Reduced fetal, placental, and amniotic fluid PTHrP in the growth-restricted spontaneously hypertensive rat

Evidence implicates pivotal roles for parathyroid hormone-related protein (PTHrP) in stimulating cell growth and differentiation, placental calcium transport, and placental vasodilatation. As spontaneously hypertensive rat (SHR) fetuses are growth restricted compared with those of its normotensive control, the Wistar Kyoto (WKY) rat, we examined intrauterine PTHrP and total and ionic calcium concentrations in these rats. Fetal plasma PTHrP concentrations, but not total calcium concentrations, were lower in the SHR compared with WKY (P < 0.05). SHR placental concentrations of PTHrP were lower than in WKY (P < 0.03) and failed to show the increase observed in WKY near term (P < 0.05). PTHrP concentrations in amniotic fluid from SHR were not raised near term and were lower compared with WKY (P < 0.0005). The increased ionic calcium concentrations in amniotic fluid in the WKY near term (P < 0.05) were not detected in the SHR. Thus SHR fetal plasma, placental, and amniotic fluid PTHrP concentrations were reduced and associated with fetal growth restriction. We suggest that PTHrP may play a role in the etiology of both growth restriction during pregnancy and hypertension later in life.     

Effect of estrogen supplementation on exercise thermoregulation in premenopausal women

This studyexamined the effects of 3 days of estrogen supplementation (ES) onthermoregulation during exercise in premenopausal (20-39 yr) adultwomen during the follicular phase of the menstrual cycle. Subjects (11 control, 10 experimental) performed upright cycle ergometer exercise at60% of maximal O₂ consumption ina neutral environment (25°C, 30% relative humidity) for 20 min. Subjects were given placebo (P) or -estradiol (2 mg/tablet, 3 tablets/day for 3 days). All experiments were conductedbetween 6:30 and 9:00 AM after ingestion of the last tablet. Heartrate, forearm blood flow (FBF), mean skin temperature, esophagealtemperature (T_es), and forearmsweat rate were measured. Blood analysis for estrogen and progesteronereflected the follicular phase of the menstrual cycle. MaximalO₂ consumption (37.1 ± 6.2 in P vs. 38.4 ± 6.3 ml · kg¹ · min¹in ES) and body weight-to-surface area ratio (35.58 ± 2.85 in P vs.37.3 ± 2.7 in ES) were similar between groups. Synthesis of 70-kDaheat shock protein was not induced by 3 days of ES. Neither thethreshold for sweating (36.97 ± 0.15 in P vs. 36.90 ± 0.22°C in ES), the threshold for an increase in FBF (37.09 ± 0.22 in P vs. 37.17 ± 0.26°C in ES), the slope ofsweat rate-T_es relationship (0.42 ± 0.16 in P vs. 0.41 ± 0.17 in ES), nor the FBF-T_es relationship (10.04 ± 4.4 in P vs. 9.61 ± 3.46 in ES) was affected(P > 0.05) by 3 days of ES. Weconclude that 3 days of ES by young adult women in the follicular phaseof their menstrual cycle have no effect on heat transfer to the skin,heat dissipation by evaporative cooling, or leukocyte synthesis of70-kDa heat shock protein.

     

Revisiting the phylogeography and demography of European badgers (Meles meles) based on broad sampling,multiple markers and simulations

Although the phylogeography of European mammals has been extensively investigated since the 1990s, many studies were limited in terms of sampling distribution, the number of molecular markers used and the analytical techniques employed, frequently leading to incomplete postglacial recolonisation scenarios. The broad-scale genetic structure of the European badger (Meles meles) is of interest as it may result from historic restriction to glacial refugia and/or recent anthropogenic impact. However, previous studies were based mostly on samples from western Europe, making it difficult to draw robust conclusions about the location of refugia, patterns of postglacial expansion and recent demography. In the present study, continent-wide sampling and analyses with multiple markers provided evidence for two glacial refugia (Iberia and southeast Europe) that contributed to the genetic variation observed in badgers in Europe today. Approximate Bayesian computation provided support for a colonisation of Scandinavia from both Iberian and southeastern refugia. In the whole of Europe, we observed a decline in genetic diversity with increasing latitude, suggesting that the reduced diversity in the peripheral populations resulted from a postglacial expansion processes. Although MSVAR v.1.3 also provided evidence for recent genetic bottlenecks in some of these peripheral populations, the simulations performed to estimate the method''s power to correctly infer the past demography of our empirical populations suggested that the timing and severity of bottlenecks could not be established with certainty. We urge caution against trying to relate demographic declines inferred using MSVAR with particular historic or climatological events.     

Effect of norgestomet on peripheral levels of progesterone and estradiol-17beta in beef cows

Peripheral levels of progesterone and estradiol 17beta were quantified in 27 cycling cows following administration of a single Hydron ear implant (G. D. Searle and Co.) containing 2, 4 or 6 mg norgestomet or controls which received no implant. Implants were inserted subcutaneously in the ear on day 15 of the estrous cycle (day of estrus = day 0) and removed 9 days later. The 4 mg (seven of seven cows) and 6 mg (six of six cows) implants suppressed estrus; however, three of eight cows in the 2 mg group exhibited estrus prior to implant removal. The 6 mg implant group had a significantly longer interval from implant removal to estrus than either the 2 or 4 mg group. Failure to detect differences in the rate at which progesterone declined indicated norgestomet treatment did not affect normal corpus luteum regression. Estradiol levels rose at a similar rate approaching estrus in all treatments. There was no indication of increased endogenous estradiol levels due to norgestomet treatment.     

Quantitative Phosphoproteomics Reveals Pathways for Coordination of Cell Growth and Division by the Conserved Fission Yeast Kinase Pom1

Complex phosphorylation-dependent signaling networks underlie the coordination of cellular growth and division. In the fission yeast Schizosaccharomyces pombe, the Dual specificity tyrosine-(Y)-phosphorylation regulated kinase (DYRK) family protein kinase Pom1 regulates cell cycle progression through the mitotic inducer Cdr2 and controls cell polarity through unknown targets. Here, we sought to determine the phosphorylation targets of Pom1 kinase activity by SILAC-based phosphoproteomics. We defined a set of high-confidence Pom1 targets that were enriched for cytoskeletal and cell growth functions. Cdr2 was the only cell cycle target of Pom1 kinase activity that we identified in cells. Mutation of Pom1-dependent phosphorylation sites in the C terminus of Cdr2 inhibited mitotic entry but did not impair Cdr2 localization. In addition, we found that Pom1 phosphorylated multiple substrates that function in polarized cell growth, including Tea4, Mod5, Pal1, the Rho GAP Rga7, and the Arf GEF Syt22. Purified Pom1 phosphorylated these cell polarity targets in vitro, confirming that they are direct substrates of Pom1 kinase activity and likely contribute to regulation of polarized growth by Pom1. Our study demonstrates that Pom1 acts in a linear pathway to control cell cycle progression while regulating a complex network of cell growth targets.The coordination of cell growth and division represents a fundamental concept in cell biology. The mechanisms that promote polarized growth and drive cell cycle progression are complex signaling networks that operate in a wide range of cell types and organisms. Understanding these networks and their molecular connections requires large-scale approaches that define the underlying biochemical reactions. Phosphorylation drives many events in both cell polarity and cell cycle signaling, and protein kinases that act in both processes represent key players in coordinated growth and division.The fission yeast S. pombe has served as a long-standing model organism for studies on cell polarity and the cell cycle. The fission yeast protein kinase Pom1 is an intriguing candidate to function in the coordination of polarized growth and cell cycle progression. This DYRK¹ family kinase was originally identified as a polarity mutant (hence the name Pom1) in a genetic screen for misshapen cells (1). Later studies revealed an additional role for Pom1 in cell cycle progression, where it delays mitotic entry until cells reach a critical size threshold (2, 3). Thus, pom1Δ mutant cells display defects in both cell polarity and cell size at mitosis, as well as misplaced division septa (1–6). Mutations that impair Pom1 kinase activity mimic these deletion phenotypes, indicating a key role for Pom1-dependent phosphorylation. The pleiotropic phenotype of pom1 mutants might result from Pom1 phosphorylating distinct substrates for cell polarity versus mitotic entry, but the targets of Pom1 kinase activity are largely unknown. Only two Pom1 substrates have been identified to date. First, Pom1 auto-phosphorylates as part of a mechanism that promotes localization in a cortical gradient enriched at cell tips (7). Second, Pom1 phosphorylates two regions of the protein kinase Cdr2. Phosphorylation of Cdr2 C terminus is proposed to prevent mitotic entry by inhibiting Cdr2 kinase activity (8, 9), while phosphorylation near membrane-binding motifs of Cdr2 promotes medial cell division by inhibiting localization of Cdr2 at cell tips (10). It has been unclear if Cdr2 represents the only cell cycle target of Pom1 kinase activity, and no cell polarity targets of Pom1 have been identified. In order to clarify how this protein kinase controls multiple cellular processes, we have comprehensively cataloged Pom1 substrates by quantitative phosphoproteomics. Such a large-scale approach also has the potential to reveal general mechanisms that operate in the coordination of cell growth and division.Stable isotope labeling of amino acids in culture (SILAC) combined with phosphopeptide enrichment and mass spectrometry has allowed the proteome-wide analysis of protein phosphorylation from diverse experimental systems (11–15). In this approach, cells are grown separately in media containing normal (“light”) or isotope-labeled (“heavy”) arginine and lysine, treated, mixed, and processed for LC-MS/MS analysis. In combination with analog-sensitive protein kinase mutants, which can be rapidly and specifically inhibited by nonhydrolyzable ATP analogs (16, 17), SILAC presents a powerful approach to identify cellular phosphorylation events that depend on a specific protein kinase. This method is particularly well suited for studies in yeast, where analog-sensitive protein kinase mutants can be readily integrated into the genome.In this study, we have employed SILAC-based phosphoproteomics to identify Pom1 substrates in fission yeast. New Pom1 targets were verified as direct substrates in vitro, and our analysis indicates that Pom1 controls cell cycle progression through a single target while coordinating a more complex network of cell polarity targets.