Interindividual variation of isolated muscle performance and fibre-type composition in the toad Bufo viridus

Interindividual analyses of physiological performance represent one of the most powerful tools for identifying functional positive and negative linkages between various performance traits. In this study we investigated functional linkages in the whole-gastrocnemius performance of juvenile Bufo viridis by examining interindividual variation in in vitro muscle performance and muscle fibre-type composition. We used the work-loop technique to investigate the maximum in vitro power output and fatigue resistance of the gastrocnemius muscle during repeated sets of three cycles at the cycle frequency of 5 Hz, simulating an intermittent style of locomotion. We found several significant correlations between different measures of in vitro muscle performance, including a negative correlation between maximum net power output and fatigue resistance of power, indicating functional trade-offs between these performance traits. We also investigated the extent of individual variation in the proportions of different fibre types, and tested for correlations between individual variation in muscle fibre-type composition and the previously measured isolated muscle performance. Fast glycolytic fibres represented 84.0+/-3.4% of the muscle, while the combined slow oxidative and fast oxidative-glycolytic fibres represented 16+/-3.4%. We found no significant correlations between measures of in vitro muscle performance and the proportion of different fibre types in the gastrocnemius muscle. However, despite this lack of correlation between whole-muscle performance and muscle fibre-type composition data, we suggest the functional linkages detected between different measures of in vitro muscular performance have important ecological and evolutionary consequences.

The Runx1 transcription factor controls CSF-1-dependent and -independent growth and survival of macrophages

Gene translocations that repress the function of the Runx1 transcription factor play a critical role in the development of myeloid leukemia. In this report, we demonstrate that Runx1 precisely regulates c-fms (CSF-1 receptor) gene expression. Runx1 controlled expression by binding to multiple sites within the mouse c-fms gene, allowing interaction between promoter and downstream enhancer elements. The runx1 and c-fms genes showed an identical pattern of expression in mature macrophages. Runx1 expression was repressed in CSF-1 stimulated, proliferating bone marrow-derived macrophages (BMM) and significantly increased in quiescent, CSF-1 starved cells. The RAW264.7 and Mono-Mac-6, macrophage-like cell lines expressed low levels of Runx1 and both showed growth arrest and cell death with ectopic expression of Runx1. The EM-3 cell line, which represents an early myeloid progenitor cell line, showed growth arrest with Runx1 expression in the absence of any detectable changes in cell differentiation. These findings suggest that Runx1 regulates growth and survival of myeloid cells and provide a novel insight into the role of Runx family gene translocations in leukemogenesis.

On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells

Monoclonal antibodies (Mab) are heterotetramers consisting of an equimolar ratio of heavy chain (HC) and light chain (LC) polypeptides. Accordingly, most recombinant Mab expression systems utilize an equimolar ratio of heavy chain (he) to light chain (lc) genes encoded on either one or two plasmids. However, there is no evidence to suggest that this gene ratio is optimal for stable or transient production of recombinant Mab. In this study we have determined the optimal ratio of hc:lc genes for production of a recombinant IgG(4) Mab, cB72.3, by Chinese hamster ovary (CHO) cells using both empirical and mathematical modeling approaches. Polyethyleneimine-mediated transient expression of cB72.3 at varying ratios of hc:lc genes encoded on separate plasmids yielded an optimal Mab titer at a hc:lc gene ratio of 3:2; a conclusion confirmed by separate mathematical modeling of the Mab folding and assembly process using transient expression data. On the basis of this information, we hypothesized that utilization of he genes at low hc:lc gene ratios is more efficient. To confirm this, cB72.3 Mab was transiently produced by CHO cells at constant he and varying lc gene dose. Under these conditions, Mab yield was increased with a concomitant increase in lc gene dose. To determine if the above findings also apply to stably transfected CHO cells producing recombinant Mab, we compared the intra- and extracellular ratios of HC and LC polypeptides for three GS-CHO cells lines transfected with a 1:1 ratio of hc:lc genes and selected for stable expression of the same recombinant Mab, cB72.3. Intra- and extracellular HC:LC polypeptide ratios ranged from 1:2 to 1:5, less than that observed on transient expression of the same Mab in parental CHO cells using the same vector. In conclusion, our data suggest that the optimal ratio of hc:lc genes used for transient and stable expression of Mab differ. In the case of the latter, we infer that optimal Mab production by stably transfected cells represents a compromise between HC abundance limiting productivity and the requirement for excess LC to render Mab folding and assembly more efficient.

Clathrin isoform CHC22, a component of neuromuscular and myotendinous junctions, binds sorting nexin 5 and has increased expression during myogenesis and muscle regeneration

The muscle isoform. of clathrin heavy chain, CHC22, has 85% sequence identity to the ubiquitously expressed CHC17, yet its expression pattern and function appear to be distinct from those of well-characterized clathrin-coated vesicles. In mature muscle CHC22 is preferentially concentrated at neuromuscular and myotendinous junctions, suggesting a role at sarcolemmal contacts with extracellular matrix. During myoblast differentiation, CHC22 expression is increased, initially localized with desmin and nestin and then preferentially segregated to the poles of fused myoblasts. CHC22 expression is also increased in regenerating muscle fibers with the same time course as embryonic myosin, indicating a role in muscle repair. CHC22 binds to sorting nexin 5 through a coiled-coil domain present in both partners, which is absent in CHC17 and coincides with the region on CHC17 that binds the regulatory light-chain subunit. These differential binding data suggest a mechanism for the distinct functions of CHC22 relative to CHC17 in membrane traffic during muscle development, repair, and at neuromuscular and myotendinous junctions.

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.

Comparative proteomic analysis of GS-NSO murine myeloma cell lines with varying recombinant monoclonal antibody production rate

We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant monoclonal antibody production (qMab) by engineered murine myeloma (NSO) cells. Four homogeneous NSO cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab clatasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant monoclonal antibody production. (C) 2004 Wiley Periodicals, Inc.

Investigation of signaling pathways that mediate the inotropic effect of urotensin-II in human heart

Objective: This study investigated signaling pathways that may contribute to the potent positive inotropic effect of human urotensin-II (hU-II) in human isolated right atrial trabeculae obtained from patients with coronary artery disease. Methods: Trabeculae were set up in tissue baths and stimulated to contract at 1 Hz. Tissues were incubated with 20 nM hU-II with or without phorbol 12-myristate 13-acetate (PMA, 10 muM) to desensitize PKC, the PKC inhibitor chelerythrine (10 muM), 10 muM 4alpha-phorbol that does not desensitize PKC, the myosin light chain kinase inhibitor wortmannin (50 nM, 10 muM), or the Rho kinase inhibitor Y-27632 (0.1 - 10 muM). Activated RhoA was determined by affinity immunoprecipitation, and phosphorylation of signaling proteins was determined by SDS-PAGE. Results: hU-II caused a potent positive inotropic response in atrial trabeculae, and this was concomitant with increased phosphorylation of regulatory myosin light chain (MLC-2, 1.8 +/- 0.4-fold, P < 0.05, n = 6) and PKCalpha/betaII (1.4 +/- 0.2-fold compared to non-stimulated controls, P < 0.05, n = 7). Pretreatment of tissues with PMA caused a marked reduction in the inotropic effect of hU-II, but did not affect hU-II-mediated phosphorylation of MLC-2. The inotropic response was inhibited by chelerythrine, but not 4alpha-phorbol or wortmannin. Although Y-27632 also reduced the positive inotropic response to hU-II, this was associated with a marked reduction in basal force of contraction. RhoA. GTP was immunoprecipitated in tissues pretreated with or without hU-II, with findings showing no detectable activation of RhoA in the agonist stimulated tissues. Conclusions: The findings indicated that hU-II increased force of contraction in human heart via a PKC-dependent mechanism and increased phosphorylation of MLC-2, although this was independent of PKC. The positive inotropic effect was independent of myosin light chain kinase and RhoA-Rho kinase signaling pathways. (C) 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Cloning and functional expression of venom prothrombin activator protease from Pseudonaja textilis with whole blood procoagulant activity

The snake venom group C prothrombin activators contain a number of components that enhance the rate of prothrombin activation. The cloning and expression of full-length cDNA for one of these components, an activated factor X (factor Xa)-like protease from Pseudonaja textilis as well as the generation of functional chimeric constructs with procoagulant activity were described. The complete cDNA codes for a propeptide, light chain, activation peptide (AP) and heavy chain related in sequence to mammalian factor X. Efficient expression of the protease was achieved with constructs where the AP was deleted and the cleavage sites between the heavy and light chains modified, or where the AP was replaced with a peptide involved in insulin receptor processing. In human kidney cells (H293F) transfected with these constructs, up to 80% of the pro-form was processed to heavy and light chains. Binding of the protease to barium citrate and use of specific antibodies demonstrated that gamma-carboxylation of glutamic acid residues had occurred on the light chain in both cases, as observed in human factor Xa and the native P. textilis protease. The recombinant protease caused efficient coagulation of whole citrated blood and citrated plasma that was enhanced by the presence of Ca2+. This study identified the complete cDNA sequence of a factor Xa-like protease from P. textilis and demonstrated for the first time the expression of a recombinant form of P. textilis protease capable of blood coagulation.

Molecular genetics of sudden cardiac death in small animals - A review

Sudden cardiac death in small animals is uncommon but often occurs due to cardiac conduction defects or myocardial diseases. Primary cardiac conduction defects are mainly caused by mutations in genes involved in impulse conduction processes (e.g., gapjunction genes and transcription factors) or repolarisation processes (e.g., ion-channel genes), whereas primary cardiomyopathies are mainly caused by defective force generation or force transmission due to gene mutations in either sarcomeric or cytoskeleton proteins. Although over 50 genes have been identified in humans directly or indirectly related to sudden cardiac death, no genetic aetiologies have been identified in small animals. Sudden cardiac deaths have been also reported in German Shepherds and Boxers. A better understanding of molecular genetic aetiologies for sudden cardiac death will be required for future study toward unveiling actiology in sudden cardiac death in small animals. (c) 2005 Elsevier Ltd. All rights reserved.

Surface-functionalized polymer nanoparticles for selective sequestering of heavy metals

Xanthate-mediated (reversible addition-fragmentation chain transfer) emulsion polymerization has been used to create novel polystyrene nanoparticles with functionalized surfaces (see Figure) for the selective sequestering of heavy metals from water below ppm levels. These nanoparticles show a high degree of selectivity for Hg-II over Co-II. This technology has potential for the selective remediation of heavy metals from the human blood system.