Leucine metabolites do not induce changes in phase angle, bioimpedance vector analysis patterns, and strength in resistance trained men.

We aimed to assess the effects of off-the-shelf leucine metabolite supplements on phase angle (PhA), bioimpedance vector analysis (BIVA) patterns and strength during an 8-week resistance training protocol. Fifty-three male participants were allocated into 4 groups: α-hydroxyisocaproic acid (n = 12, age = 30.9 ± 9.3 years), ß-hydroxy-ß-methylbutyrate free acid (n = 12, age = 31.0 ± 9.3 years), calcium ß-hydroxy-ß-methylbutyrate (n = 15, age = 32.1 ± 5.2 years) or placebo (n = 14, age = 28.9 ± 6.6 years). Bioimpedance parameters and 1-repetition maximum (1RM) for back squat and bench press were assessed at baseline and at the end of weeks 4 and 8. Additionally, fat-free mass and fat mass were evaluated by dual-energy X-ray absorptiometry. No statistically group by time interactions were found, even adjusting for age. PhA and vector did not change over the training period, while time-dependent increases were observed for 1RM back squat and 1RM bench press. A direct association was observed between PhA and 1RM bench press changes (whole sample), while PhA and strength were correlated throughout the study, even when adjusting for fat-free mass and percentage of fat mass. Leucine metabolites have no effect on PhA, BIVA patterns or strength during an 8-week resistance training program, in resistance trained subjects. The trial was registered at ClincicalTrials.gov: NCT03511092. Novelty: Supplementation with leucine metabolites is not a supplementation strategy that improves bioelectrical phase angle, cellular health, and strength after an 8-week resistance training program. When consuming a high protein diet, none of the α-hydroxyisocaproic acid, ß-hydroxy-ß-methylbutyrate free acid, and calcium ß-hydroxy-ß-methylbutyrate metabolites resulted in an ergogenic effect in resistance trained men.

Regulation of prostaglandin endoperoxide H2 synthase in term human gestational tissues.

Increased production of prostaglandins by the gestational tissues is pivotal for the initiation and maintenance of human labour. A major source of prostaglandins in the pregnant human uterus is the amnion membrane, which synthesizes increased amounts of prostaglandin E2 (PGE2) at parturition. We have found that the activity of prostaglandin endoperoxide H2 synthase (PGHS), the enzyme catalyzing the committing step of prostanoid biosynthesis, increases significantly in the amnion at term and preterm labour, and also prior to the onset of clinical labour at term. Furthermore, the abundance of the mRNA encoding the inducible PGHS-2 isoenzyme was higher in the amnion after spontaneous delivery that before labour. The level of the constitutive PGHS-1 mRNA remained unchanged. In addition, we found a significant positive correlation between PGHS activity and the level of PGHS-2 mRNA, but not of PGHS-1 mRNA, in the individual tissue samples, also indicating that PGHS-2 was selectively induced in the amnion membrane at labour. The regulation of PGHS expression by agonists was studied using primary cultures of amnion cells. Glucocorticoid treatment enhanced the activity of PGHS and the level of PGHS-2 mRNA in the cultured cells, without affecting PGHS-1 mRNA abundance. The stimulation was glucocorticoid specific and was blocked by the glucocorticoid receptor antagonist RU486, suggesting that it was mediated by the glucocorticoid receptor. Inhibition of protein synthesis did not block the accumulation of PGHS-2 mRNA showing that the steroid acted directly, without inducing an intervening protein. Protein kinase C activator and protein phosphatase inhibitor compounds and epidermal growth factor also promoted PGHS-2 mRNA expression, demonstrating the involvement of protein kinase dependent mechanisms in PGHS-2 regulation. However, the role of these effectors in the in vivo control of PGHS-2 expression remains to be determined.

Prostaglandin endoperoxide-H synthase-1 and -2 messenger ribonucleic acid levels in human amnion with spontaneous labor onset.

Increased prostaglandin (PG) production within the uterine compartment has a pivotal role in the processes leading to labor onset in women. Two PG endoperoxide-H synthase (PGHS) isoenzymes have been identified in a number of cell types. PGHS-1 is constitutively expressed in most cases, whereas PGHS-2 expression is rapidly induced by several agonists. The aims of this study were to determine the levels of PGHS-1 and PGHS-2 expression before and after spontaneous labor (SL) onset in the amnion and to assess the contribution of PGHS-1 and PGHS-2 to enzyme activity. We established and validated ribonuclease protection assays to quantify PGHS-1 and PGHS-2 messenger ribonucleic acid (mRNA) levels in the amnion. PGHS enzyme activity was measured with an established assay. The antisense RNA probes used in the protection assays were generated using human PGHS-1 and PGHS-2 complementary DNAs. These probes specifically detected the 2.8-kilobase mRNA of PGHS-1 and the 4.8-kilobase mRNA of PGHS-2 in amnion RNA samples on Northern blots. We measured mRNA levels in amnion from patients after SL at term and from patients not in labor undergoing elective cesarean section (CS) at term. PGHS-2 mRNA levels were markedly higher after SL compared to levels in CS amnion [5.18 +/- 1.08 (n = 16) and 2.27 +/- 0.50 (n = 15), densitometric units, respectively; P < 0.02], whereas there was no difference in PGHS-1 mRNA levels after labor compared with CS samples. PGHS-2 mRNA levels were also positively correlated with PGHS enzyme activity in 4 separate assays with a total of 25 patients (r = 0.65-0.88; P < 0.05). There was no correlation between PGHS-1 mRNA levels and enzyme activity. We conclude that PGHS-2 mRNA is present in human amnion; its levels are elevated after SL onset, and they are correlated with enzyme activity. The stimulation of PGHS activity at labor onset probably involves increased expression of PGHS-2. The expression of PGHS-1 does not change in association with labor in human amnion.

Prostaglandin endoperoxide-H synthase (PGHS) activity and immunoreactive PGHS-1 and PGHS-2 levels in human amnion throughout gestation, at term, and during labor.

Prostaglandins (PGs) are of primary importance in the initiation and maintenance of labor in women. A major intrauterine source of prostaglandins is the amnion, which synthesizes increased amounts of PGE2 at term labor. Because PG endoperoxide-H synthase (PGHS) catalyzes the rate-limiting step of PG synthesis from arachidonic acid, we investigated the changes in amniotic PGHS specific activity during gestation and at term and preterm labor. Also, we determined the level of immunoreactive PGHS protein in the amnion to evaluate the mechanisms by which PGHS activity may be regulated. PGHS specific activity, measured at the amount of PGE2 produced by amnion microsomes under optimal conditions, was 18.2 +/- 3.7 pg PGE2/micrograms protein.min (mean +/- SE; n = 19) at term (37-42 weeks gestation) before the spontaneous onset of labor. PGHS specific activity was significantly higher after spontaneous term labor (38.9 +/- 6.0 pg PGE2/micrograms protein.min; n = 19; P < 0.05). Amnion samples from preterm (< 36 weeks gestation) nonlaboring patients contained low levels of PGHS specific activity (5.9 +/- 1.8 pg PGE2/micrograms protein.min; n = 9), which increased significantly with spontaneous preterm labor (28.3 +/- 6.8 pg PGE2/micrograms protein.min; n = 10; P < 0.05). Longitudinal analysis of the data showed that PGHS specific activity was low in the first and second trimesters of gestation, but increased dramatically before labor onset at term. We detected PGHS protein in all microsomal samples, with an antiovine PGHS antibody recognizing both PGHS-1 and -2 isoforms of the enzyme. However, there was no correlation between PGHS specific activity and the amount of immunoreactive PGHS protein. Using an antibody specific for PGHS-2, we detected immunoreactive protein in only 9 of the 25 tissues examined and found no correlation between PGHS specific activity and the amount of PGHS-2 protein. These results suggest that 1) PGHS specific activity in the amnion increases sharply before the onset of labor at term; 2) further increases in specific activity occur during term and preterm labor; and 3) the specific activity of PGHS in the amnion is not related directly to the amount of immunoreactive enzyme protein.

Regulation of prostaglandin endoperoxide H synthase by glucocorticoids and activators of protein kinase C in the human amnion.

Since glucocorticoids decrease and protein kinase C (PKC) activators increase amniotic PGE2 production, the possibility that they regulate the activity of prostaglandin endoperoxide H synthase (PGHS), the rate-limiting enzyme of prostaglandin synthesis from arachidonate, was investigated. Glucocorticoids inhibited the production of PGE2 from exogenous arachidonate specifically and in a concentration dependent fashion. Furthermore, cortisol decreased PGHS activity and the amount of PGHS protein in amnion microsomes, and reduced the rate of recovery of PGHS after acetylsalicylic acid (ASA) pretreatment. Actinomycin D blocked the inhibition of PGHS recovery by cortisol, but did not suppress the spontaneous recovery of the enzyme, indicating that the glucocorticoid induced a post-transcriptional inhibitor of PGHS synthesis. PKC-activating phorbol esters, such as 12-tetradecanoyl phorbol 13-acetate (TPA) increased the synthesis of PGE2 from exogenous arachidonate, also in a specific and concentration dependent manner. PGHS recovery after ASA treatment was enhanced by TPA. PGHS activity and protein concentrations were increased by phorbol ester treatment; however, this was apparent only in tissues in which the concentrations of PGHS were initially low. These results show that the synthesis of PGHS is positively and negatively regulated in the human amnion by PKC and glucocorticoids, respectively, and suggest that effectors using these pathways may regulate the enzyme in vivo.

Postnatal maturation of phrenic nerve in children.

The phrenic nerve at the pericardial level was examined postmortem in 17 children, ages 3 days to 8 years. Detailed macroscopic and histologic examination of the central nervous system in all patients disclosed no abnormalities. Quantitative developmental studies demonstrated that myelinated axons doubled in number from birth to age 1 year when a plateau was reached. The main period of growth in diameter of myelinated axons also corresponded to the first year when median diameters increased from 1.75 microns at 3 days of age to 3.0 microns by 8 months of age. Unmyelinated axons also grew significantly in the first 11 months when median diameters reached 1.4 microns. There was no significant increase in axonal diameter at later ages. The slope of the regression line for the number of myelin lamellae on axonal diameters increased with age until 6 months of age, whereas the dispersion around the regression lines decreased in the same period. This finding suggests a direct relationship between myelination and axonal growth. Significant maturation of the phrenic nerve occurs during the first year of life.

Prostaglandin- and isoproterenol-stimulated cyclic AMP accumulation in rat perinatal lung fibroblasts: effects of developmental age.

The fibroblast of the fetal and neonatal lung is intimately involved with lung development and function. Additionally, the perinatal rat lung fibroblast is a significant source of prostaglandins (PG) I2 and E2, which in turn affect lung development and function. Their effects may be mediated by cAMP. We, therefore, tested both the relative effectiveness of PG and the beta-adrenergic agonist, isoproterenol, and the developmental age sensitivity to these agonists on rat perinatal lung fibroblast cAMP accumulation. Confluent monolayer cultures of 3rd-passage fibroblasts (greater than 95% purity) from d-3 newborn rats responded in a concentration-dependent fashion to several PG (10(-8)-10(-4) M) and isoproterenol (10(-7)-2 x 10(-6) M) by increasing cAMP accumulation. The rank order of responsiveness, in terms of maximum accumulated cAMP, were carba PGI2 greater than PGE1 = PGI2 Na salt = PGI2 methyl ester much greater than PGE2 greater than isoproterenol. At the 3 developmental d tested [d 20 fetus, d 1 newborn, and d 3 newborn (term = d 22)], PGE2, carba PGI2, and isoproterenol each elicited concentration-dependent increases in cAMP accumulation. Unstimulated cAMP levels were 2-5 fmol/micrograms protein/15 min at all three ages. On d 20 of gestation, the highest accumulation achieved at the highest concentration tested was 30-70 fmol/micrograms protein/15 min for each agonist. There was no age-dependent change in responsiveness to PGE2. Carba PGI2-stimulated cAMP accumulation increased from d 20 of gestation with each advancing age tested to approximately 15-fold by d 3 newborn.(ABSTRACT TRUNCATED AT 250 WORDS)