Successful control of methicillin-resistant Staphylococcus aureus in a spinal cord injury center: a 10-year prospective study including molecular typing.

STUDY DESIGN: Prospective cohort study with medical record review. OBJECTIVE: To evaluate the clinical utility of an infection control program in a patient cohort at high risk for methicillin-resistant Staphylococcus aureus (MRSA) infection and to identify risk factors interfering with successful decolonization of MRSA. SETTING: All spinal cord injured (SCI) patients hospitalized at the Swiss Paraplegic Center (SPC) Nottwil from April 1991 to April 2001. METHODS: Patients whose medical records indicated laboratory-confirmed MRSA colonization or infection were included. Incidence of MRSA colonization or infection was classified as community acquired, nosocomial or transferred based on standardized criteria. Risk factors for community-acquired MRSA colonization in SCI patients were determined. MRSA subtyping and identification of nosocomial spread was performed through pulse-field gel electrophoresis (PFGE). RESULTS: Of 5992 admissions, 100 episodes of MRSA (colonization 22 cases, infection 78 cases) were identified among 76 patients. Overall incidence (1991-2001) per 1000 patient days was 0.26 cases on admission compared to 0.08 at discharge (P<0.001). Community-acquired MRSA was most frequent (56%) followed by nosocomial acquisition (34%). PFGE subtyping identified two nosocomial clusters with six and three cases, respectively. Most of community-acquired MRSA isolates were genetically unrelated and also distinct from epidemic strains identified in Switzerland during the study period. Decolonization was successful in 60 of 76 (78.9%) MRSA-positive patients. CONCLUSION: In the largest European SCI center, MRSA controlling is feasible if infection control policies are vigorously applied.

Influence of chronic supplementation of arginine aspartate in endurance athletes on performance and substrate metabolism - a randomized, double-blind, placebo-controlled study.

The intake of arginine aspartate has been shown to increase anabolic hormones like human growth hormone (hGH) and glucagon. The aim of our study was to investigate whether daily intake of two different dosages of arginine asparate during four weeks affects selected parameters of overtraining syndrome like performance, metabolic and endocrine parameters. Thirty male endurance-trained athletes were included in a randomized, double-blind, placebo-controlled study and divided into three groups. During four weeks, they ingested either arginine aspartate with a high concentration (H) of 5.7 g arginine and 8.7 g aspartate, with a low concentration (L) of 2.8 g arginine and 2.2 g aspartate or placebo (P).VO(2)peak and time to exhaustion were determined on a cycling ergometer in an incremental exercise test before and after supplementation. Before and after each incremental exercise test, concentrations of hGH, glucagon, testosterone, cortisol, ferritine, lactate, and urea were measured. Compared to placebo, no significant differences on endurance performance (VO(2)peak, time to exhaustion), endocrine (concentration of hGH, glucagon, cortisol, and testosterone) and metabolic parameters (concentration of lactate, ferritine, and urea) were found after chronic arginine aspartate supplementation. The chronic intake of arginine asparate during four weeks by male endurance athletes showed independent of dosage no influence on performance, selected metabolic or endocrine parameters. Consequently, there seems to be no apparent reason why the supplementation of arginine aspartate should be an effective ergogenic aid. The practice of using arginine aspartate as potential ergogenics should be critically reevaluated. Further investigations with higher dosage and extended supplementation periods should be performed.

Effects of different sodium concentrations in replacement fluids during prolonged exercise in women.

OBJECTIVE: To investigate the effect of different sodium concentrations in replacement fluids on haematological variables and endurance performance during prolonged exercise. METHODS: Thirteen female endurance athletes completed three four hour runs on a 400 m track. Environmental conditions differed between the three trials: 5.3 degrees C and snow (trial 1), 19.0 degrees C and sunny weather (trial 2), 13.9 degrees C and precipitation (trial 3). They consumed 1 litre of fluid an hour during the trials with randomised intake of fluids: one trial (H) with high sodium concentration (680 mg/l), one trial (L) with low sodium concentration (410 mg/l), and one trial with only water (W). Before and after the trials, subjects were weighed and blood samples were taken for analysis of [Na(+)](plasma), packed cell volume, and mean corpuscular volume. RESULTS: The mean (SD) decrease in [Na(+)](plasma) over the whole trial was significantly (p<0.001) less in trial H (2.5 (2.5) mmol/l) than in trial W (6.2 (2.1) mmol/l). Mild hyponatraemia ([Na(+)](plasma) = 130-135 mmol/l) was observed in only six women (46%) in trial H compared with nine (69%) in trial L, and 12 (92%) in trial W. Two subjects (17%) in trial W developed severe hyponatraemia ([Na(+)](plasma)<130 mmol/l). No significant differences were found in performance or haematological variables with the three different fluids. There was no significant correlation between[Na(+)](plasma) after the run and performance. There was a significant correlation between changes in [Na(+)](plasma) and changes in body weight. CONCLUSIONS: Exercise induced hyponatraemia in women is likely to develop from fluid overload during prolonged exercise. This can be minimised by the use of replacement fluids of high sodium concentration. Sodium replacement of at least 680 mg/h is recommended for women in a state of fluid overload during endurance exercise of four hours. However, higher [Na(+)](plasma) after the run and smaller decreases in [Na(+)](plasma) during the trials were no indication of better performance over four hours.

The intracompartmental sorting of myosin alkali light chain isoproteins reflects the sequence of developmental expression as determined by double epitope-tagging competition.

In order to compare within the same cell the various degrees of specificity of myosin alkali light chain (MLC) isoproteins sorting to sarcomeres, a competition assay was established using double epitope tagging. Various combinations of two different MLC isoform cDNAs tagged with either a vesicular stomatitis virus VSV-G (VSV) or a medium T (mT) protein epitope were co-expressed in cultured cardiomyocytes from adult and neonatal rat ventricles. Expressed isoproteins were detected by means of anti-VSV and anti-mT antibodies and their sorting patterns were analyzed by confocal microscopy. The sorting specificity of MLC isoforms to sarcomeric sites was shown to increase in the order MLC3nm, to ML1sa, to MLC1sb, to MLC1f and MLC3f following the sequence of developmental expression. Expressed fast skeletal muscle isoforms (MLC1f and MLC3f) were always localized at the A-bands of myofibrils, while nonmuscle type (MLC3nm) was distributed throughout the cytoplasm. The slow skeletal muscle type (MLC1sa) showed a weak sarcomeric pattern if it was co-expressed with MLC3nm, but it was distributed throughout the cytoplasm when expressed in combination with MLC1f, MLC3f or the slow skeletal/ventricular muscle isoform (MLC1sb). The MLC1sb was localized at the A-bands when it was co-expressed with MLC3nm or MLC1sa, while it was also distributed to the cytoplasm if co-expressed with MLC1f or MLC3f. Further, expression of chimeric cDNAs revealed that the N-terminal lobe of each isoprotein is responsible for the isoform-specific sorting pattern.

Dominant negative effect of cytoplasmic actin isoproteins on cardiomyocyte cytoarchitecture and function.

The intracompartmental sorting and functional consequences of ectopic expression of the six vertebrate actin isoforms was investigated in different types of cultured cells. In transfected fibroblasts all isoactin species associated with the endogenous microfilament cytoskeleton, even though cytoplasmic actins also showed partial localization to peripheral submembranous sites. Functional and structural studies were performed in neonatal and adult rat cardiomyocytes. All the muscle isoactin constructs sorted preferentially to sarcomeric sites and, to a lesser extent, also to stress-fiber-like structures. The expression of muscle actins did not interfere with cell contractility, and did not disturb the localization of endogenous sarcomeric proteins. In sharp contrast, ectopic expression of the two cytoplasmic actin isoforms resulted in rapid cessation of cellular contractions and induced severe morphological alterations characterized by an exceptional outgrowth of filopodia and cell flattening. Quantitative analysis in neonatal cardiomyocytes indicated that the levels of accumulation of the different isoactins are very similar and cannot be responsible for the observed isoproteins-specific effects. Structural analysis revealed a remodeling of the cytoarchitecture including a specific alteration of sarcomeric organization; proteins constituting the sarcomeric thin filaments relocated to nonmyofibrillar sites while thick filaments and titin remained unaffected. Experiments with chimeric proteins strongly suggest that isoform specific residues in the carboxy-terminal portion of the cytoplasmic actins are responsible for the dominant negative effects on function and morphology.

Remodelling of cardiomyocyte cytoarchitecture visualized by three-dimensional (3D) confocal microscopy.

The break-down and reassembly of myofibrils in long-term cultures of adult rat cardiomyocytes was investigated by a novel combination of confocal laser scanning microscopy and three-dimensional image reconstruction, referred to as FTCS, to visualize the morphological changes these cells undergo in culture. FTCS is discussed as an alternative imaging mode to low-magnification scanning electron microscopy. The three-dimensional shape of the cells are correlated with the assembly state of myofibrils in different stages. Based on immunofluorescence and confocal laser scanning microscopy it was shown that myofibrils are degraded within a few days after plating and that newly assembled myofibrils are predominantly confined to the continuous area in the perinuclear region close to the membrane in contact with the substratum. The localization of myofibrils along the cell's vertical axis has been investigated both by optical sectioning using confocal light microscopy and by physical sectioning followed by transmission electron microscopy. Based on the distribution of myofibrillar proteins we propose a model of myofibrillar growth locating the putative assembly sites to a region concentric around the nuclei. We provide evidence that the cell shape is dominated by the myofibrillar apparatus.

Molecular analysis of protein sorting during biogenesis of muscle cytoarchitecture.

Isolated, rod-shaped adult rat cardiomyocytes (ARC) were kept in long-term cell cultures and the changes of the cardiomyocyte structure were investigated by confocal microscopy. The cells round up and make contact with the substrate by very flat, foot-like structures. After prolonged culture the amorphous cells regenerate a cardiomyocyte-like cytoarchitecture and myofibrils reemerge. In the perinuclear region myofibrils form continuously while in other cells discontinuous myofibrillogenesis was observed, where short sarcomeric segments occur all over the cytoplasmic space. During the regeneration of myofibrils certain proteins like a smooth muscle actin sort to non sarcomeric region, while myomesin or heart C-protein localize on myofibrils with high specificity. This culture system combined with method of epitope-tagging of contractile proteins are ideally suited to monitor the intracellular localization sites of exogenously introduced constructs to different cytoskeletal, since ARC exhibit at the same time stress fiber-like filaments (SFLF) and nascent myofibrils. The molecular properties of the different members of the myosin light chain isoprotein family were investigated by transfection experiments using epitope-tagged myosin light chain (MLC) cDNA. The sorting of the different types of MLC was shown to be isoprotein specific and with chimeric constructs it was shown that the isoprotein-specific incorporation into myofibrils was dependent on the presence of the middle domain of MLC-1f/3f. These MLC isoproteins can be arranged into a sequence of increasing affinity to myofibrils. A hierarchical order of myofibrillar assembly is postulated based on the association affinity. Similar experiments with constructs containing alpha-cardiac, alpha-smooth muscle and gamma-cytoplasmic actins have shown that expression of epitope-tagged actins in ARC result in different epitope staining patterns. While the alpha-cardiac actin showed a marked preference for sarcomeres, the alpha-smooth muscle isoproteins had an intermediate specificity and could either be preferentially incorporated into stress fiber-like filaments (SFLF) and in some cells to a lesser extent into myofibrils as well. Most striking results were obtained with gamma-cytoplasmic actin carrying a 5 or 11-mer epitope. This actin gave rise to large cells, induced the formation of filopodia filled with the transfected actin and depletion of the transfected actin from the perinuclear myofibrillar region.