Pollination context alters female advantage in gynodioecious Silene vulgaris.

Gynodioecy, the co-occurrence of females and hermaphrodites, is arguably the most common angiosperm gender polymorphism in many florae. Females' ability to invade and persist among hermaphrodites depends, in part, on pollinators providing adequate pollination to females. We directly measured diurnal and nocturnal pollinators' contributions to female and hermaphrodite seed production in artificial populations of gynodioecious Silene vulgaris by experimentally restricting pollinator access. We found that female relative seed production in this system depended strongly on pollination context: females produced more than twice as many seeds as hermaphrodites in the context of abundant, nectar-collecting moths. Conversely, females showed no seed production advantage in the context of pollen-collecting syrphid flies and bees due to acutely hermaphrodite-biased visitation. We infer that variation in pollinator type, behaviour and abundance may be important for achieving the female relative fitness thresholds necessary for the maintenance of gynodioecy. Generally, our study illustrates how pollinator-mediated mechanisms may influence the evolution of breeding systems and associated suites of floral traits. Segments of a pollinator community may facilitate gynodioecy by selecting for plant characteristics that increase the attractiveness of both sexes to pollinators, such as nectar rewards. Conversely, discriminating visitors in search of pollen may restrict gynodioecy in associated plant lineages by reducing male steriles' fitness below threshold levels.

Characterization of rat liver subcellular membranes. Demonstration of membrane-specific autoantigens.

The induction of acute hepatocellular necrosis in rats resulted in the production of complement fixing, IgM autoantibodies directed toward inner and outer mitochondrial membranes, microsomal membrane, lysosomal membrane, nuclear membrane, cytosol, but not to plasma membrane. Utilizing selective absorption procedures it was demonstrated that each subcellular membrane fraction possessed unique autoantigenic activity with little or no cross-reactivity between the various membrane fractions. It is proposed that the development of membrane-specific autoantibodies may provide an immunological marker useful in the differential characterization of various subcellular membranes.

The EMG of conventional abdominal exercise and exercise with a semi-upright commercial device: comparative effects and technique considerations.

AIM: The purpose of this study was to measure and compare the muscle activity of the rectus abdominis (RA) and external obliques (EO) with conventional exercises and while using an upright commercial abdominal training device (the CoreMaster). It was hypothesized that the upright device would elicit higher electromyography (EMG) values compared to conventional abdominal exercises. METHODS: Fifteen subjects (8 males, 7 females) participated in this study. Each subject performed 10 repetitions for 5 exercises: truck lift (TL); trunk rotation to opposite knee (TROK); trunk lift on the CoreMaster (TLCM); trunk rotation on the CoreMaster (TRCM); and trunk rotation with a leg lift on the CoreMaster (TRLLCM). Muscle activity was measured for the RA and EO using surface EMG. A Biopac system (Goleta, CA, USA) processed the EMG signals. A repeated measures analysis of variance (ANOVA) determined any difference in the root mean square values and Bonferroni comparisons were used to clarify the order of differences (P<0.05). RESULTS: For the RA, all exercises on the CoreMaster produced significantly higher EMG values compared to the conventional TL. For the EO, TRCM elicited the highest EMG values. However, no significant difference was found for EO between TROK and TRLLCM. CONCLUSION: The CoreMaster elicited a greater challenge to the RA. For the EO, the CoreMaster yielded optimal effects for exercises that required pronounced rotation.

Consumption of classical complement components by heart subcellular membranes in vitro and in patients after acute myocardial infarction.

Experiments were conducted to characterize the antibody-independent activation of complement in human serum by isolated human heart mitochondrial membranes in vitro and to determine whether similar patterns of complement consumption occurred in patients after acute myocardial infarction. Direct evidence for the interaction of C1 and heart mitochondrial membranes was obtained by mitochondria-C1 binding and elution experiments. Exposure of normal human sera to isolated human heart mitochondria at 37 degrees C resulted in the consumption of C1, C4, C2, and C3 without significant consumption of the terminal components of the complement system (C6 through C9). The consumption occurred in the absence of detectable anti-heart mitochondria autoantibody, was demonstrated to be calcium dependent, and was inhibited by either 0.01 M EDTA or ethylene glycol bis(bets-aminoethyl ether) N,N,N',N',-tetraacetic acid (EDTA). Although specific absorption of C1q from human sera inhibited the mitochondria-dependent activation of C4, C3 donsumption was not affected. These data indicate that the consumption of C4 and C2 likely occurred due to the mitochondrial membrane-mediated activation of C1, but that the consumption of the C3 did not necessarily involve either the classical or alternative complement pathways. After the in vitro characterization of the mitochondria-dependent activation of the complement system, additional studies were performed to determine whether similar consumption occurred in patients after acute myocaridal infarction. During a 72-h period after hospital admission significant decreases in C1, C4, and C3 occurred in six patients with recent chest pain but no evidence of acute myocardial infarction. These studies suggest that myocardial cell necrosis results in the release of subcellular membrane constituents capable of activating the complement system in the absence of detectable anti-heart autoantibodies; such activation may be responsible in part for the development of acute inflammation and evolution of the infarct size following coronary artery occulusion.

Complement localization and mediation of ischemic injury in baboon myocardium.

We sought to determine whether the third component of complement (C3) is localized in ischemic baboon myocardium after coronary artery ligation. Furthermore, we assessed the effects of prior C3 depletion on myocardial necrosis. We studied seven control baboons (group I) and seven C3-depleted (group II) baboons that were killed 24 h after ligation of the anterior descending coronary artery. Multiple tissue samples were obtained from infarct, intermediate, and normal myocardial sites as defined by serial unipolar epicardial ECG mapping. In group I baboons, myocardial creatine kinase content from infarct sites was reduced as compared with normal sites (12.6+/-0.92 [SE] vs. 24.4+/-0.75 IU/mg protein, P < 0.001). The intermediate sites from group I contained more creatine kinase (19.0+/-1.25 IU/mg protein) than infarct sites (P < 0.001), but less (P < 0.025) than normal sites. In group II, intermediate sites showed no significant reduction in creatine kinase from normal sites and there was significantly less creatine kinase depletion in infarct sites when compared with group I animals (33.7+/-4.6 and 51.4+/-1.8% depletion, respectively, P < 0.001). In all seven group I baboons, uniform C3 localization was observed in infarct sites by direct immunofluorescence but appeared in mosaic patterns in intermediate sites. C3 was not demonstrated in any normal sites, nor in any site from group II baboons. Additional studies on baboons killed at earlier times after ligation indicated that C3 was localized focally on swollen myocytes in infarct sites as early as 4 h after coronary ligation. These results strongly implicate the active participation of the complement system of inflammatory proteins in the pathogenesis of myocardial tissue injury following coronary occlusion.

Regulation of gluconeogenesis from pyruvate and lactate in the isolated perfused rat liver.

The effects of glucagon and the alpha-adrenergic agonist, phenylephrine, on the rate of 14CO2 production and gluconeogenesis from [1-14C]lactate and [1-14C]pyruvate were investigated in isolated perfused livers of 24-h-fasted rats. Both glucagon and phenylephrine stimulated the rate of 14CO2 production from [1-14C]lactate but not from [1-14C]pyruvate. Neither glucagon nor phenylephrine affected the activation state of the pyruvate dehydrogenase complex in perfused livers derived from 24-h-fasted rats. 3-Mercaptopicolinate, an inhibitor of the phosphoenolpyruvate carboxykinase reaction, inhibited the rates of 14CO2 production and glucose production from [1-14C]lactate by 50% and 100%, respectively. Furthermore, 3-mercaptopicolinate blocked the glucagon- and phenylephrine-stimulated 14CO2 production from [1-14C]lactate. Additionally, measurements of the specific radioactivity of glucose synthesized from [1-14C]lactate, [1-14C]pyruvate and [2-14C]pyruvate indicated that the 14C-labeled carboxyl groups of oxaloacetate synthesized from 1-14C-labeled precursors were completely randomized and pyruvate----oxaloacetate----pyruvate substrate cycle activity was minimal. The present study also demonstrates that glucagon and phenylephrine stimulation of the rate of 14CO2 production from [1-14C]lactate is a result of increased metabolic flux through the phosphoenolpyruvate carboxykinase reaction, and phenylephrine-stimulated gluconeogenesis from pyruvate is regulated at step(s) between phosphoenolpyruvate and glucose.

Studies of mitochondrial calcium movements using chlorotetracycline.

1. The association of calcium with isolated rat liver mitochondrial membranes under various metabolic conditions was monitored using the fluorescent chelate probe, chlorotetracycline. Chlorotetracycline fluorescence increased markedly during energized calcium uptake in the absence of a permeant anion. Uncoupler and a respiratory chain inhibitor caused a rapid decrease in chlorotetracycline fluorescence when added either before or after calcium. During calcium uptake experiments concentrations of calcium exceeding 100 muM caused a transient fluorescence increase followed by an extensive decrease in fluorescence. 2. Changes in the chlorotetracycline-associated fluorescence of the mitochondrial suspensions were correlated with the uptake of exogenous 45Ca. A positive correlation was observed between fluorescence and energized 45Ca uptake in the absence of permeant anions. Addition of the permeant anion, phosphate, caused an extensive decrease in chloretetracycline fluorescence but an enhanced uptake of exogenous 45Ca. 3. The interaction of endogenous mitochondrial calcium with the fluorescent chelate probe was studied under a number of experimental conditions using mitochondria labeled during preparation with 45Ca. Endogenous 45Ca was lost rapidly from the mitochondria upon treatment with uncoupler, antimycin A, and A23187. Potassium phosphate and EGTA had no effect on the endogenous calcium as measured by either the 45Ca content of the mitochondria or the fluorescence of the probe. 4. Mitochondria treated with antimycin A lost most of their endogenous 45Ca within 3 min; subsequent energization of the mitochondria resulted in a partial uptake of the released 45Ca but caused nearly a complete return of the chlorotetracycline fluorescence to the original level. Addition of phosphate did not change the fluorescence level but resulted in an almost complete accumulation of the 45Ca previously released. 5. Following this energized uptake of 45Ca, EGTA, p-trifluoromethoxyphenyl hydrazone of carbonyl cyanide, A23187 and calcium chloride all caused a nearly complete loss of the 45Ca from the mitochondria and, with the exception of calcium chloride, caused an extensive decrease in the fluorescence level. Hence, the apparent location and/or properties of the endogenous calcium in this rat liver mitochondrial system were altered significantly by manipulation of the energetic state of the mitochondrial membrane.

Beta-adrenergic inhibition of AGEPC-stimulated Na+/Ca2+ exchange and AGEPC-induced platelet activation.

Recently, AGEPC (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was found to initiate contraction of ileal smooth muscle strips and to enhance Na+/Ca2+ exchange in ileal plasmalemmal vesicles. In the present study, the effects of the smooth muscle relaxant, isoproterenol, on Na+/Ca2+ exchange in rat ileal plasmalemmal vesicles was examined. In this preparation, Na+/Ca2+ exchange was stimulated 131 +/- 8% and 264 +/- 19% by addition of 50 nM and 100 nM AGEPC, respectively. Isoproterenol, a beta-adrenergic agonist, inhibited AGEPC stimulation of Na+/Ca2+ exchange in a dose- and time-dependent manner but had no effect on basal rates of Na+/Ca2+ antiport. At 1 microM, isoproterenol inhibited 86% of the Na+/Ca2+ exchange stimulated by 50 nM AGEPC. Vesicular cAMP levels were increased over 100% following the addition of 1 microM isoproterenol for 30 s. Inhibition of AGEPC-stimulated vesicular Na+/Ca2+ exchange and elevation of vesicular cAMP levels by isoproterenol was prevented by the beta-receptor antagonist propranolol (5 microM), demonstrating that these effects of isoproterenol were mediated by interaction with vesicular beta-adrenergic receptors. Additional studies with washed rabbit platelets demonstrated that isoproterenol inhibited AGEPC-induced aggregation and serotonin release. These effects of isoproterenol were dose- and time-dependent and were antagonized by propranolol. Isoproterenol had no effect on thrombin-induced aggregation and did not change appreciably platelet cAMP levels. Moreover, dibutyryl cAMP could not mimic the effect of isoproterenol to inhibit an AGEPC-induced aggregation. On a molar basis, the inhibitory effects of isoproterenol toward AGEPC action were greater in the ileal preparation than in the platelets. It is suggested that beta-adrenergic agonists may modulate AGEPC-induced ileal Na+/Ca2+ exchange and AGEPC-induced platelet aggregation through cAMP-dependent and-independent mechanisms, respectively.

Platelet-activating factor-mediated synthesis of prostaglandins in rat Kupffer cells.

Synthesis of prostaglandins was stimulated in rat Kupffer cells upon challenge with platelet-activating factor (PAF). PAF-mediated synthesis of prostaglandins was inhibited by the Ca2+ ion chelator (EGTA), the Ca2+ channel antagonist (nifedipine) and U66985, a structural analogue and antagonist of the biological effects of PAF in other cellular systems. Inhibitors of protein kinase C, staurosporine and polymixin B, did not affect PAF-induced prostaglandin synthesis. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, stimulated synthesis of prostaglandins in Kupffer cells; PAF and PMA exerted additive actions on this process. Both PAF- and PMA-stimulated prostaglandin production was inhibited by TMB-8. PAF-stimulated synthesis of prostaglandins also was inhibited upon treatment of Kupffer cells with pertussis toxin. Cholera toxin, in contrast, stimulated the production of prostaglandins in a concentration-dependent manner; cholera toxin and PAF together had an additive effect. These results suggest that PAF-induced synthesis of prostaglandins is stimulated via a specific receptor coupled to a pertussis toxin-sensitive G-protein, is dependent upon extracellular Ca2+ and is not influenced by protein Kinase C activation. Since PAF and prostaglandins are produced in the liver under conditions such as endotoxemia, PAF-mediated synthesis of these lipid autacoids may be of importance in the regulation of hepatic function during pathophysiological episodes.

Characterization of autoantigenic sites on isolated dog heart mitochondria.

1. Anti-heart mitochondria autoantibodies were developed in serum from dogs following experimental myocardial infarction. 2. Heart mitochondria frozen and thawed repeatedly in a sucrose/Tris-chloride buffer retained both their functional integrity as measured by the respiratory control ratio and their ability to serve as an antigen in a complement fixation test. Mitochondria frozen and thawed in a potassium chloride/Tris-chloride buffer lost both their functional integrity and their autoantigenic activity after one freeze-thaw cycle. 3. Extraction of the heart mitochondria with acetone/water mixtures to remove phospholipids from the membrane led to a complete loss of the ability of the mitochondria to react in the complement fixation test but did not affect the ability of the membranes to bind autoantibody in absorption experiments. 4. Treatment of the mitochondrial membranes with increasing concentrations of trypsin caused a loss of up to approximately 50% of the membrane protein with a gradual decrease in the autoantigenic activity of the membrane without impairment of the ability of the membrane to bind autoantibody. 5. Removal of up to 90% of the sialic acid of the mitochondrial membrane with neuraminidase resulted in a considerable increase in the complement-fixing autoantigenic activity of the membrane without changing the apparent ability of the membrane to bind autoantibody in absorption experiments. 6. Exposure of mitochondrial membranes to autoantibody and complement caused an inhibition of both an inner mitochondrial membrane enzyme, i.e. cytochrome oxidase (48%) and an outer mitochondrial membrane enzyme, i.e. NADH cytochrome c reductase (rotenone insensitive) (37%).

Fluorocarbon perfusion of the isolated rat brain: measurement of tissue spaces, EEG and oxygen uptake.

Previously, we have used the isolated perfused rat brain (IPRB) to demonstrate authentic cerebral synthesis of the lipid mediator platelet-activating-factor (Kumar, R., Harvey, S.A.K., Kester, M., Hanahan, D.J. and Olson, M.S. (1988) Biochim. Biophys. Acta 963, 375-383). The present study demonstrates that this fluorocarbon perfusion technique maintains the integrity of the blood-brain barrier (BBB), as evidenced by the small volume (1.77-3.33%) accessible to [carboxyl-14C]inulin. 51-66% of the brain was accessible to 3H2O, except for the spinal cord which is poorly perfused (16% accessible to 3H2O). There is no effective perfusion of muscle tissue associated with the preparation (less than 6% accessible to 3H2O). Fast Fourier Transform analysis of digitized EEG data showed that in low frequency bands (less than 7.5 Hz) the IPRB had reduced electrical activity relative to the whole conscious animal. The GABA antagonist bicuculline, which has convulsant effects in vivo, causes a 3-4-fold increase in overall (root-mean-square) electrical activity, but decreases further the relative amplitude of low frequencies. With appropriate corrections, measurement of the oxygen consumption of the IPRB can be made without the necessity for venous cannulation. Oxygen consumption of the IPRB is flow-dependent. At a perfusion rate of 1.54 ml/min per g, unstimulated oxygen consumption of the IPRB is 2.07-2.23 mumol/min per g, or 67-72% of the consumption of the brain in vivo. Administration of bicuculline to the IPRB causes a 31% increase in lactate efflux, but only a 15% increase in oxygen uptake, suggesting that the preparation becomes functionally ischemic. Measurement of ATP/ADP levels in control and bicuculline-treated IPRBs confirms this. Other workers have used the IPRB as a model for the cerebral effects of pharmacological agents and of metabolic insult. The present study shows that under various experimental conditions oxygen uptake, analytical EEG measurements, and the integrity of the blood-brain barrier all can be monitored.

Endothelin association with cultured rat hepatic endothelial cells: functional characterization.

Endothelin is a potent vasoactive peptide whose concentration increases in a number of pathophysiological states. In the intact animal, the liver is known to sequester approximately 12% of an injected bolus of [125I]endothelin-1 ([125I]ET-1). Endothelial cells (ECs) isolated from rat liver were maintained in culture in order to examine their role in ET sequestration. LECs were shown to express predominantly ET(B) receptors both by association assays and by Northern blot analysis. In these cells the reaction between [125I]ET-1 and its receptor was essentially irreversible. Ligand binding experiments performed at 4 degrees C showed that LECs in early culture (approximately 3 h) had 4.3 +/- 0.8 fmol of ET receptors per 10(6) cells; this number fell progressively to < or = 1 fmol/10(6) cells during 24 h of culture. The decrease in receptor numbers could be blocked by maintaining the cells at 4 degrees C. Northern blot analysis showed that relative to freshly isolated cells, mRNA for the ET(B) receptor decreased 4-fold in early culture, and recovered somewhat at 24 h. At 37 degrees C [125I]ET-1 bound by the cells was rapidly internalized, with concomitant down-regulation of ET receptors. Recovery of down-regulated ET receptors was sensitive to cycloheximide, making short-term receptor recycling unlikely. Metabolism of [125I]ET-1 was low at short (< 4 h) exposure times, and at 24 h showed a concentration dependence similar to that of ligand association, suggesting that ET-1 metabolism primarily was intracellular. ET stimulation of Kupffer cells and other hepatic cell types is known to activate phosphoinositide signaling, but no such activation was seen in LECs. Moreover, ET did not appear to stimulate protein tyrosine kinase activity in LECs. While hepatic LECs may lack some of the ET-dependent responses seen in other cell types, they likely contribute substantially to the liver's previously reported ability to sequester systemically administered ET.

Regulation of platelet-activating factor (PAF) biosynthesis via coenzyme A-independent transacylase in the macrophage cell line IC-21 stimulated with lipopolysaccharide.

The regulation of PAF synthesis by the macrophage cell line IC-21 challenged with bacterial endotoxin was investigated. The LPS-induced increase in cellular PAF levels was rapid, sustained and attained maximal levels within 30 min following LPS stimulation. PAF accumulation was accompanied by the activation of the CoA-independent transacylase and acetyl-CoA: lyso-PAF acetyltransferase, whereas the release of free [3H]arachidonic acid in prelabeled cells reflecting the activation of phospholipase A2, occurred primarily within the initial 1-5 min of treatment with LPS. Cell lysates from LPS-stimulated macrophages exhibited a markedly increased enzymatic activity that was capable of both acylation of 1-[3H]alkyl-2-lyso-GPC (lyso-PAF) and deacylation of 1-[3H]alkyl-2-acyl-GPC generating [3H]lyso-PAF via CoA-independent transacylation of exogenous lysoplasmenylethanolamine compared with extracts from resting macrophages. Pretreatment of the cells with LPS for 5 and 30 min enhanced significantly the transfer of [14C]arachidonic acid from 1-[3H]alkyl-2-[14C]arachidonoyl-GPC into plasmenylethanolamine in prelabeled cell homogenates following the addition of exogenous lysoplasmenylethanolamine. Taken together, these data suggest that the CoA-independent transacylase, but not phospholipase A2, is a key enzyme responsible for the prolonged generation of lyso-PAF and that the increased capability of CoA-independent transacylation followed by CoA-dependent acetylation of lyso-PAF can sustain the biosynthesis of PAF in LPS-stimulated IC-21 macrophages.

Production and effects of platelet-activating factor in the rat brain.

The synthesis of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in rat brain was evaluated. Extracted PAF was characterized using standard HPLC and TLC techniques, and by correlation of its bioactivity with the acetylation state of the 2-position of the molecule. PAF was quantified by bioassay, its ability to cause [3H]serotonin release from washed rabbit platelets. The low basal level of PAF (0.25 +/- 0.15 pmol/g wet wt., mean +/- S.E.) in the brain of the intact rat was greatly increased by intraperitoneal injection of the chemoconvulsant drugs picrotoxin or bicuculline, to levels of 10.68 +/- 2.18 and 4.97 +/- 0.75 pmol/g wet wt., respectively. Electroconvulsion also increased brain PAF, to 1.76 +/- 0.30 pmol/g wet wt. Equivalent experiments using bicuculline in the isolated perfused rat brain yielded qualitatively similar results, indicating that the production of PAF in the brain is independent of systemic metabolism. When a 32P-labeled nerve-ending (synaptosome) preparation from rat brain was challenged with synthetic PAF (denoted AGEPC) at 0.1 nM concentration, responses were observed consistent with accelerated turnover of polyphosphoinositides. AGEPC also caused an increase in the Na+-Ca2+ exchange of synaptic membrane vesicles. Furthermore, AGEPC infused into the vasculature of the isolated perfused rat brain caused changes consistent with an increase in blood-brain barrier permeability, although AGEPC did not itself significantly penetrate the blood-brain barrier. It is concluded from these studies that PAF is synthesized within the rat brain in response to convulsant stimuli and that one of its effects is to accelerate synaptic polyphosphoinositide turnover. In addition, circulating PAF can influence blood-brain barrier permeability without itself penetrating the blood-brain barrier.

Bayesian procedures for discriminating among hypotheses with discrete distributions: inheritance in the tetraploid Astilbe biternata.

Discrimination between disomic and tetrasomic inheritance aids in determining whether tetraploids originated by allotetraploidy or autotetraploidy, respectively. Past assessments of inheritance in tetraploids have used analyses whereby each inheritance hypothesis is tested independently. I present a Bayesian analysis that is appropriate for discriminating among several inheritance hypotheses and can be used in any case where hypotheses are defined by discrete distributions. The Bayesian approach incorporates prior knowledge of the probability of occurrence of disomic and tetrasomic hypotheses so that the results of the analysis are not biased by the fact that there is a single tetrasomic hypothesis and multiple disomic hypotheses. This analysis is used to interpret data from crosses in the tetraploid Astilbe biternata, a herbaceous plant native to the southern Appalachians. The progeny ratios from all crosses favored the hypothesis of disomic inheritance at both the PGM and slow-PGI loci. These results support earlier cytogenetic evidence for the allotetraploid origin of Astilbe biternata.

A quantitative genetic analysis of nuclear-cytoplasmic male sterility in structured populations of Silene vulgaris.

Gynodioecy, the coexistence of functionally female and hermaphroditic morphs within plant populations, often has a complicated genetic basis involving several cytoplasmic male-sterility factors and nuclear restorers. This complexity has made it difficult to study the genetics and evolution of gynodioecy in natural populations. We use a quantitative genetic analysis of crosses within and among populations of Silene vulgaris to partition genetic variance for sex expression into nuclear and cytoplasmic components. We also use mitochondrial markers to determine whether cytoplasmic effects on sex expression can be traced to mitochondrial variance. Cytoplasmic variation and epistatic interactions between nuclear and cytoplasmic loci accounted for a significant portion of the variation in sex expression among the crosses. Source population also accounted for a significant portion of the sex ratio variation. Crosses among populations greatly enhanced the dam (cytoplasmic) effect, indicating that most among-population variance was at cytoplasmic loci. This is supported by the large among-population variance in the frequency of mitochondrial haplotypes, which also accounted for a significant portion of the sex ratio variance in our data. We discuss the similarities between the population structure we observed at loci that influence sex expression and previous work on putatively neutral loci, as well as the implications this has for what mechanisms may create and maintain population structure at loci that are influenced by natural selection.

Compartmental analysis of 45Ca2+ efflux in perfused rat liver: effects of hormonal stimulation.

The kinetics of calcium movements in the isolated perfused rat liver were examined using compartmental analysis of the efflux profiles of 45Ca2+ from 45Ca(2+)-equilibrated livers under a variety of calcium concentrations and hormonal treatments. From the 45Ca2+ efflux profiles, we determined that a three compartment model was appropriate to describe the movements of calcium in the liver on the time scale of the experiments. Hormonal treatment with the alpha-adrenergic agonist, phenylephrine, or the vasoactive peptide, vasopressin, during the efflux period lowered significantly the rate of transfer of Ca2+ between the internal compartments at all of the calcium concentrations employed. Also, phenylephrine treatment leads to increased transfer of Ca2+ into the liver from the perfusate. The temporal characteristics of the phenylephrine and vasopressin sensitive Ca2+ pools were examined by pulsing livers, loaded for variable periods of time with 45Ca2+, with the two hormones during the efflux of 45Ca2+ to measure the kinetics of Ca2+ exchange in the hormone-sensitive pools. Results from these experiments indicate that the rate of unstimulated Ca2+ efflux, k2, for the phenylephrine and vasopressin sensitive Ca2+ pools, modeled as a one compartment system, are the same, 0.074 and 0.078 min-1 for phenylephrine and vasopressin respectively, corresponding to half times for turnover of the pool(s) of 9.3 and 8.9 min, respectively.

Changes in the structure of pyruvate dehydrogenase complex induced by mono- and divalent ions.

The activity of pyruvate dehydrogenase complex purified from pig kidney cortex was affected by various mono- and di-valent ions and changes in ionic strength. The fluorescence emission spectrum of PDC exposed to 0.04 M ionic strength and excited at 280 nm exhibited a maximum at 334 nm; the fluorescence intensity of PDC appeared to depend upon the ionic strength and the K+ and Na+ content of the incubation buffer. Alteration of ionic strength to which the enzyme complex was exposed produced a change in the absorption of the complex at 230 nm. The presence of HPO4(2-) ions prevented changes in the UV absorption spectrum of PDC induced by the variation in ionic strength. The K+ and Na+ ions alone had no effect on the UV spectrum of PDC. Upon increasing the ionic strength to which the enzyme complex was exposed, dramatic changes in the circular dichroism (CD) pattern were observed. At 0.04 M ionic strength PDC exhibited a CD spectrum with minima at 216, 218 and 222 nm and a cross-over point at 215 nm. At 0.15 M ionic strength the CD spectrum of PDC exhibited minima at 223, 226, 228 nm and a cross-over point at 221 nm. The presence of HPO4(2-) ions prevented alterations in the CD spectrum of PDC induced by variations in ionic strength. The K+ and Na+ ions had no effect on the CD spectrum of PDC.

Stimulation and homologous desensitization of calcitonin gene-related peptide receptors in cultured beating rat heart cells.

Addition of calcitonin gene-related peptide (CGRP), 1 X 10(-7) M, to cultured neonatal rat heart cells resulted in rapid increases in beating rate and cellular concentrations of cAMP. Calcitonin (1 X 10(-7) M), in contrast, had no significant effect on heart cell beating rate or cAMP content. CGRP-stimulated increases in heart cell cAMP content were rapid, transient, dose dependent, and potentiated by isobutyl-methylxanthine (1 X 10(-4) M). Half-maximal increases in heart cell cAMP content occurred at 1 X 10(-8) M CGRP. Heart cell adenylate cyclase responses to CGRP were desensitized in a rapid (i.e. within 5 min) and dose-dependent manner by prior exposure to CGRP. Complete and half-maximal desensitization of heart cells to CGRP occurred at 1 X 10(-8) and 3 X 10(-10) M CGRP, respectively. Desensitization of heart cells to CGRP did not modify the cAMP response of heart cells to beta-adrenergic agonist stimulation, and beta-adrenergic agonist desensitization of heart cells did not modify responses to CGRP. Heart cell cAMP responses to CGRP were additive to those of the beta-adrenergic agonist isoproterenol and occurred in the presence of beta-adrenergic blockade. These observations demonstrate that CGRP exerts specific and potent agonist actions in cardiac myocytes and that regulation of myocardial responses to CGRP may occur by mechanisms involving increases in cAMP and receptor desensitization.

Osteogenic protein-1 and insulin-like growth factor I synergistically stimulate rat osteoblastic cell differentiation and proliferation.

Previous studies have shown that osteogenic protein-1 (OP-1; also known as BMP-7) alters the steady state levels of messenger RNA (mRNA) encoding insulin-like growth factor I (IGF-I), IGF-II, and IGF-binding proteins (IGFBPs) in primary cultures of fetal rat calvaria (FRC) cells. In the present study, the effects of exogenous IGF-I on bone cell differentiation and mineralized bone nodule formation induced by OP-1 were examined. Exogenous IGF-I synergistically and dose dependently enhanced OP-1 action in stimulating [3H]thymidine incorporation, alkaline phosphatase activity, PTH-dependent cAMP level, and bone nodule formation. Maximal synergism between OP-1 and IGF-I was observed when both factors were added simultaneously. Synergism was not observed when FRC cells were pretreated with IGF-I for 24 h, followed by OP-1 treatment. These findings suggest that IGF-I acted on OP-1-sensitized FRC cells. To examine the mechanism(s) by which this sensitization may occur, levels of mRNA encoding OP-1 receptor, IGF-I receptor, and IGFBPs were measured. The mRNA levels of both type I and II OP-1 receptors were elevated by OP-1, but were not changed further by combined OP-1 and IGF-I treatment. IGF-I receptor gene expression was not changed by OP-1, IGF-I, or a combination of both factors. OP-1 alone or together with IGF-I increased the steady state IGFBP-3 mRNA level and reduced the steady state mRNA levels of IGFBP-4, -5, and -6. IGF-I alone did not change the steady state mRNA levels of IGFBP-3, -4, and -6, but elevated that of IGFBP-5. Des(1-3)-IGF-I, which has a lower affinity for IGFBPs, was more effective than the full-length IGF-I in enhancing the OP-1-induced alkaline phosphatase activity. Exogenous IGFBP-5 inhibited the OP-1-induced alkaline phosphatase activity and reduced the synergistic stimulatory effect of IGF-I and OP-1. These findings strongly suggest that the OP-1-induced down-regulation of IGFBPs, especially that of IGFBP-5, is an important mechanism by which OP-1 and IGF-I synergize to stimulate FRC cell differentiation.