Sustained swimming performance and muscle structure are altered by thermal acclimation in male mosquitofish

Males of the eastern mosquito fish (Gambusia holbrooki) possess one of the widest reproductively active temperature ranges for any ectotherm, ranging across seasons from at least 18degreesC to 34degreesC. In this study, we tested the ability of male G. holbrooki to acclimate their sustained swimming performance following long-term exposure to 18degreesC or 30degreesC. We also investigated some of the possible physiological mechanisms associated with thermal acclimation responses in swimming performance, including changes in slow muscle fibre size and abundance and the expression of myosin heavy chains (MyHC). We found that U-crit, of 18degreesC-acclimated G. holbrooki was 20% greater at 18degreesC than 30degreesC-acclimated fish, and the Ucrit of the 30degreesC-acclimated group was more than 15% greater at 30degreesC. Slow, fast and intermediate muscle fibres were identified on the basis of their myosin ATPase staining reaction. Although the number of slow and intermediate muscle fibres was similar between groups, the total cross-sectional area of aerobic fibre types was 40% greater in 18degrees-than 30degreesC-acclimated fish, reflecting an increase in the average fibre diameter. An S58 antibody raised against chicken slow skeletal muscle myosin stained a sub-set of the slow fibres identified by myosin ATPase staining. The number of S58-positive muscle fibres was 50% greater in 30degreesC-than 18degreesC-acclimated fish, implying that different MyHCs are being expressed in cold and warm acclimated individuals. Given the sexually coercive mating system of this species, increases in the sustained swimming performance via thermal acclimation may benefit the ability of males to maintain a high rate of sneaky copulations. (C) 2004 Elsevier Ltd. All rights reserved.

Membrane insertion of anthrax protective antigen and cytoplasmic delivery of lethal factor occur at different stages of the endocytic pathway

The protective antigen (PA) of anthrax toxin binds to a cell surface receptor, undergoes heptamerization, and binds the enzymatic subunits, the lethal factor (LF) and the edema factor (EF). The resulting complex is then endocytosed. Via mechanisms that depend on the vacuolar ATPase and require membrane insertion of PA, LF and EF are ultimately delivered to the cytoplasm where their targets reside. Here, we show that membrane insertion of PA already occurs in early endosomes, possibly only in the multivesicular regions, but that subsequent delivery of LF to the cytoplasm occurs preferentially later in the endocytic pathway and relies on the dynamics of internal vesicles of multivesicular late endosomes.

AAA ATPases regulate membrane association of yeast oxysterol binding proteins and sterol metabolism

The yeast genome encodes seven oxysterol binding protein homologs, Osh1p-Osh7p, which have been implicated in regulating intracellular lipid and vesicular transport. Here, we show that both Osh6p and Osh7p interact with Vps4p, a member of the AAA ( ATPases associated with a variety of cellular activities) family. The coiled-coil domain of Osh7p was found to interact with Vps4p in a yeast two-hybrid screen and the interaction between Osh7p and Vps4p appears to be regulated by ergosterol. Deletion of VPS4 induced a dramatic increase in the membrane-associated pools of Osh6p and Osh7p and also caused a decrease in sterol esterification, which was suppressed by overexpression of OSH7. Lastly, overexpression of the coiled-coil domain of Osh7p (Osh7pCC) resulted in a multi-vesicular body sorting defect, suggesting a dominant negative role of Osh7pCC possibly through inhibiting Vps4p function. Our data suggest that a common mechanism may exist for AAA proteins to regulate the membrane association of yeast OSBP proteins and that these two protein families may function together to control subcellular lipid transport.

NmlR of Neisseria gonorrhoeae: a novel redox responsive transcription factor from the MerR family

A MerR-like regulator (NmlR -Neisseria merR-like Regulator) identified in the Neisseria gonorrhoeae genome lacks the conserved cysteines known to bind metal ions in characterized proteins of this family. Phylogenetic analysis indicates that NmlR defines a subfamily of MerR-like transcription factors with a distinctive pattern of conserved cysteines within their primary structure. NmlR regulates itself and three other genes in N. gonorrhoeae encoding a glutathione-dependent dehydrogenase (AdhC), a CPx-type ATPase (CopA) and a thioredoxin reductase (TrxB). An nmlR mutant lacked the ability to survive oxidative stress induced by diamide and cumene hydroperoxide. It also had > 50-fold lower NADH-S-nitrosoglutathione oxidoreductase activity consistent with a role for AdhC in protection against nitric oxide stress. The upstream sequences of the NmlR regulated genes contained typical MerR-like operator/promoter arrangements consisting of a dyad symmetry located between the -35 and -10 elements of the target genes. The NmlR target operator/promoters were cloned into a beta-galactosidase reporter system and promoter activity was repressed by the introduction of NmlR in trans. Promoter activity was activated by NmlR in the presence of diamide. Under metal depleted conditions NmlR did not repress P-AdhC (or P-CopA) promoter activity, but this was reversed in the presence of Zn(II), indicating repression was Zn(II)-dependent. Analysis of mutated promoters lacking the dyad symmetry revealed constitutive promoter activity which was independent of NmlR. Gel shift assays further confirmed that NmlR bound to the target promoters possessing the dyad symmetry. Site-directed mutagenesis of the four NmlR cysteine residues revealed that they were essential for activation of gene expression by NmlR.

Thapsigargin modulates osteoclastogenesis through the regulation of RANKL-induced signaling pathways and reactive oxygen species production

Introduction: Apoptosis and differentiation are among the consequences of changes in intracellular Ca2+ levels. In this study, we investigated the effects of the endoplasmic reticular Ca2+-ATPase inhibitor, thapsigargin (TG), on osteoclast apoptosis and differentiation. Materials and Methods: Both RAW264.7 cells and primary spleen cells were used to examine the effect of TG on RANKL-induced osteoclastogenesis. To determine the action of TG on signaling pathways, we used reporter gene assays for NF-kappa B and activator protein-1 (AP-1) activity, Western blotting for phosphoextracellular signal-related kinase (ERK), and fluorescent probes to measure changes in levels of intracellular calcium and reactive oxygen species (ROS). To assess rates of apoptosis, we measured changes in annexin staining, caspase-3 activity, and chromatin and F-actin microfilament structure. Results: At concentrations that caused a rapid rise in intracellular Ca2+, TG increased caspase-3 activity and promoted apoptosis in osteoclast-like cells (OLCs). Low concentrations of TG, which were insufficient to measurably alter intracellular Ca2+, unexpectedly suppressed caspase-3 activity and enhanced RANKL-induced osteoclastogenesis. At these lower concentrations, TG potentiated ROS production and RANKL-induced NF-kappa B activity, but suppressed RANKL-induced AP-1 activity and had little effect on ERK phosphorylation. Conclusion: Our novel findings of a biphasic effect of TG are incompletely explained by our current understanding of TG action, but raise the possibility that low intensity or local changes in subcellular Ca2+ levels may regulate intracellular differentiation signaling. The extent of cross-talk between Ca2+ and RANKL-mediated intracellular signaling pathways might be important in determining whether cells undergo apoptosis or differentiate into OLCs.

Physiological role of carnosine in contracting muscle

High-intensity exercise leads to reductions in muscle substrates (ATP, PCr, and glycogen) and a subsequent accumulation of metabolites (ADP, Pi, H+, and M2+) with a possible increase in free radical production. These factors independently and collectively have deleterious effects on muscle, with significant repercussions on high-intensity performance or training sessions. The effect of carnosine on overcoming muscle fatigue appears to be related to its ability to buffer the increased H+ concentration following high-intensity work. Carnosine, however, has other roles such as an antioxidant, a metal chelator, a Ca2+ and enzyme regulator, an inhibitor of protein glycosylation and protein-protein cross-linking. To date, only 1 study has investigated the effects of carnosine supplementation (not in pure form) on exercise performance in human subjects and found no improvement in repetitive high-intensity work. Much data has come from in vitro work on animal skeletal muscle fibers or other components of muscle contractile mechanisms. Thus further research needs to be carried out on humans to provide additional understanding on the effects of carnosine in vivo.

Altered fungal sensitivity to a plant antimicrobial peptide through over-expression of yeast cDNAs

A yeast cDNA expression library was screened to identify genes and cellular processes that influence fungal sensitivity to a plant antimicrobial peptide. A plasmid-based, GAL1 promoter-driven yeast cDNA expression library was introduced into a yeast genotype susceptible to the antimicrobial peptide MiAMP1 purified from Macadamia integrifolia. Following a screen of 20,000 cDNAs, three yeast cDNAs were identified that reproducibly provided transformants with galactose-dependent resistance to MiAMP1. These cDNAs encoded a protein of unknown function, a component (VMA11) of the vacuolar H+-ATPase and a component (cytochrome c oxidase subunit VIa) of the mitochondrial electron transport chain, respectively. To identify genes that increased sensitivity to MiAMP1, the yeast cDNA expression library was introduced into a yeast mutant with increased resistance to MiAMP1. From 11,000 cDNAs screened, two cDNA clones corresponding to a ser/thr kinase and a ser/thr phosphatase reproducibly increased MiAMP1 susceptibility in the mutant in a galactose-dependent manner. Deletion mutants were available for three of the five genes identified but showed no change in their sensitivity to MiAMP1, indicating that these genes could not be detected by screening of yeast deletion mutant libraries. Yeast cDNA expression library screening therefore provides an alternative approach to gene deletion libraries to identify genes that can influence the sensitivity of fungi to plant antimicrobial peptides.

Calcium transport and signaling in the mammary gland: Targets for breast cancer

The mammary gland is subjected to extensive calcium loads during lactation to support the requirements of milk calcium enrichment. Despite the indispensable nature of calcium homeostasis and signaling in regulating numerous biological functions, the mechanisms by which systemic calcium is transported into milk by the mammary gland are far from completely understood. Furthermore, the implications of calcium signaling in terms of reaulating proliferation, differentiation and apoptosis in the breast are currently uncertain. Deregulation of calcium homeostasis and signaling is associated with mammary gland pathophysiology and as such, calcium transporters, channels and binding proteins represent potential drug targets for the treatment of breast cancer. (c) 2005 Elsevier B.V. All rights reserved.

CIC-5: A chloride channel with multiple roles in renal tubular albumin uptake

CIC-5 is a chloride (Cl-) channel expressed in renal tubules and is critical for normal tubular function. Loss of function nonsense or missense mutations in CIC-5 are associated with Dent's disease, a condition in which patients present with low molecular weight (LMW) proteinuria (including albuminuria), hypercalciuria and nephrolithiasis. Several key studies in CIC-5 knockout mice have shown that the proteinuria results from defective tubular reabsorption of proteins. CIC-5 is typically regarded as an intracellular Cl- channel and thus the defect in this receptor-mediated uptake pathway was initially attributed to the failure of the early endosomes to acidify correctly. CIC-5 was postulated to play a key role in transporting the Cl- ions required to compensate for the movement of H+ during endosomal acidification. However, more recent studies suggest additional roles for CIC-5 in the endocytosis of albumin. CIC-5 is now known to be expressed at low levels at the cell surface and appears to be a key component in the assembly of the macromolecular complex involved in protein endocytosis. Furthermore, mutations in CIC-5 affect the trafficking of v-H+-ATPase and result in decreased expression of the albumin receptor megalin/cubulin. Thus, the expression of CIC-5 at the cell surface as well as its presence in endosomes appears to be essential for normal protein uptake by the renal proximal tubule. (c) 2005 Elsevier Ltd. All rights reserved.

The effects of freshwater to seawater transfer on circulating levels of angiotensin II, C-type natriuretic peptide and arginine vasotocin in the euryhaline elasmobranch, Carcharhinus leucas

This study examined the effect of transfer to increased environmental salinity on the circulating levels of angiotensin II (ANG II), C-type natriuretic peptide (CNP), and arginine vasotocin (AVT) in the euryhaline elasmobranch, Carcharhinus letteas. Plasma levels of ANG 11 and CNP were significantly increased in C. leucas chronically acclimated to seawater (SW) in comparison to freshwater (FW) acclimated fish. There was no difference in plasma AVT levels. Acute transfer of FW fish to 75% SW induced an increase in plasma ANG II levels within 12 h, and subsequent transfer from 75 to 100% SW further increased plasma ANG 11 levels at both 24 and 72 h. No change in plasma CNP was observed during acute transfer to increased salinity. However, a significant increase in plasma AVT levels was observed following 96 h in 75% SW and 24 h in 100% SW. In chronically SW acclimated C leucas plasma osmolality, sodium, chloride, and Urea were all significantly higher than FW acclimated fish but there was no difference in haematocrit. Acute transfer of C letteas to 75% SW induced a significant increase in plasma osmolality, sodium and urea concentrations within 96 h of transfer. Subsequent transfer from 75 to 100% SW induced a further increase in these variables within 24 h in addition to a significant increase in plasma chloride above control levels. Haematocrit did not differ between the experimental and control groups throughout the acute study. Circulating levels of ANG 11 were significantly correlated to plasma, sodium, chloride, and urea concentrations during acclimation to SW. Conversely, circulating levels of CNP and AVT did not correlate to plasma osmolytes, however, CNP was significantly correlated to haematocrit during acclimation to seawater. (c) 2005 Elsevier Inc. All rights reserved.

Electrolyte transport in the mammalian colon: mechanisms and implications for disease Kunzelmann and Marcus Mall

Electrolyte Transport in the Mammalian Colon: Mechanisms and Implications for Disease. Physiol. Rev. 82: 245-289, 2002.The colonic epithelium has both absorptive and secretory functions. The transport is characterized by a net absorption of NaCl, short-chain fatty acids (SCFA), and water, allowing extrusion of a feces with very little water and salt content. In addition, the epithelium does secret mucus, bicarbonate, and KCl. Polarized distribution of transport proteins in both luminal and basolateral membranes enables efficient salt transport in both directions, probably even within an individual cell. Meanwhile, most of the participating transport proteins have been identified, and their function has been studied in detail. Absorption of NaCl is a rather steady process that is controlled by steroid hormones regulating the expression of epithelial Na+ channels (ENaC), the Na+-K+-ATPase, and additional modulating factors such as the serum- and glucocorticoid-regulated kinase SGK. Acute regulation of absorption may occur by a Na+ feedback mechanism and the cystic fibrosis transmembrane conductance regulator (CFTR). Cl- secretion in the adult colon relies on luminal CFTR, which is a cAMP-regulated Cl- channel and a regulator of other transport proteins. As a consequence, mutations in CFTR result in both impaired Cl- secretion and enhanced Na+ absorption in the colon of cystic fibrosis (CF) patients. Ca2+- and cAMP-activated basolateral K+ channels support both secretion and absorption of electrolytes and work in concert with additional regulatory proteins, which determine their functional and pharmacological profile. Knowledge of the mechanisms of electrolyte transport in the colon enables the development of new strategies for the treatment of CF and secretory diarrhea. It will also lead to a better understanding of the pathophysiological events during inflammatory bowel disease and development of colonic carcinoma.

Isoform specific changes in calcium transporters in colon and breast cancer cell lines

Calcium transporters play vital roles in the transport of calcium ions across cells of the mammary gland and the intestine. One such transporter is the plasma membrane Ca2+-ATPase (PMCA), of which there are 4 different genes (PMCA1-4). In these studies we investigated the hypothesis that the expression of PMCA is altered in HT-29 colon cancer cells during sodium butyrate and post-confluence mediated differentiation. We also investigated if PMCA expression is altered in breast cancer cell lines in an isofrom specific manner. Our results indicate isoform specific changes in PMCA mRNA and protein levels in HT-29 cells during differentiation, using real time RT-PCR and western blotting, respectively. We also observed pronounced alterations in the mRNA levels of the PMCA isoform linked to lactation (PMCA2) in a bank of breast cancer cell lines compared to normal cell lines. Changes in other isoforms were less pronounced. To further study the role of specific calcium transporters we have optimised conditions for the reverse transfection of MCF-7 breast cancer cells using NeoFX (Ambion). Using real time RT-PCR we have confirmed gene knockdown for specific isoforms and have studied the time course of knockdown over 96 hours. We see approximately 68 % inhibition at 24 hours increasing to 84 % 96 hours post-reverse transfection. Our studies suggest that the expression of specific calcium transporter isoforms can be significantly altered in cancer cell lines and that isoform specific inhibition of calcium transporters is possible using reverse transfection of siRNA