Calcium level in organelles of the slime mold Physarum polycephalum was monitored by chlortetracycline, a low-affinity calcium indicator. It was found that 2,5'-di(tertbutyl)-1,4,-benzohydroquinone (BHQ) at a concentration of 100 microM, but not the highly specific inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), thapsigargin (1-10 microM), elicited calcium release from the CTC-stained intracellular calcium pool. Ionomycin also caused a calcium release (23.7+/-5.1%), which was less than that induced by BHQ (30.1+/-6.0%). Procaine (10 mM), a blocker of ryanodine receptor, completely abolished the responses to BHQ and ionomycin. Another blocker, ryanodine (100 microM), only slightly diminished the responses to ionomycin and BHQ. Apparently, BHQ and ionomycin acting as a Ca2+-ATPase inhibitor and an ionophore, respectively, elicit an increase in [Ca2+]i, which in turn triggers a calcium-induced calcium release (CICR) via the ryanodine receptor. Caffeine, an activator of ryanodine receptor, at a concentration of 25-50 mM produced a Ca2+-release (5.6-16.0%), which was not similar in magnitude to CICR. The response to 25 mM caffeine was only moderately inhibited by 25 mM procaine, and almost completely abolished by 50 mM procaine and 100 microM ryanodine.
Callosobruchus maculatus (Cm) and Zabrotes subfasciatus (Zs) were reared on resistant (IT81D-1045) and on susceptible (Epace 10) cowpea seeds. The emergence of adult insects, total developmental period (TDP) and excretion of trypsin inhibitor and vicilin were determined for both bruchid populations. Parameter evaluation showed that the Zs populations emerged from both seeds had no significant differences in emergence and TDP. The Cm population raised from resistant seeds had lower emergence (5.6+/-1.3%) and delayed TDP (46+/-1.25 days) than those emerged from susceptible seeds. The excretion of defense proteins showed that Zs reared in resistant seeds excreted 1.7 times more trypsin inhibitor, but this did not affect emergence or TDP. Furthermore, Cm population emerged from resistant seeds excreted 7 times higher vicilin and 0.4 times less trypsin inhibitor than that emerged from susceptible seeds. These results indicate that vicilins from resistant seeds are involved to significantly longer TDP (46 days) and also drastic reduction of insect emergence ( approximately 5%) of C. maculatus.
In natural spawning grounds, breeding round goby, Neogobius melanostomus, males are exposed to various social stimuli, including high density of same-sex competitors and separation from females. We hypothesize that breeding males subjected to overcrowding in the wild experience high stress that affects their socio-sexual behavior and their relationships among conspecifics. We designed an experiment to mimic natural stimulation when highly aggregated breeding males are subjected to same-sex opponents. Males were sampled sequentially from experimental tank stocked at decreasing fish densities of 15 fish/m(2), 9 fish/m(2) and 4 fish/m(2). We studied the effects of overcrowding on male behavior and selected hormones, brain arginine vasotocin (AVT) and isotocin (IT) and plasma 11-ketotestosterone (11-KT) and cortisol as these are known to play roles in reproduction and related social interactions. The highest brain AVT and plasma cortisol levels were measured in non-aggressive males kept in the overcrowded group of 15 fish/m(2). IT level was elevated in fish kept at the lower density of 9 fish/m(2), and at which the males began to display territoriality and aggression. The plasma level of 11-KT was similar in all the males. Brain AVT and IT and plasma cortisol along with behavioral observations can be applied as species-specific indicators of the well-being of round goby males.
Cortisol, the main corticosteroid in fish, is frequently described as a modulator of fish immune system. Moreover, 11-deoxycorticosterone (DOC) was shown to bind and transcriptionally activate the mineralocorticoid receptor and may act as a mineralocorticoid in fish. Immune modulations induced by intraperitoneal injections of these two corticosteroids were assessed in Eurasian perch juveniles. Cortisol and DOC were injected at 0.8mgkg-1 and 0.08mgkg-1 body weight respectively. Cortisol increased plasma lysozyme activity 72h post-injection, C-type lysozyme expression in spleen from 1 to 72h post-injection, and favoured blood neutrophils at the expense of a mixture of lymphocytes and thrombocytes. Moreover, 6h after injection, cortisol reduced expression levels of the pro-inflammatory cytokine TNF-α in spleen.DOC had no effects on the immune variables measured in plasma, but increased expression levels of C-type lysozyme and apolipoprotein A1 mRNA in both gills and spleen. Meanwhile, DOC stimulated its putative signalling pathway by increasing expression of mineralocorticoid receptor and 11β-hydroxysteroid dehydrogenase-2 in spleen. These results confirmed the role of cortisol as an innate, short term immune stimulator. For the first time, DOC is described as a possible immune stimulator in fish.
The developmental transition from a residential, immature 'yellow' eel to a migratory, maturing adult 'silver' eel is accompanied by many morphological changes that appear to be under endocrine control. High circulating levels of the teleost, and usually male-specific, androgen 11-ketotestosterone (11-KT) are found in migrating female short-finned eels, Anguilla australis. We examined the role of this steroid in silvering by implanting immature, female short-finned eels either with blank vehicles or with vehicles containing 11-KT. Six weeks after they had received the implants, eels treated with 11-KT had developed 'chisel-shaped' snouts and black pectoral fins with tapered ends, and the size of their eyes had increased significantly. 11-KT treated eels had a thicker dermis than control eels and an epidermis with fewer or no mucous cells. Ventricular mass at the end of the experiment was two-fold larger than in control eels. 11-KT treated eels also had larger livers and gonads. Ovaries contained predominantly cortical alveolus stage III oocytes, as opposed to the smaller gonads of control eels containing previtellogenic stage II oocytes. All of these changes correspond to changes during the developmental transition from yellow to silver eels in the wild. This demonstrates that silvering in eels is under endocrine control and that the presumed male-specific steroid 11-KT is capable of inducing silvering-related changes in a female teleost. We discuss how species-specific responses to 11-KT may differ depending on tissue-specific androgen receptor abundance and how a dual demand on liver function can explain the apparently positive effects of 11-KT on liver growth.
We have studied in vivo, the effects of physiological androgen (11-ketotestosterone: 11-KT and testosterone: T) concentrations on the growth of cod previtellogenic oocytes and steroidogenic gene expression patterns. Immature female Atlantic cod were injected three times (days 0, 7 and 14) with 0.05, 0.5 and 5 mg/kg of 11-KT and T. The control group was injected with the carrier solvent (ethanol diluted 1:10 in sunflower oil). Quantitative histological analyses demonstrated growth and development of previtellogenic oocytes after exposure to androgens. The oocyte developmental effect of androgens was more pronounced in fish receiving 11-KT. Quantitative PCR analysis demonstrated dose- and androgen-specific modulation of mRNA expression for genes involved in steroidogenesis (StAR (steroidogenic acute regulatory) protein, P450scc (P450-mediated cholesterol side-chain cleavage), 20beta-HSD (20beta-hydroxysteroid dehydrogenase)) and cell growth control, namely--opioid growth factor receptor (OGF-R), progesterone receptor protein p23 (PR23P) and apoptosis-inducing TAF9-like domain 1 (TAF9). Messenger RNA species associated with the zona pelucida, namely--the zona pellucida protein A domain (ZPA) and egg envelope glycoprotein (EeG) were modulated based on dose and androgen type. Cyclin-B mRNA expression was not affected by androgen exposure. Interestingly, we showed recently that these transcripts were responsive to in vitro androgen exposure in previtellogenic cod ovary. In conclusion, the present study adds further information regarding the effects of androgens on the development of previtellogenic oocytes, suggesting androgen control of early oocyte growth in cod. The enhanced effects of 11-KT on oocyte growth support our hypothesis that non-aromatizable androgens play significant roles in the regulation of early previtellogenic oocyte growth and development.
Sex steroid hormones are important for reproduction in all vertebrates, but few studies examine inter-individual, temporal, and population-level variations, as well as environmental influences on circulating steroid levels within the same species. In this study we analyzed plasma 11-ketotoestosterone (11-KT) and 17beta-estradiol (E(2)) levels in the oyster toadfish to test for 1) individual and temporal variations by serially sampling the same individuals during the reproductive and post-reproductive period, 2) variations in steroid levels among toadfish obtained from different sources or maintained under different holding conditions, and 3) correlations with environmental parameters. Results from serial sampling showed marked inter-individual variations in male 11-KT levels in two separate groups of toadfish, but no temporal differences from June to September. Females also showed inter-individual variations in E(2) concentrations, but most had elevated levels late in the reproductive season coincident with oocyte growth prior to winter quiescence. E(2) concentration, but not 11-KT, was positively correlated with water temperature, and negatively correlated with daylength and lunar phase. Maricultured toadfish held under constant conditions had elevated levels of E(2) and 11-KT that should be considered when using these fish for experimentation. This study provides important comparative information on the relationship between individual variations in steroid levels, and how they relate to physiological and environmental correlates in a model marine teleost.
The effect of 11-ketoandrostenedione (OA) on plasma concentrations of sexual steroids and spermatogenesis of Senegalese sole (Solea senegalensis) implanted with gonadotropin-releasing hormone agonist (GnRHa) was investigated. Males were treated with saline (control) or with GnRHa implants (50 mug kg(-1)) in the presence or absence of OA (2 or 7 mg kg(-1)) during twenty eight days. Treatment with GnRHa alone slightly stimulated spermatogenesis and milt production with respect to controls, and this was associated with a transient elevation of plasma 11-ketotestosterone (11-KT) at day seven and an increase of 5beta-reduced metabolite(s) of 17,20beta-dihydroxy-pregn-4-en-3-one (17,20betaP) at day twenty eight. However, treatment with GnRHa+OA increased plasma concentrations of 11-KT and free+sulphated 5beta-reduced metabolites of 17,20betaP at days seven, fourteen and twenty one. After twenty eight days, the testis of GnRHa+OA-treated fish showed a lower number of spermatogonia B and spermatocytes I, and a higher number of spermatids, than fish treated with GnRHa alone. In addition, the motility of spermatozoa produced by GnRHa+OA males was enhanced by 2-fold with respect to controls or GnRHa males. These results suggest that treatment of Senegalese sole with GnRHa+OA stimulates spermatogenesis resulting in more motile sperm. Such effects could be mediated by an increased synthesis of 11-KT and/or 17,20betaP in the testis but further studies will be required to elucidate the specific mechanism involved.
The present study investigated, for the first time in a perciform teleost, the effects of in vivo 11-ketotestosterone (11-KT) treatment using slow-release implants on ovarian development and gonadotropin receptor mRNA levels in captive previtellogenic females of hapuku (Polyprion oxygeneios). At the cellular/functional level, ovarian development and ovarian and hepatic total lipid levels were examined. At the molecular level, transcript abundance of ovarian follicle-stimulating hormone receptor (FSH-R) and luteinizing hormone receptor (LH-R) were measured. Additionally, cyclic adenosine monophosphate (cAMP) levels in ovarian fragments from placebo and 11-KT implanted fish incubated with or without human chorionic gonadotropin (hCG) in vitro were compared between groups. There were no significant differences between treatments with regard to oocyte size and lipid contents of liver and ovary. Messenger RNA levels of FSH-R and LH-R were significantly lower in the treated females. Similarly, cAMP levels were significantly lower in the ovarian fragments of the 11-KT implanted females. These results suggest that 11-KT specifically, but possibly androgens in general, may not have an important function in regulating gonadal development of previtellogenic female hapuku; indeed, if anything, 11-KT appeared to have a detrimental effect and its use will not be beneficial in advancing sexual maturity of hapuku in aquaculture.
FKBPs are cytosolic receptors for the immunosuppressive drug FK506. FKBP12.6 and FKBP12 associate with cardiac and skeletal muscle isoforms of ryanodine receptors and thereby regulate intracellular Ca(2+) release. The amino acid sequences of FKBP12.6 and FKBP12 are highly conserved among mammals and chicken. The expression profiles of FKBP12.6 and FKBP12 genes during chicken development were compared by Northern blot and in situ hybridization analyses. Transcripts of the FKBP12 gene were abundant throughout the embryo at early stages of development but subsequently underwent gradual down-regulation. Expression of the FKBP12.6 gene was mostly restricted to the heart during early embryogenesis and also underwent subsequent down-regulation, becoming localized to the atrium after hatching. Treatment of early embryos with FK506 had no apparent effect on embryogenesis. Retinoic acid induced marked abnormalities in cardiogenesis, but it did not affect FKBP gene expression. These results suggest that, even though FKBP12.6 and FKBP12 genes are expressed in chick embryos, FK506-sensitive functions of the encoded proteins do not appear to contribute to early embryogenesis or cardiogenesis.
This paper reviews the general mechanisms by which leptin acts as a regulator of lipid reserves through changes in food intake, energy expenditure and fuel selection, with an emphasis on its direct effects on cellular lipid metabolism. Briefly, when leptin levels increase, food consumption decreases via modulation of hypothalamic neuropeptides. As well, normal decreases in energy expenditures (e.g. with diurnal cycles or reduced caloric intake) do not occur. This is probably caused by an increase in mitochondrial proton leak mediated by leptin via increases in sympathetic nervous system stimulation and thyroid hormone release. The decrease in caloric input coupled with relatively higher energy expenditure, therefore, leads to negative energy balance. Leptin also changes the fuel source from which ATP is generated. Fuel preference switches from carbohydrate (glucose) to lipid (fatty acids). This effect arises through stimulation of triacylglycerol catabolism by leptin. In vitro studies show that leptin is a potent stimulator of lipolysis and fatty acid oxidation in adipocytes and other cell types. Consequently, leptin is also a regulator of cellular triacylglycerol content. Hormonal regulation of leptin, as well as its role in fasting and seasonal weight gain and energy expenditure are also briefly discussed.
No organism can survive across the entire temperature range found in the biosphere, and a given species can rarely support active metabolism across more than a few tens of degrees C. Nevertheless, life can be maintained at surprisingly extreme temperatures, from below -50 to over 110 degrees C. That proteins, which are assembled with the same 20 amino acids in all species, can function well at both extremes of this range illustrates the plasticity available in the construction of these macromolecules. In studying proteins from extremophiles, researchers have found no new amino acids, covalent modifications or structural motifs that explain the ability of these molecules to function in such harsh environments. Rather, subtle redistributions of the same intramolecular interactions required for protein stabilization at moderate temperatures are sufficient to maintain structural integrity at hot or cold extremes. The key to protein function, whether in polar seas or hot springs, is the maintenance of an appropriate balance between molecular stability on the one hand and structural flexibility on the other. Stability is needed to ensure the appropriate geometry for ligand binding, as well as to avoid denaturation, while flexibility is necessary to allow catalysis at a metabolically appropriate rate. Comparisons of homologous proteins from organisms spanning a wide range of thermal habitats show that adaptive mutations, as well as stabilizing solutes, maintain a balance between these two attributes, regardless of the temperature at which the protein functions.
It is well established that the release of surfactant phospholipids into the alveolar lumen proceeds by the exocytosis of lamellar bodies (LBs), the characteristic storage organelles of surfactant in alveolar type II cells. Consequently, the fusion of LBs with the plasma membrane and the formation of exocytotic fusion pores are key steps linking cellular synthesis of surfactant with its delivery into the alveolar space. Considering the unique structural organization of LBs or LB-associated aggregates which are found in lung lavages, and the roughly 1-microm-sized dimensions of these particles, we speculated whether the fusion pore diameter of fused LBs might be a specific hindrance for surfactant secretion, delaying or even impeding full release. In this mini-review, we have compiled published data shedding light on a possibly important role of fusion pores during the secretory process in alveolar type II cells.
Pattle, who provided some of the initial direct evidence for the presence of pulmonary surfactant in the lung, was also the first to show surfactant was susceptible to proteases such as trypsin. Pattle concluded surfactant was a lipoprotein. Our group has investigated the roles of the surfactant proteins (SP-) SP-A, SP-B, and SP-C using a captive bubble tensiometer. These studies show that SP-C>SP-B>SP-A in enhancing surfactant lipid adsorption (film formation) to the equilibrium surface tension of approximately 22-25 mN/m from the 70 mN/m of saline at 37 degrees C. In addition to enhancing adsorption, surfactant proteins can stabilize surfactant films so that lateral compression induced through surface area reduction results in the lowering of surface tension (gamma) from approximately 25 mN/m (equilibrium) to values near 0 mN/m. These low tensions, which are required to stabilize alveoli during expiration, are thought to arise through exclusion of fluid phospholipids from the surface monolayer, resulting in an enrichment in the gel phase component dipalmitoylphosphatidylcholine (DPPC). The results are consistent with DPPC enrichment occurring through two mechanisms, selective DPPC adsorption and preferential squeeze-out of fluid components such as unsaturated phosphatidylcholine (PC) and phosphatidylglycerol (PG) from the monolayer. Evidence for selective DPPC adsorption arises from experiments showing that the surface area reductions required to achieve gamma near 0 mN/m with DPPC/PG samples containing SP-B or SP-A plus SP-B films were less than those predicted for a pure squeeze-out mechanism. Surface activity improves during quasi-static or dynamic compression-expansion cycles, indicating the squeeze-out mechanism also occurs. Although SP-C was not as effective as SP-B in promoting selective DPPC adsorption, this protein is more effective in promoting the reinsertion of lipids forced out of the surface monolayer following overcompression at low gamma values. Addition of SP-A to samples containing SP-B but not SP-C limits the increase in gamma(max) during expansion. It is concluded that the surfactant apoproteins possess distinct overlapping functions. SP-B is effective in selective DPPC insertion during monolayer formation and in PG squeeze-out during monolayer compression. SP-A can promote adsorption during film formation, particularly in the presence of SP-B. SP-C appears to have a superior role to SP-B in formation of the surfactant reservoir and in reinsertion of collapse phase lipids.
Surface temperatures (Ts) of eight 13-lined ground squirrels and seven yellow-bellied marmots were measured during arousal from hibernation using infrared thermography (IRT) and recorded on videotape. Animals aroused normally in 5 degrees C cold rooms. Body temperatures were recorded during arousal using both cheek pouch and interscapular temperature probes. Warming rate in arousal was exponential. Mean mass specific warming rates show the squirrels warm faster (69.76 degrees C/h/kg) than the marmots (4.49 degrees C/h/kg). Surface temperatures (Ts) for 11 regions were measured every few minutes during arousal. The smaller ground squirrel shows the ability to perfuse distal regions without compromising rise in deep body temperature (Tb). All squirrel Ts's remained low as Tb rose to 18 degrees C, at which point, eyes opened, squirrels became more active and all Ts's rose parallel to Tb. Marmot Ts remained low as Tb rose initially. Each marmot showed a plateau phase where Tb remained constant (mean Tb 20.3+/-1.0 degrees C, duration 9.4+/-4.1 min) during which time all Ts's rose, and then remained relatively constant as Tb again began to rise. An anterior to posterior Ts gradient was evident in the ground squirrel, both body and feet. This gradient was only evident in the feet of the marmots.
Spider silk has been evolutionarily optimized for contextual mechanical performance over the last 400 Ma. Despite precisely balanced mechanical properties, which have yet to be reproduced, the underlying molecular architecture of major ampullate spider silk can be simplified being viewed as a versatile block copolymer. Four primary amino acid motifs: polyalanine, (GA)(n), GPGXX, and GGX (X = G,A,S,Q,L,Y) will be considered in this study. Although synthetic mimetics of many of these amino acid motifs have been produced in several biological systems, the source of spider silk's mechanical integrity remains elusive. Mechanical robustness may be a product not only of the amino acid structure but also of the tertiary structure of the silk. Historically, solid state nuclear magnetic resonance (ssNMR) has been used to reveal the crystalline structure of the polyalanine motif; however, limitations in amino acid labeling techniques have obscured the structures of the GGX and GPGXX motifs thought to be responsible for the structural mobility of spider silk. We describe the use of metabolic pathways to label tyrosine for the first time as well as to improve the labeling efficiency of proline. These improved labeling techniques will allow the previously unknown tertiary structures of major ampullate silk to be probed.
Global climate change is associated with a progressive rise in ocean CO(2) concentrations (hypercapnia) and, consequently, a drop in seawater pH. However, a comprehensive picture of the physiological mechanisms affected by chronic CO(2) stress in marine biota is still lacking. Here we present an analysis of protein biosynthesis rates in isolated muscle of the marine invertebrate Sipunculus nudus, a sediment dwelling worm living at various water depths. We followed the incorporation of (13)C-labelled phenylalanine into muscular protein via high-resolution NMR spectroscopy. Protein synthesis decreased by about 60% at a medium pH of 6.70 and a consequently lowered intracellular pH (pHi). The decrease in protein synthesis rates is much stronger than the concomitant suppression of protein degradation (60% versus 10-15%) possibly posing a threat to the cellular homeostasis of structural as well as functional proteins. Considering the progressive rise in ocean CO(2) concentrations, permanent disturbances of cellular protein turnover might seriously affect growth and reproductive performance in many marine organisms with as yet unexplored impacts on species density and composition in marine ecosystems.
Metabolic effects of dietary macronutrients on diet-tissue isotopic discrimination factors were investigated in harbor seals. Three seals were fed either high fat/low protein herring (H), or low fat/high protein pollock (P), and switched to the alternative every 4 months. This allowed each seal to be subjected to two dietary treatments in each of three metabolically defined seasons (breeding from May to September, molting from September to January, and late winter/early spring period from January to May) over a 2 year cycle, and function as its internal control regardless of physiological changes over season. One seal was fed a constant equal mix of H and P over the entire trial. Up to 1 per thousand differences in serum delta15N values of one seal fed alternatively on H and P were observed. Progressively more enriched serum delta15N values as diet switching from H to P might link to changes in seal digestive physiology and protein metabolism in response to very high protein intake on P diet. These findings demonstrate that dietary macronutrients of prey species and protein intake level of consumers also play important roles in shaping isotopic patterns of a consumer's tissues, and thus influence accurate data interpretation of stable isotope techniques in ecological applications.
The (13)C-labelled Na-bicarbonate technique uses stable isotopes to measure energy expenditure in birds. After administration, the isotopes reach equilibrium within the body's bicarbonate pools at a fast rate due to the small size of the bicarbonate pool in relation to CO(2) flux. This technique is therefore ideal for measuring energy expenditure over short-term activities. The major advantage of this technique is that it can be applied without the animal having to wear a respirometry mask or being enclosed in a respirometry chamber. Despite the technique's suitability for use in birds and other animals, there have been few studies that have used it to date and so its potential is not fully understood. Here we discuss the methodology and review previous applications.
The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies of environmental toxicants and osmoregulation. Previous research in our laboratory has shown that acute acclimation to seawater is mediated by an increase in SGK1. SGK1 promotes the trafficking of CFTR chloride channels from intracellular vesicles to the plasma membrane of the gill within the first hour in seawater resulting in increased chloride secretion. Although we have shown that the increase in gill SGK1 does not require activation of the glucocorticoid receptor, the mechanisms that mediate the rise SGK1 during acute acclimation is unknown. To test the hypothesis that mitogen activated protein kinase (MAPK14) is responsible for the rise in SGK1 we identified the coding sequence of killifish MAPK14-1 and designed a translational blocking vivo-morpholino targeting MAPK14-1. Injection of the MAPK14-1 vivo-morpholino resulted in a 30% reduction of MAPK14-1 and a 45% reduction in phosphorylated-MAPK14-1 protein in the gill of killifish transitioned from freshwater to seawater. Knock down of phosphorlyated-MAPK14-1 completely blocked the rise in SGK1 mRNA and protein in the killifish gill, providing the first direct and in vivo evidence that MAPK14-1 is necessary for acute seawater acclimation.
Fat sand rats Psammomys obesus feed exclusively on plants of the family Chenopodiaceae, which contain high concentrations of chloride salts (NaCl, KCl) and oxalate salts. Ingestion of large quantities of oxalate is challenging for mammals because oxalate chelates Ca(2+) cations, reducing Ca(2+) availability. Oxalate is a metabolic end-point in mammalian metabolism, however it can be broken-down by intestinal bacteria. We predicted that in fat sand rats microbial breakdown of oxalate will be substantial due to the high dietary load. In addition, since a high concentration of soluble chloride salts increases the solubility of calcium oxalate in solution, we examined whether a change in the intake of chloride salts affects microbial oxalate breakdown and calcium excretion in fat sand rats. We measured oxalate, calcium and other inorganic matter (ash) intake and excretion in fat sand rats feeding on two different diets: saltbush (Atriplex halimus), their natural diet, and goose-foot (Chenopodium album), a non-native chenopod on which fat sand rats will readily feed and that has a similar oxalate content to saltbush but only 2/3 of the ash content. In animals feeding on both diets, 65-80% of the oxalate ingested did not appear in urine or faeces. In animals consuming the more saline saltbush, significantly more oxalate was apparently degraded (p<0.001), while significantly less oxalate was excreted in urine (p<0.01) and in faeces (p<0.05). We propose, therefore, that fat sand rats rely on symbiotic bacteria to remove a large portion of the oxalates ingested with their diet, and that the high dietary salt intake may play a beneficial role in their oxalate and calcium metabolism.
The postprandial morphological changes of the intestinal epithelium of Burmese pythons were examined using fasting pythons and at eight time points after feeding. In fasting pythons, tightly packed enterocytes possess very short microvilli and are arranged in a pseudostratified fashion. Enterocyte width increases by 23% within 24 h postfeeding, inducing significant increases in villus length and intestinal mass. By 6 days postfeeding, enterocyte volume had peaked, following as much as an 80% increase. Contributing to enterocyte hypertrophy is the cellular accumulation of lipid droplets at the tips and edges of the villi of the proximal and middle small intestine, but which were absent in the distal small intestine. At 3 days postfeeding, conventional and environmental scanning electron microscopy revealed cracks and lipid extrusion along the narrow edges of the villi and at the villus tips. Transmission electron microscopy demonstrated the rapid postprandial lengthening of enterocyte microvilli, increasing 4.8-fold in length within 24 h, and the maintaining of that length through digestion. Beginning at 24 h postfeeding, spherical particles were found embedded apically within enterocytes of the proximal and middle small intestine. These particles possessed an annular-like construction and were stained with the calcium-stain Alizarine red S suggesting that they were bone in origin. Following the completion of digestion, many of the postprandial responses were reversed, as observed by the atrophy of enterocytes, the shortening of villi, and the retraction of the microvilli. Further exploration of the python intestine will reveal the underlying mechanisms of these trophic responses and the origin and fate of the engulfed particles.
Water is usually thought to be required for the living state, but several organisms are capable of surviving complete dehydration (anhydrobiotes). Elucidation of the mechanisms of tolerance against dehydration may lead to development of new methods for preserving biological materials that do not normally support drying, which is of enormous practical importance in industry, in clinical medicine as well as in agriculture. One of the molecular mechanisms of damage leading to death in desiccation-sensitive cells upon drying is free-radical attack to phospholipids, DNA and proteins. This review aims to summarize the strategies used by anhydrobiotes to cope with the danger of oxygen toxicity and to present our recent results about the importance of some antioxidant defense systems in the dehydration tolerance of Saccharomyces cerevisiae, a usual model in the study of stress response.
Nutritional response to different diet quality was examined in the subterranean rodent Ctenomys talarum (tuco-tuco). Animals maintained in captive conditions were fed with three plant species that differed in their fibre content. Tuco-tucos showed the ability to perform adjusts in short time lapse in response to diet quality; food ingestion, egestion and feces ingestion changed in animals under different plant species diets. Time budget, mainly time devoted to feeding and activity accompanied such changes. Coprophagy was practiced along the day and night following the arrhythmic activity pattern found for this species. Feces reingestion was not associated to resting. Furthermore, it was observed during fresh food ingestion, being pellets chewed. Soft and hard feces differed in morphological and nutritional characteristics.
The freshwater turtle Trachemys scripta is among the most anoxia-tolerant of vertebrates, a true facultative anaerobe able to survive without oxygen for days at room temperature to weeks or months during winter hibernation. Our good friend and colleague Peter Lutz devoted nearly 25 years to the study of the physiology of anoxia tolerance in these and other model organisms, promoting not just the basic science but also the idea that understanding the physiology and molecular mechanisms behind anoxia tolerance provides insights into critical survival pathways that may be applicable to the hypoxic/ischemic mammalian brain. Work by Peter and his colleagues focused on the factors which enable the turtle to enter a deep hypometabolic state, including decreases in ion flux ("channel arrest"), increases in inhibitory neuromodulators like adenosine and GABA, and the maintenance of low extracellular levels of excitatory compounds such as dopamine and glutamate. Our attention has recently turned to molecular mechanisms of anoxia tolerance, including the upregulation of such protective factors as heat shock proteins (Hsp72, Hsc73), the reversible downregulation of voltage gated potassium channels, and the modulation of MAP kinase pathways. In this review we discuss three phases of anoxia tolerance, including the initial metabolic downregulation over the first several hours, the long-term maintenance of neuronal function over days to weeks of anoxia, and finally recovery upon reoxygenation, with necessary defenses against reactive oxygen stress.
Melatonin is a hormone that is released from the pineal gland into the blood stream and is controlled by nerve impulses from the suprachiasmatic nuclei. Melatonin synthesis, which is inhibited by light on the mammalian retina, peaks in plasma concentrations during the night. Though still a subject of intense research, melatonin in mammals is known to effect the reproductive system, thyroid function, and adaptations to seasonal changes. Sled dogs in Fairbanks, Alaska (65 degrees N) can be exposed to anywhere from 21 h of daylight in the summer to 4 h in the winter. While light may be the primary factor influencing melatonin production, we hypothesized that exercise may also affect melatonin production. In the current study, sled dogs were used to study seasonal and diurnal variation in melatonin production. Sled dogs by nature are elite athletes and therefore exercise was a focus in the study. Both exercise and non exercise dogs from 2 distinct latitudes were used. The peak in melatonin production was prolonged in high latitude dogs (65 degrees N), compared with lower latitude dogs (45 degrees N). Dogs at both latitudes show a reduction in peak melatonin levels with exercise, and winter melatonin levels in both locations were higher than the summer. Surprisingly, sled dogs in Alaska had lower melatonin levels than sled dogs in New York.
The transport of D-glucose into rainbow trout (Oncorhynchus mykiss) and river lamprey (Lampetra fluviatilis) hepatocytes, as well as into rainbow trout hepatoblastoma cell line RTH-149 was studied using tracer methods. The half-time for D-glucose equilibration was 15 s for rainbow trout. The half-times for the non-metabolizable D-glucose analog, 3-O-methyl-D-glucose equilibration were 8 s, 37 s and 38 s for rainbow trout, lamprey and RTH-149 cells, respectively. The 3-O-methyl-D-glucose was taken up by rainbow trout hepatocytes by facilitated diffusion in addition to simple diffusion. The uptake showed saturation kinetics with the K(m) of 37 mM and V(max) of 62 mmol kg(-1) cells min(-1). The uptake was sensitive to phloretin and cytochalasin B, but not affected by ouabain. The 3-O-methyl-D-glucose uptake by lamprey hepatocytes and RTH-149 cells showed no indication of saturation up to 160 mM, and was not affected by phloretin, cytochalasin B or ouabain, which suggests the mode of transport to be by passive diffusion. However, immunocytochemical stainings revealed the existence of mammalian type GLUT1 and GLUT2 transporters in all cells studied. The lack of a functioning carrier-mediated glucose uptake in lamprey hepatocytes might be due to its physiological state (prespawning starvation). The minor 3-O-methyl-D-glucose uptake into RTH-149 cells compared to freshly isolated rainbow trout hepatocytes might reflect low metabolic activity of the cell lines. Under the conditions applied the RTH-149 cell line is no suitable in vitro model for glucose transport in fish cells.
Glutathione (GSH) is an important antioxidant that is involved in a multitude of cellular processes. However, in fish, GSH levels, turnover, and activity of associated enzymes are low when compared to those of mammals. To determine whether temperature influences the GSH antioxidant system in fish, and can explain the differences in GSH between fish and mammals, we examined the effects of acclimation temperature on total GSH (tGSH) levels and apparent half-life (as an estimate of turnover) in a rainbow trout hepatoma cell line (RTH-149), and GSH levels, and glutathione peroxidase (GPx) and reductase (GR) activity in the eurythermal killifish. Increasing incubation temperature decreased half-life and transiently increased levels of tGSH in RTH-149 cells. In killifish, increased acclimation temperature increased tGSH levels in the liver, brain and muscle, and increased hepatic GPx and GR activities. When the relationships between temperature and GSH half-life, levels and enzyme activity were extrapolated to 37 degrees C, temperature could only partially accounted for differences in the GSH antioxidant system in fish compared to mammals. The differences in the GSH antioxidant system between fish and mammals may not be solely due to temperature effects, but also to the increased metabolic cost of endothermy in mammals.
A lanosterol 14alpha-demethylase (CYP51) cDNA, which consisted of a 1419 bp open reading frame encoding 472 amino acids and a 918 bp 3'-untranslated region, was isolated from the chicken testis. The sequence corresponding to exon 1 of this cDNA was completely different from those of CYP51 cDNAs in other tissues, including the liver. The expression level of the CYP51 gene with the testis-specific exon 1 was much higher in mature (2-year-old) male chickens than in immature (5-week-old) chickens. In addition, a CYP51 transcript common to several tissues, including the liver, adrenal gland, cerebellum, cerebrum, lobus opticus, kidney, lung, heart, muscle, spleen, small intestine and ovary, was also detected in the testis by RT-PCR. Furthermore, testis-specific shorter transcripts, which have been observed in mammals such as humans, rats and pigs, were not detected in the chicken testis. The results of this study demonstrate for the first time the presence of a chicken testis-specific CYP51 transcript and its sexual maturation-related expression, although its biological significance remains unclear.
Small mammals that remain active throughout the year at a constant body temperature have a much greater energy and food requirement in winter. Lower body temperatures in winter may offset the increased energetic cost of remaining active in the cold, if cellular metabolism is not constrained by a negative thermodynamic effect. We aimed to determine whether variable body temperatures can be advantageous for small endotherms by testing the hypothesis that body temperature fluctuates seasonally in a wild rat (Rattus fuscipes); conferring an energy saving and reducing food requirements during resource restricted winter. Additionally we tested whether changes in body temperature affected tissue specific metabolic capacity. Winter acclimatized rats had significantly lower body temperatures and thicker fur than summer acclimatized rats. Mitochondrial oxygen consumption and the activity of enzymes that control oxidative (citrate synthase, cytochrome c-oxidase) and anaerobic (lactate dehydrogenase) metabolism were elevated in winter and were not negatively affected by the lower body temperature. Energy transfer modeling showed that lower body temperatures in winter combined with increased fur thickness to confer a 25 kJ day(-1) energy saving, with up to 50% owing to reduced body temperature alone. We show that phenotypic plasticity at multiple levels of organization is an important component of the response of a small endotherm to winter. Mitochondrial function compensates for lower winter body temperatures, buffering metabolic heat production capacity.
In crustaceans, vitellogenesis is known to be controlled by eyestalk neuropeptides, biogenic amines, ecdysteroids and a juvenile hormone-like compound, methyl farnesoate. In recent years, the occurrence of vertebrate steroid hormones, estradiol 17beta (E2) and progesterone (PG) has also been reported in a few decapods, although their precise role in female reproduction is yet to be determined. The levels of E2 and PG in the ovary, hepatopancreas and the hemolymph of the red mud crab, Scylla serrata were analyzed in different vitellogenic stages in order to establish a correlation between hormone profile and stages of vitellogenesis. It was observed that the levels of both the steroids increased steeply in the tissues at the onset of vitellogenesis (vitellogenic stage I). Maximum levels of estradiol were present in the hepatopancreas whereas the highest concentration of progesterone was seen in the ovary, suggesting dichotomous roles for these hormones in vitellogenesis. Furthermore, levels of these hormones were estimated in different embryonic stages of the eggs of the sand crab Emerita asiatica and mud crab S. serrata. Their levels fluctuated, following a definite pattern in the different stages, suggesting a possible functional role as morphogenetic hormones. This study, in addition, also reports the presence of E2 and PG on lipovitellin purified from ovary and eggs as well as vitellogenin purified from the hemolymph implicating a role for these lipoproteins as steroid carriers.
The effects of 17 beta-estradiol on induction of vitellogenin synthesis and oocyte development were investigated in previtellogenic ovary of immature kuruma prawn (Marsupenaeus japonicus) incubated with Medium 199. After three days incubation of previtellogenic ovary, Vg concentrations in media containing 3.6 nM, 36.7 nM, 367 nm and 3671 nM 17 beta-estradiol were significantly (p<0.01) greater than that of Ringer solution or the pure ethanol vehicle. Furthermore, a more advanced stage of oocyte development at oil globule stage (primary vitellogenic stage), which is surrounded by round and greatly expanded follicle cells, was observed in previtellogenic ovarian pieces incubated in media containing 3.6, 36.7, 367 and 3671 nM 17 beta-estradiol. The results of these studies show that 17 beta-estradiol induces Vg synthesis and appearance of primary vitellogenic oocyte in the ovary of immature prawns.
The soluble matrix of the sagittal otolith of the cod Gadus morhua was analyzed using UV and IR spectroscopy, liquid chromatography and electrophoresis. This matrix is a complex mixture of proteins and glycoproteins, with a large range of molecular weights. High weights (>1000 kDa) are shown for the first time in water-soluble matrix of otolith. However, the 2D denaturing electrophoresis and large range of sorting used in high performance liquid chromatography columns do not separate the soluble matrix to well-defined molecular weights. The IR data indicate that several conformations are present and the main part of the sugars is not sulfated. Additionally, electrophoresis data show that the acidity of the sugar components is higher than that of the proteins. Despite the relative scarcity of literature data, our study of G. morhua suggests that the chemical composition of otolith soluble organic matrix may differ among species.
An 11-day trial was conducted to investigate the osmoregulatory capacity (OC) and regulation of K(+), Na(+), Ca(2+) and Mg(2+) of Penaeus monodon juveniles when exposed to K(+) deficient inland saline water (ISW) of four different salinities (5, 15, 25 and 35 ppt). The survival of juveniles showed a positive linear relationship (R(2) ranging from 0.72 to 0.98) with salinity. At the end of the trial, juveniles were able to survive only in 5 ppt of ISW and showed no changes in OC when transferred from ocean water (OW) to ISW. Further, the OC of juveniles in 5 ppt of ISW was significantly different (P<0.05) from the OC of juveniles exposed to 15, 25 and 35 ppt and exhibited strong serum K(+), Na(+), Ca(2+) and Mg(2+) regulation monitored over 16 h. In contrast, at 35 ppt, significant decrease (P<0.05) in serum K(+) and Mg(2+) concentrations and accumulation of serum Na(+) concentration occurred after 16 h of exposure to ISW. At higher salinity, an increase in serum Na(+) concentration leads to an increase in the serum osmolality of the juveniles, which in turn causes decrease in the OC of the juveniles. The results of this study suggest that K(+) deficiency in ISW has a negative effect on survival, OC and the ability of P. monodon juveniles to regulate serum Na(+), K(+), Ca(2+) and Mg(2+) concentrations. These effects are compounded as salinity increases.
This study directly tested the hypothesis that the induction of oocyte maturation in the catfish Clarias batrachus is followed by a transient decrease in oocyte cyclic AMP (cAMP) level that is due to an increase in phosphodiesterase (PDE) activity. Further, the PDE inhibitor theophylline was used to investigate the possible role of PDE in the maturation-inducing action of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), the physiological maturation-inducing steroid of this catfish species. The results obtained from batches of oocytes taken from the same donor at the same time clearly show a close relationship between dose-dependent induction of germinal vesicle breakdown (GVBD) and PDE activity with a concomitant decrease in cAMP in the oocytes treated with different concentrations of 17alpha,20beta-DP. In contrast, theophylline prevents GVBD and inhibits PDE activity by promoting cAMP accumulation in oocytes. A time-dependent decrease in PDE activity and an increase in cAMP content with a marked inhibition of GVBD were recorded even in oocytes pre-stimulated with 1 microgram/ml 17alpha,20beta-DP for 6 h and then treated with 1 mM theophylline for various times. These results suggest that cAMP plays a key role in the regulation of oocyte maturation in C. batrachus which may be mediated by PDE activity.
cDNA clones were isolated as expressed sequence tags (ESTs) from the ovarian cDNA library of Macropodus opercularis. The EST sequences showed similarity with many housekeeping genes and ribosomal proteins. One of the ESTs showed similarity to beta-thymosin, a 5-kDa polypeptide expressed under different physiological conditions. The cDNA corresponding to beta-thymosin of M. opercularis is 368 bp in length and codes for a putative polypeptide of 42 amino acids. Multiple alignment of the deduced amino acid sequence showed 61% similarity with piscine beta-thymosins and 56% similarity with mammalian beta-thymosins. Administration of a gonadotropin releasing hormone analog or estradiol-17beta induced an increase in the gonadosomatic index, oocyte diameter and also enhanced expression of beta-thymosin m-RNA in the recrudizing ovary. This report indicates that both GnRH analog and E(2) might induce similar pathways for the differentiation of ovarian cells for the maturation of oocytes.
Two distinct vitellogenins (VTG) were purified from the blood of estradiol-17beta (E(2))-injected tilapia, Oreochromis mossambicus. Enzyme-linked immunosorbent assays (ELISA) of each VTG were developed to examine effects of E(2) treatment on induction of VTG synthesis in the primarily cultured tilapia hepatocytes. Two VTG molecules (VTG210 and VTG140) had apparent molecular masses of 370 and 220 kDa by gel filtration and 210 and 140 kDa by SDS-PAGE, respectively. Western blot analyses showed that antibodies raised against the purified VTG210 and VTG140 reacted only with each protein band. Furthermore, ELISA for each VTG was specific for target VTG. When E(2) was added into the media of primarily cultured tilapia hepatocytes, VTG210 and VTG140 were both detected from E(2) concentrations of 1x10(-7) M and 5x10(-7) M, respectively. Time course experiments showed that there was a difference in the detection time of VTG210 and VTG140 after the hormone treatment. Although the injection of different E(2) doses induced both VTGs in the plasma of male tilapia, the concentration of VTG210 was nearly five to eight times higher than that of VTG140. These results suggest that E(2) is a direct inducer of both VTGs in the tilapia hepatocytes in vitro and in vivo, and that there is difference in the hormone response in inducing the VTGs in the tilapia hepatocytes.
In vitro effects of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, calphostin C (PKC inhibitor) and okadaic acid [OA, a protein phosphatase (PP; PP1 and PP2A) inhibitor] on 2-hydroxyestradiol-17beta (2-OHE(2))-induced oocyte maturation were investigated in the catfish Heteropneustes fossilis. Incubations of postvitellogenic follicles with PMA or OA alone did not induce oocyte maturation. However, co-incubations with 2-OHE(2) and PMA (0.05, 0.5 and 5 microM) or 2-OHE(2) and OA (0.5, 1.0 or 2.0 microM) increased germinal vesicle breakdown (GVBD) significantly over that of 2-OHE(2). Incubation of follicles with calphostin C elicited varied effects on GVBD, low (0.005 and 0.01 microM) and high (5.0 and 10.0 microM) concentrations did not affect GVBD, but medium concentrations (0.05, 0.1, 0.5, 1.0 and 2.5 microM) stimulated it. The medium concentrations elicited a biphasic stimulatory response with peak GVBD at 0.1 microM (54%). Calphostin C (>or=2.5 microM) inhibited the 2-OHE(2)-induced GVBD in a concentration-dependent manner during the 24 h incubation. Pre- or post-treatment with calphostin C inhibited the steroid-induced GVBD only at 6 h. In co-incubation studies, both PMA and OA reversed the inhibitory effect of calphostin C: the former partially and the latter fully. The results of the present study show that PKC appears to modulate the 2-OHE(2)-induced oocyte maturation. The OA-sensitive PP may be involved in the PKC modulation of steroid-induced oocyte maturation.
Previous studies demonstrated that native and recombinant growth hormone from mammalian and fish species potentiate the estrogenic induction of vitellogenin synthesis by cultured eel hepatocytes. In the present study, the metabolic competence (respiratory activity and estradiol catabolism) of cultured hepatocytes and their functional capacity to synthesize a specific protein, vitellogenin, in the presence of estradiol and/or bovine growth hormone was investigated. In addition, we examined the possible role of insulin-like growth factors as mediators of growth hormone. Hepatocytes retain a high level of metabolic activity under the primary culture conditions applied. Estradiol has a half life of several hours in the hepatocyte culture, and is metabolized into conjugated forms. Estradiol and/or growth hormone had no effects on respiratory activity of the cultured hepatocytes. Moreover, the estradiol catabolic parameters were not affected by growth hormone. Finally, human and trout recombinant insulin-like growth factors do not potentiate vitellogenin synthesis induced by estradiol.
The effects of follicle-stimulating hormone (FSH) and 17beta-estradiol (E2) on chicken ovarian germ cell proliferation were evaluated through a germ-somatic cell coculture model. Ovarian cells from the left ovaries of 18-day-old chicken embryos were cultured in serum-free McCoy's 5A medium at 39 degrees C and challenged with FSH (0.25-1.0 IU/mL) or E2 (10(-8)-10(-5) M) alone and in combination for 48 h. The number of germ cells was counted, and the proliferating cells were immunolocalized by a specific antibody against proliferating cell nuclear antigen (PCNA). The labeling index (LI) was determined for germ cells. Results revealed that germ cells could survive and kept proliferating under support of somatic cells. Germ cells were localized by expression of a specific antibody for stem cell factor receptor c-kit. Both FSH (0.25-1.0 IU/mL) and E2 (10(-7)-10(-5) M) alone induced a marked increase in germ cell number (P<0.05), and PCNA-LI of germ cells was greater in FSH-treated groups (0.25-1.0 IU/mL) and E2-treated groups (10(-8)-10(-5) M), compared with vehicle-treated group (P<0.05). Furthermore, FSH manifested a synergistic effect with E2 (10(-6)-10(-5) M) in stimulating germ cell proliferation. These results indicate that FSH might interact with estrogen to promote ovarian germ cell proliferation in embryonic chickens near hatching.
The hypothesis of this study was that 17β-estradiol (estradiol) stimulates turkey skeletal muscle growth by influencing myogenic satellite cell proliferation, differentiation, and the gene expression of selected proteins important in regulating growth and development. Increasing levels of estradiol were administered in basal medium containing additional nutrients. Female-derived pectoralis major (PM) satellite cell proliferation was stimulated by estradiol at a level of 10(-9)M following 4days of treatment. Male PM and biceps femoris (BF) satellite cell proliferation was increased at 10(-12)M estradiol. Turkey embryonic myoblast proliferation, however, decreased with 10(-9)M and 10(-5)M estradiol following 3days under these conditions. Estradiol had no effect on the differentiation of any of the 4 groups of cells. Likewise, glypican-1 expression was unaffected by estradiol treatment. MyoD expression decreased in male PM but not BF cells. MyoD expression in female PM cells and embryonic myoblasts were also unaffected by estradiol administration. Estradiol decreased myogenin expression in male satellite cells, but had no effect on female cells. There was a slight decrease in myogenin expression in embryonic myoblasts. The results demonstrate a direct effect of estradiol on avian satellite cell proliferation independent of glypican-1, and decreased expression of MyoD and myogenin in some myogenic cells, coinciding with increased cellular proliferation.
In the marine polychaete Nereis virens, the yolk protein precursor vitellogenin (Vg) is synthesized in specialized coelomic cells (eleocytes) during oogenesis. This process was visualized by immunohistochemistry using antibodies raised against the yolk protein. Transversal sections from male and female worms confirmed that eleocytes from females but not from males produce Vg. In order to investigate the hormonal regulation of Vg synthesis, eleocytes were incubated in vitro with estradiol-17beta (E(2)) at a concentration of 1 microg/l for up to three days. A strong increase in Vg secretion was detected by ELISA in culture media of treated eleocytes from vitellogenic females. In contrast, no response to the hormonal treatment was detectable in immature worms. Our results showed that Vg synthesis is under a complex regulation, which involves endocrine factors like estrogens. The role of E(2) in vitellogenesis of N. virens rather resembles the situation found in vertebrate than the one in insects.