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.

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.

Practical considerations for the measurement of free light chains in serum

Background: A new immunoassay for free light chain measurements has been reported to be useful for the diagnosis and monitoring of monoclonal light chain diseases and nonsecretory myeloma. We describe experience with and some potential pitfalls of the assay. Methods: The assay was assessed for precision, sample type and stability, recovery, and harmonization of results between two analyzers on which the reagents are used. Free-light-chain concentrations were measured in healthy individuals (to determine biological variation), patients with monoclonal gammopathy of undetermined significance, myeloma patients after autologous stem cell transplants, and patients with renal disease. Results: Analytical imprecision (CV) was 6-11% for kappa and A free-light-chain measurement and 16% for the calculated kappa/lambda ratio. Biological variation was generally insignificant compared with analytical variation. Despite the same reagent source, values were not completely harmonized between assay systems and may produce discordant free-light-chain ratios. In some patients with clinically stable myeloma, or post transplantation, or with monoclonal gammopathy of undetermined significance, free-light-chain concentration and ratio were within the population reference interval despite the presence of monoclonal intact immunoglobulin in serum. In other patients with monoclonal gammopathy of undetermined significance, values were abnormal although there was no clinical evidence of progression to multiple myeloma. Conclusions: The use of free-light-chain measurements alone cannot differentiate some groups of patients with monoclonal gammopathy from healthy individuals. As with the introduction of any new test, it is essential that more scientific data about use of this assay in different subject groups are available so that results can be interpreted with clinical certainty. (C) 2003 American Association for Clinical Chemistry.

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.

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.

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.

Rho and vascular disease

The Rho family GTPases are regulatory molecules that link surface receptors to organisation of the actin cytoskeleton and play major roles in fundamental cellular processes. In the vasculature Rho signalling pathways are intimately involved in the regulation of endothelial barrier function, inflammation and transendothelial leukocyte migration, platelet activation, thrombosis and oxidative stress, as well as smooth muscle contraction, migration, proliferation and differentiation, and are thus implicated in many of the changes associated with atherogenesis. Indeed, it is believed that many of the beneficial, non-lipid lowering effects of statins occur as a result of their ability to inhibit Rho protein activation. Conversely, the Rho proteins can have beneficial effects on the vasculature, including the promotion of endothelial repair and the maintenance of SMC differentiation. Further identification of the mechanisms by which these proteins and their effectors act in the vasculature should lead to therapies that specifically target only the adverse effects of Rho signalling. (c) 2005 Elsevier Ireland Ltd. 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.

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.

Periocular and Orbital Amyloidosis. Clinical Characteristics, Management, and Outcome

Objective: To present the clinical features and management outcome in a large series of patients with periocular and orbital amyloidosis. Design: Retrospective, noncomparative, interventional case series. Patients: All patients diagnosed with periocular and orbital amyloidosis in 6 oculoplastic and orbital units. Methods: Clinical records of all patients were reviewed. Main Outcome Measures: Clinical presentation, radiological and histological findings, treatment modalities, and outcome. Results. The study included 24 patients (15 female, 9 male) with a mean age of 57 17 years. Nineteen cases were unilateral, and 5 were bilateral. Clinical signs and symptoms included a visible or palpable periocular mass or tissue infiltration (95.8%), ptosis (54.2%), periocular discomfort or pain (25%), proptosis or globe displacement (21%), limitations in ocular motility (16.7%), recurrent periocular subcutaneous hemorrhages (12.5%), and diplopia (8.3%). Seven cases had orbital involvement, and 17 were periocular. Immunohistochemistry in 7 patients showed B cells or plasma cells producing monoclonal immunoglobulin chains that were deposited as amyloid light chains. Only 1 patient was diagnosed with systemic amyloid light chain amyloidosis. Treatment modalities were mainly observation and surgical debulking. During a mean follow-up period of 39 months, 21% showed significant progression after treatment, whereas 79% were stable or showed no recurrence after treatment. Conclusion: Periocular and orbital amyloidosis may present with a wide spectrum of clinical findings and result in significant ocular morbidity. Complete surgical excision is not feasible in many cases, and the goal of treatment is to preserve function and to prevent sight-threatening complications.

Functional recycling of C2 domains throughout evolution: A comparative study of synaptotagmin, protein kinase C and phospholipase C by sequence, structural and modelling approaches

The C2 domain is one of the most frequent and widely distributed calcium-binding motifs. Its structure comprises an eight-stranded beta-sandwich with two structural types as if the result of a circular permutation. Combining sequence, structural and modelling information, we have explored, at different levels of granularity, the functional characteristics of several families of C2 domains. At the coarsest level,the similarity correlates with key structural determinants of the C2 domain fold and, at the finest level, with the domain architecture of the proteins containing them, highlighting the functional diversity between the various subfamilies. The functional diversity appears as different conserved surface patches throughout this common fold. In some cases, these patches are related to substrate-binding sites whereas in others they correspond to interfaces of presumably permanent interaction between other domains within the same polypeptide chain. For those related to substrate-binding sites, the predictions overlap with biochemical data in addition to providing some novel observations. For those acting as protein-protein interfaces' our modelling analysis suggests that slight variations between families are a result of not only complementary adaptations in the interfaces involved but also different domain architecture. In the light of the sequence and structural genomic projects, the work presented here shows that modelling approaches along with careful sub-typing of protein families will be a powerful combination for a broader coverage in proteomics. (C) 2003 Elsevier Ltd. All rights reserved.