The effects of immersion and exercise on prolactin during pregnancy

Prolactin is an important hormone during pregnancy, affecting mother, fetus, and amniotic fluid volume. Immersion is known to affect prolactin levels significantly. To determine the effect of immersion and exercise on the prolactin response during pregnancy, we examined serum prolactin levels at 15, 25, and 35 weeks' gestation and 10 weeks post partum. Twelve women completed 20 min land rest, 20 min immersion in 30 degrees C water to the xiphoid, and 20 min exercise in the water at 60% VO2max. Resting prolactin levels were 1.91 +/- 0.32, 4.55 +/- 0.5, and 5.85 +/- 0.27 nmol.l-1 +/- standard error of the mean at 15, 25, and 35 weeks' gestation, respectively. Postpartum lactating women had a resting mean prolactin level of 3.95 +/- 1.6 versus 0.22 +/- 0.4 nmol.l-1 in non-lactating women. Prolactin levels declined significantly during immersion even after correction for dilution by plasma volume shifts. The immersion response was inversely related to the duration of pregnancy with 29%, 22%, and 12% drops during 15-, 25- and 35-week trials, respectively. Compared to rest, exercise prolactin levels remained depressed during the 15th and 25th week trials. We hypothesize that immersion in water caused prolactin levels to decline.     

Cell-free synthesis of a putative precursor to the rat liver mitochondrial glycerol-3-phosphate dehydrogenase

Antibodies to purified glycerol-3-phosphate dehydrogenase were raised in rabbits and purified from serum by affinity chromatography on enzyme-bound Sepharose columns. RNA from membrane-free polyribosomes, or poly(A)+ RNA (total cellular RNA) of rat liver, was translated in a rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, and the glycerol-3-phosphate dehydrogenase synthesized was isolated by immunoprecipitation using the antibody. The in vitro product moved on sodium dodecyl sulfate-polyacrylamide gels as a polypeptide that was about 5,000 daltons larger than the subunit of the mature enzyme (74,000 daltons). Digestion of both the mature and the in vitro newly synthesized forms of the enzyme yielded respective sets of peptide fragments which had similar patterns upon sodium dodecyl sulfate-gel electrophoresis. When the presumptive precursor that had been synthesized in vitro was incubated with isolated intact rat liver mitochondria, it was converted to "mature" subunits that were no longer susceptible to externally added proteases. Import of the presumptive precursor is dependent upon an electrochemical potential across the inner mitochondrial membranes. The mature form of the protein is assembled in its native location (the outer surface of the inner mitochondrial membrane).     

The reproducibility of resting and post exercise plasma beta-endorphins

This investigation examined the reproducibility of resting and post exercise plasma beta-endorphin levels. Twenty subjects (10 men and 10 women) had their resting endorphin levels measured under controlled conditions on four separate occasions. Concomitantly, the endorphin response of eight trained runners completing three similar ten mile runs was also determined. For the resting data, there was no significant overall variation among trials, but the intra-subject variability was substantial; the within subject variance was 6.16, and it corresponded to an intra-class reliability coefficient of r = 0.239. No gender effect was noted for the average beta-endorphin values for the four occasions (men = 4.6 +/- 1.7; women = 4.4 +/- 2.1 pM/l); however, the males' within-subject variance of 8.548 (r = 0.080) was significantly larger than that of 3.719 (r = 0.485) for females. Of the runners, one outlier subject had a uniquely high average beta endorphin level of 85.67. Analysis including and excluding the outlier subject yielded within-subject variances of 29.61 (r = 0.960) and 34.47 (r = 0.176), respectively; variances for differences in confidence limits for random variation, they must exceed 7 pM/l at rest, 17 pM/l post exercise, and 20 pM/L difference from rest to post exercise.     

Characterization of CDPdiacylglycerol hydrolase in mitochondrial and microsomal fractions from rat lung

CDPdiacylglycerol pyrophosphatase (E.C. 3.6.1.26) activity has been examined in rat lung mitochondrial and microsomal fractions. While the mitochondrial hydrolase exhibited a broad pH optimum from pH 6-8, the microsomal activity decreased rapidly above pH 6.5. Apparent Km values of 36.2 and 23.6 microM and Vmax values of 311 and 197 pmol.min-1.mg protein-1 were observed for the mitochondrial and microsomal preparations, respectively. Addition of parachloromercuriphenylsulphonic acid led to a marked inhibition of the microsomal fraction but slightly stimulated the mitochondrial activity at low concentrations. Mercuric ions were inhibitory with both fractions. Although biosynthetic reactions utilizing CDPdiacylglycerol require divalent cations, addition of Mg2+, Mn2+, Ca2+, Zn2+, Co2+, and Cu2+ all inhibited the catabolic CDPdiacylglycerol hydrolase activity in both fractions. EDTA and EGTA also produced an inhibitory effect, especially with the mitochondrial fraction. Although addition of either adenine or cytidine nucleotides led to a decrease in activity with both fractions, the marked susceptibility to AMP previously reported for this enzyme in Escherichia coli membranes, guinea pig brain lysosomes, and pig liver mitochondria was not observed. These results indicate that rat lung mitochondria and microsomes contain specific CDPdiacylglycerol hydrolase activities, which could influence the rate of formation of phosphatidylinositol and phosphatidylglycerol for pulmonary surfactant.     

Regulation of prodynorphin gene expression in the ovary: distal DNA regulatory elements confer gonadotropin regulation of promoter activity.

Examination of the regulation of prodynorphin (pro-DYN) promoter activity is limited by the absence of a good cell model. The discovery of pro-DYN mRNA and derived peptides in the reproductive tract led us to examine the cellular localization and hormonal regulation of ovarian pro-DYN expression and to evaluate normal granulosa cells as a model for studying pro-DYN gene regulation. Ovarian pro-DYN mRNA levels were significantly elevated in PMSG-primed immature rats 12-24 h after receiving an ovulatory dose of hCG. Levels peaked 2 days after hCG, remained elevated throughout the ensuing pseudopregnancy, and rose again at the end of pseudopregnancy. In situ hybridization localized pro-DYN expression predominantly to granulosa and luteal cells. Transfection of primary cultures of granulosa cells revealed that the activity of the rat pro-DYN promoter [-1858 to 133 base pairs (bp)] was increased 18- to 19-fold by hCG and human FSH treatments and 7-fold by cAMP analog treatment. Deletion analysis identified a 358 bp fragment as the primary hormone-responsive sequence (-1858 to -1500 bp; containing three potential cAMP-responsive elements); its deletion resulted in severely reduced FSH responsiveness, and its ligation to hormone-unresponsive basal promoter sequences completely restored FSH responsiveness. This is an unusually distal position for cAMP-responsive elements compared to other cAMP-regulated genes. These data demonstrate specific expression of pro-DYN in granulosa and luteal cells, which is under sensitive gonadotropin regulation, and identify a distal hormone-responsive sequence within the promoter.     

Evidence for hemiketals as intermediates in the inactivation of serine proteinases with halomethyl ketones

The mechanism of inactivation of serine proteinases by peptide halomethyl ketone inhibitors was studied through the inhibition of trypsin with a series of model peptide ketones (Lys-Ala-LysCH2X). In this series, X is a poor leaving group with increasing electron-withdrawing capacity (X = H, CH2CO2CH3, COCH3, OCOCH3, and F), and as expected, the peptide ketones are reversible, competitive inhibitors of trypsin. The strength of binding of these inhibitors to trypsin increases with the electron-withdrawing ability of X, indicating that the inhibition constant Ki obtained is a measure of reversible hemiketal formation between the inhibitor ketone carbonyl group and the hydroxyl group of the active site serine. A Hammett plot of -log Ki vs. sigma I, the inductive substituent constant of X, reveals a linear relationship between the free energy of binding and the electron-withdrawing power of X. The reversible binding constant obtained for the corresponding chloromethyl ketone Lys-Ala-LysCH2Cl falls on this line, indicating that the reversible binding involves hemiketal formation, which is followed by alkylation of the enzyme.     

Purification and characterization of glycerol-3-phosphate dehydrogenase (flavin-linked) from rat liver mitochondria

We have purified the membrane-intrinsic glycerol-3-phosphate dehydrogenase from both normal and hyperthyroid rat liver mitochondria by extraction with Triton X-100, hydrophobic affinity chromatography, ion exchange chromatography, gel filtration, and FAD-linked Sepharose 4B affinity chromatography. The yields in both cases were over 20%, and purification ranged from 800- to 650-fold in mitochondria from hyperthyroid and normal rats, respectively. The final preparations appeared to be greater than 95% pure by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate. The pure enzyme focused at pH 5.5 and produced a biphasic thermal inactivation plot at 50 degrees C. The holoenzyme was found to have a molecular mass of 250,000 daltons on gel filtration. The subunit molecular mass was found to be 74,000 daltons +/- 3,000 by sodium dodecyl sulfate-gel electrophoresis and high-performance liquid chromatography gel filtration in 0.1% sodium dodecyl sulfate. 1 mol of the holoenzyme preparation contains 1.1 mol of non-heme iron and 0.7-0.9 mol of noncovalently bound FAD. The absorption spectrum has a maximum at 375 nm and a shoulder at 450 nm which is bleached on treatment with sodium dithionite. The enzymatic reaction is competitively inhibited by glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate, and phosphoglycolic acid. The apparent Km for DL-alpha-glycerol 3-phosphate and noncovalently bound FAD were found to be 6 mM and 7 microM, respectively.     

Influenza vaccination in the elderly: 2. The economics of sending reminder letters

Reminder letters and follow-up telephone calls were used to increase influenza vaccination acceptance by 273 well elderly registered at an urban community health centre. The net effect of the reminder letters was to increase overall coverage to 43%, from 17% in the previous year. Follow-up telephone calls to patients who had not responded to the letters increased coverage to only 55%. Calculation of costs per additional vaccination given revealed that the use of reminder letters alone was much more cost-effective than follow-up telephone calls in increasing coverage. However, with the current fee-for-service reimbursement by medical care insurance in Ontario, neither means of improving vaccination coverage would result in net practice earnings. The implications for an effective and efficient annual influenza program in Canada are discussed.