14 to 16 post calving. Eighty-four herd mates served as saline controls in the double blind study. The reproductive parameters used to measure fertility were mean days to first service on all cows and mean days open, first service conception rates and services per pregnancy on cows that became pregnant. Mean days to first service was similar in both groups (71.8 +/- 27, treated; 68.5 +/- 28.6, controls; P = 0.5352). The mean days open was 98.6 +/- 52.0 d for treated cows compared to 118.8 +/- 71.2 d for controls (P = 0.0680). First service pregnancy rates (treated, 41.3%; control, 35.7%) were not significantly different (P = 0.0630); however, the mean services per pregnancy (treated, 1.64; control, 2.33) were significantly (P = 0.0021) improved in the treated group. Ten cows in the treated group and twelve cows in the control group were diagnosed as having retained fetal membranes and/or metritis. For treated and control cows mean days to first service were 82.2 +/- 34.8 d and 82.8 +/- 58.7 d (P = 0.5652). Days open were 97.0 +/- 32.5 d and 133.4 +/-58.4 d (P = 0.3636); services per conception were 1.83 and 2.50 (P = 0.3178), respectively. In all treated cows reproductive parameters were similar whether cows had high serum progesterone ( > 1 ng/ml) or low serum progesterone ( < 1 ng/ml) on the treatment day. This study suggests that early postpartum treatment of lactating dairy cows with prostaglandin produces an improvement in fertility.     

Evolutionary conservation of homeodomain-binding sites and other sequences upstream and within the major transcription unit of the Drosophila segmentation gene engrailed.

The engrailed (en) gene functions throughout Drosophila development and is expressed in a succession of intricate spatial patterns as development proceeds. Normal en function relies on an extremely large cis-acting regulatory region (70 kilobases). We are using evolutionary conservation to help identify en sequences important in regulating patterned expression. Sequence comparison of 2.6 kilobases upstream of the en coding region of D. melanogaster and D. virilis (estimated divergence time, 60 million years) showed that 30% of this DNA occurs in islands of near perfect sequence conservation. One of these conserved islands contains binding sites for homeodomain-containing proteins. It has been shown genetically that homeodomain-containing proteins regulate en expression. Our data suggested that this regulation may be direct. The remaining conserved islands may contain binding sites for other regulatory proteins.     

Reversible cellular senescence: implications for immortalization of normal human diploid fibroblasts.

IMR-90 normal human diploid fibroblasts, transfected with a steroid inducible mouse mammary tumor virus-driven simian virus 40 T antigen, were carried through crisis to yield an immortal cell line. Growth was dependent on the presence of the inducer (dexamethasone) during both the extended precrisis life span of the cells and after immortalization. After dexamethasone removal, immortal cells divided once or twice and then accumulated in G1. These results are best explained by a two-stage model for cellular senescence. Mortality stage 1 (M1) causes a loss of mitogen responsiveness and arrest near the G1/S interface and can be bypassed or overcome by the cellular DNA synthesis-stimulating activity of T antigen. Mortality stage 2 (M2) is an independent mechanism that is responsible for the failure of cell division during crisis. The inactivation of M2 is a rare event, probably of mutational origin in human cells, independent of or only indirectly related to the expression of T antigen. Under this hypothesis, T-antigen-immortalized cells contain an active but bypassed M1 mechanism and an inactivated M2 mechanism. These cells are dependent on the continued expression of T antigen for the maintenance of immortality for the same reason that precrisis cells are dependent on T antigen for growth: both contain an active M1 mechanism.     

An endogenous 'hypertensive factor' enhances the voltage-dependent calcium current

The effects of an immunoaffinity-purified putative endogenous hypertensive factor (HF) on voltage-dependent calcium current in frog cardiac myocytes were assessed. In 9 out of 10 cells, HF reversibly increased the peak amplitude of the calcium current. HF increased peak calcium current density at -5 mV from a control level of 1.8 +/- 1.3 pA/pF (mean +/- SD) to 4.4 +/- 2.0 pA/pF. HF shifted the peak of the calcium current-voltage relationship in the hyperpolarizing direction. HF shifted the voltage dependence of the inactivation of the calcium current to more negative potentials with prepulses from -80 to 0 mV, but the inactivation was not affected with prepulses more positive than 0 mV. Modulation of the voltage-dependent calcium current by HF may be the mechanism underlying its pressor effects.     

Characterization of third chromosome dominant alpha-methyl dopa resistant mutants (Tcr) and their interactions with l(2)amd alpha-methyl dopa hypersensitive alleles in Drosophila melanogaster

In Drosophila melanogaster two alleles at the Third chromosome resistance locus (Tcr; 3-39-6) were isolated in a screen of EMS mutagenized third chromosomes for dominant resistance to dietary alpha-methyl dopa, alpha-MD, a structural analogue of DOPA. Both alleles of Tcr are recessive lethals exhibiting partial complementation. Almost half (48.3%) of the Tcr40/Tcr45 heterozygotes die as embryos but some survive past adult eclosion. Both the embryonic lethal phenotype and the adult phenotype suggest that Tcr is involved in cuticle synthesis. Tcr mutants suppress the lethality of partially complementing alleles at the alpha-MD hypersensitive locus, l(2)amd. The viability of Tcr40/Tcr45, however, is not increased by the presence of a l(2)amd allele. The possibility that the Tcr and l(2)amd mutations reveal a catecholamine metabolic pathway involved in cuticle structure is discussed.     

The order of loci in the pericentric region of chromosome 17, based on evidence from physical and genetic breakpoints.

Previous genetic analyses of chromosome 17 markers and NF1 (Fain et al. 1987) were extended in an attempt to order marker loci that map physically to 17cen----17q12. Three additional markers (HHH202, CRI-L581, and CRI-L946) were included in the analyses. Recombinants within the cluster of seven unordered marker loci were identified by pairwise analyses for each family and by examining the within-sibship segregation patterns for different markers. Changes in the segregation pattern for different loci define genetic breakpoints. Given that interference is complete in the region, markers with the same segregation pattern lie on one side of the breakpoint, while markers with different segregation patterns lie on opposite sides of the breakpoint. If the order of boundary markers is known, markers on each side of a breakpoint can be oriented in relation to the centromere. The order cen-(HHH202/NF1)-(EW207)-(EW203/CRI-L581)- (CRI-L946)-(HOX-2/NGFR)-qter was inferred by combining information from physical breakpoints in a panel of mouse/human hybrids and information from genetic breakpoints found in 16 NF1 families.     

Generation of nitric oxide by human neutrophils

Human neutrophils were evaluated for their ability to generate nitric oxide. Neutrophils incubated with superoxide dismutase at 37 degrees C produce nitrite anion at a rate of 1.8 nmols/2 x 10(6) cells/30 min, providing indirect evidence of nitric oxide production. Incubation of the neutrophils with concentrations of serum-opsonized zymosan, N-formyl-methionyl-leucyl-phenylalanine, or phorbol myristate acetate sufficient to stimulate the respiratory burst and lysosomal enzyme release caused no additional nitrite anion production. Glass wool-adherent neutrophils exhibited a similar dissociation of nitrite anion production from the respiratory burst and lysosomal enzyme release. Direct evidence for nitric oxide production was also obtained using nitric oxide-specific chemiluminescence. These results demonstrate that human neutrophils are capable of generating nitric oxide.     

Lysophosphatidylcholine metabolism to 1,2-diacylglycerol in lymphoblasts: involvement of a phosphatidylcholine-hydrolyzing phospholipase C

We have previously described the chemoattraction of lymphoblasts by lysophosphatidylcholine [Hoffman, R. D., et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3285-3289]. In studying the mechanism of chemoattraction it was found that lysophosphatidylcholine was metabolized to 1,2-diacylglycerol by the lymphoblastic cell line 6C3HED. One route of metabolism involves the acylation of lysophosphatidylcholine to phosphatidylcholine with subsequent hydrolysis to 1,2-diacylglycerol and phosphocholine by the action of phospholipase C. The increase in cellular 1,2-diacylglycerol was established by metabolic experiments using [14C]glycerol-labeled lysophosphatidylcholine and by mass measurements of 1,2-diacylglycerol. The presence of a phosphatidylcholine-hydrolyzing phospholipase C was confirmed in 6C3HED cell homogenates. In intact cells, lysophosphatidylcholine induced a pattern of protein phosphorylation similar to those of 1,2-dioctanoylglycerol and phorbol 12-myristate 13-acetate, two known activators of protein kinase C. This pathway of lysophosphatidylcholine metabolism, which involves a phosphatidylcholine-hydrolyzing phospholipase C, may be important in the activation of protein kinase C independent of inositol phospholipid hydrolysis.