Comparative Biochemistry and Physiology Part A Molecular & Integrative Physiology

Published by Elsevier BV

Print ISSN: 1095-6433


CDNA cloning, characterization and expression of lipopolysaccharide and β-1,3-glucan binding protein (LGBP) gene from the Indian white shrimp Fenneropenaeus indicus
  • Article

May 2012


51 Reads

J Sivakama Valli



Ionomycin and 2,5′-di(tertbutyl)-1,4,-benzohydroquinone elicit Ca2+-induced Ca2+ release from intracellular pools in Physarum polycephalum

March 2001


39 Reads


S.I. Beylina


N.B. Matveeva




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.

Performance of bean bruchids Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae) reared on resistant (IT81D-1045) and susceptible (Epace 10) Vigna unguiculata seeds: Relationship with trypsin inhibitor and vicilin excretion

January 2006


235 Reads

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.

First evidence of the possible implication of the 11-deoxycorticosterone (DOC) in immune activity of Eurasian perch (Perca fluviatilis, L.): Comparison with cortisol

February 2013


115 Reads

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.

Changes in brain arginine vasotocin, isotocin, plasma 11-ketotestosterone and cortisol in round goby, Neogobius melanostomus, males subjected to overcrowding stress during the breeding season

March 2013


151 Reads

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.

11-Ketotestosterone induces silvering-related changes in immature female short-finned eels, Anguilla australis

December 2001


176 Reads

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.

Fig. 1. Temporal and sex differences in circulating 11-KT and E 2 concentrations in serially 
Fig. 1. Temporal and sex differences in circulating 11-KT and E 2 concentrations in serially sampled 2007 outdoor oyster toadfish Opsanus tau. Data are plotted as medians (bar) and 25th-75th quartiles (error bars). Asterisks indicate sex differences within a sampling time (Mann-Whitney rank sum tests, p b 0.05). Bars with different letters indicate temporal statistical differences for females only (KW one-way ANOVA on ranks, p b 0.050; Dunn's tests, p b 0.050). There was no difference among sample times for males.
Table 1 Summary of toadfish used in this study
Fig. 2. Inter-individual and temporal variation in circulating 11-KT and E 2 concentrations in serially sampled 2007 outdoor toad fi sh. Each graph represents a different individual measured across the six sampling times and shows both 11-KT (solid line) and E 2 (dashed line). Note that the y-axis scales differ to illustrate the temporal variations within each individual. Inset shows the ovary of Fish H one week after the Sept 13 sampling and illustrates eggs of various sizes, including large ones 3 – 4 mm in diameter. SB, swim bladder; SM, 
Fig. 2. Inter-individual and temporal variation in circulating 11-KT and E 2 concentrations in serially sampled 2007 outdoor toadfish. Each graph represents a different individual measured across the six sampling times and shows both 11-KT (solid line) and E 2 (dashed line). Note that the y-axis scales differ to illustrate the temporal variations within each individual. Inset shows the ovary of Fish H one week after the Sept 13 sampling and illustrates eggs of various sizes, including large ones 3-4 mm in diameter. SB, swim bladder; SM, sonic muscle; Scale bar = 3 mm.


Individual, temporal, and population-level variations in circulating 11-ketotestosterone and 17 beta-estradiol concentrations in the oyster toadfish Opsanus tau
  • Article
  • Full-text available

February 2009


91 Reads

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.

Previtellogenic oocyte growth and transcriptional changes of steroidogenic enzyme genes in immature female Atlantic cod (Gadus morhua L.) after exposure to the androgens 11-ketotestosterone and testosterone

December 2008


61 Reads

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.

Treatment of GnRHa-implanted Senegalese sole (Solea senegalensis) with 11-ketoandrostenedione stimulates spermatogenesis and increases sperm motility

September 2007


64 Reads

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 effects of 11-ketotestosterone on ovarian physiology of previtellogenic captive hapuku (Polyprion oxygeneios)

August 2013


34 Reads

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.

Gene expression of FK506-binding proteins 12.6 and 12 during chicken development

November 2003


31 Reads

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.

Leptin: an essential regulator of lipid metabolism. Comp Biochem Physiol A 125: 285-298

April 2000


43 Reads

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.

Fields, P. A. Protein function at thermal extremes: balancing stability and flexibility. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 129, 417-431

July 2001


92 Reads

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.

The conception of fusion pores as rate-limiting structures for surfactant secretion. Comp Biochem Physiol A Mol Integr Physiol 129(1): 227-231
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.

Surface activity in vitro: role of surfactant proteins. Comp Biochem Physiol A 129(1):209-220

May 2001


23 Reads

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.

Comparison of surface temperature in 13-lined ground squirrel (Spermophilus tridecimlineatus) and yellow-bellied marmot (Marmota flaviventris) during arousal from hibernation

September 2004


42 Reads

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.

Elucidating metabolic pathways for amino acid incorporation into dragline spider silk using C-13 enrichment and solid state NMR

February 2011


91 Reads

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.

Effects of environmental hypercapnia on animal physiology: A 13C NMR study of protein synthesis rates in the marine invertebrate Sipunculus nudus

September 2006


161 Reads

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.

Dietary macronutrients influence 13C and 15N signatures of pinnipeds: Captive feeding studies with harbor seals (Phoca vitulina)

May 2006


84 Reads

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.

Measuring energy expenditure in birds using bolus injections of 13C-labelled Na-bicarbonate

March 2011


89 Reads

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.

Figure 2. Demonstration that the MAPK14 antibody recognized killifish MAPK14-1. Different concentrations of a plasmid containing cDNA of the complete coding sequence of killifish MAPK14-1 were transfected into HEK-293 cells. A. Representative western blot of MAPK14, B. Summary of MAPK14-1 western blot experiments. N=3/group.
Figure 3. MAPK14-1 and phosphorylated MAPK14-1 protein levels in gills of killifish injected with a control vivo-morpholino or a translational blocking vivo-morpholino targeting MAPK14-1. A. Representative western blot of MAPK14-1. B. Summary of MAPK14-1 western blot experiments. C. Representative western blot of phosphorylated-MAPK14-1. D. Summary of phosporylated-MAPK14-1 western blot experiments. Freshwater acclimated killifish were injected with control vivo-morpholino or MAPK14-1 vivo-morpholino. FW-C: Freshwater control vivo-morpholino, FSW-C: Freshwater to seawater control vivo-morpholino, FSW-M: Freshwater to seawater MAPK14-1 vivo-morpholino. N=9/group. Different letters indicated statistically significant differences between groups p<0.05.
Figure 4. SGK1 mRNA and protein levels in gills of killifish injected with control vivo-morpholino or translational blocking vivo-morpholino targeting MAPK14-1. A: SGK1 mRNA abundance. B: Representative western blot of SGK1. C: Summary of SGK1 western blot experiments. Freshwater acclimated killifish were injected with control vivo-morpholino or MAPK14-1 vivo-morpholino. FW-C: Freshwater control vivo-morpholino, FSW-C: Freshwater to seawater control vivo-morpholino, FSW-M: Freshwater to seawater MAPK14-1 vivomorpholino. N=9/group. Different letters indicated statistically significant differences between groups p<0.05.
Figure 5. Na + ,K + -ATPase protein levels in gills of killifish injected with control vivo-morpholino or translational blocking vivo-morpholino targeting MAPK14-1. A: Representative western blot of Na + ,K + -ATPase. B: Summary of Na + ,K + -ATPase western blot experiments. Freshwater acclimated killifish were injected with control vivo-morpholino or MAPK14-1 vivo-morpholino. FW-C: Freshwater control vivo-morpholino, FSW-C: Freshwater to seawater control vivo-morpholino, FSW-M: Freshwater to seawater MAPK14-1 vivomorpholino. N=9/group. Different letters indicated statistically significant differences between groups p<0.05.
Mitogen Activated Protein Kinase 14-1 Regulates Serum Glucocorticoid Kinase 1 During Seawater Acclimation in Atlantic Killifish, Fundulus heteroclitus

May 2012


59 Reads

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.

of food, water and electrolyte intake in excretion in fat sand rats feeding on two different diets
Palgi, N. , Vatnick, I. & Pinshow, B. Oxalate, calcium and ash intake and excretion balances in fat sand rats (Psammomys obesus) feeding on two different diets. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 141, 48-53

May 2005


130 Reads

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.

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