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In vitro experiments indicate a non-metabolic role of muscle glycogen in contracting skeletal muscles. Since the sequence of events in excitation\#8211;contraction (E\#8211;C) coupling is known to be located close to glycogen granules, at specific sites on the fibre, we hypothesized that the distinct compartments of glycogen have specific effects on muscle fibre contractility and fatigability. Single skeletal muscle fibres (n = 19) from fed and fasted rats were mechanically skinned and divided into two segments. In one segment glycogen localization and volume fraction were estimated by transmission electron microscopy. The other segment was mechanically skinned and, in the presence of high and constant myoplasmic ATP and PCr, electrically stimulated (10 Hz, 0.8 s every 3 s) eliciting repeated tetanic contractions until the force response was decreased by 50% (mean +/- S.E.M., 81 +/- 16, range 22-252 contractions). Initially the total myofibrillar glycogen volume percentage was 0.46 +/- 0.07%, with 72 +/- 3% in the intermyofibrillar space and 28 +/- 3% in the intramyofibrillar space. The intramyofibrillar glycogen content was positively correlated with the fatigue resistance capacity (r(2) = 0.32, P = 0.02). Intermyofibrillar glycogen was inversely correlated with the half-relaxation time in the unfatigued tetanus (r(2) = 0.25, P = 0.03). These results demonstrate for the first time that two distinct subcellular populations of glycogen have different roles in contracting single muscle fibres under conditions of high myoplasmic ATP.

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Fluoroacetate-specific defluorinase (FSD) is a critical enzyme in the detoxication of fluoroacetate. This study investigated whether FSD can be classed as a glutathione S-transferase (GST) isoenzyme with a high specificity for fluoroacetate detoxication metabolism. The majority of FSD and GST activity, using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as GST substrates, in rat liver was cytosolic. GSTT1 specific substrate, EPNP caused a slight non-competitive inhibition of FSD activity. CDNB, a general substrate of GST isoenzyme, was a more potent non-competitive inhibitor of FSD activity. The fluoroacetate defluorination activity by GST isoenzymes was determined in this study. The results showed that the GSTZ1C had the highest fluoroacetate defluorination activity of the various GST isoenzymes studied, while GSTA2 had a limited activity toward fluoroacetate. The human GSTZ1C recombinant protein then was purified from a human GSTZ1C cDNA clone. Our experiments showed that GSTZ1C catalysed fluoroacetate defluorination. GSTZ1 shares many of the characteristics of FSD; however, it accounts only for 3% of the total cytosolic FSD activity. GSTZ1C based enzyme kinetic studies has low affinity for fluoroacetate. The evidence suggests that GSTZ1 may not be the major enzyme defluorinating fluoroacetate, but it does detoxify the fluoroacetate. To clarify the identity of enzymes responsible for fluoroacetate detoxication, further studies of the overall FSD activity are needed.

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Two forms of fluoroacetate-specific defluorinase (FSD) were purified from rat hepatic cytosol. The first form, FSD1 (molecular weight 38 kDa), contained 81% of the total cytosolic fluoroacetate defluorination activity and did not bind to the glutathione-affinity, orange A or mono P columns used in the purification procedures. The second form, FSD2 (molecular weight 27 kDa), contained only 13% of the fluoroacetate defluorination activity, had a pI = 7.8, and exhibited a high glutathione S-transferase (GST)-like activity towards dichloroacetic acid. The FSD1 proteins were identified from peptide mass data and best matched with rat sorbitol dehydrogenase (SDH) (short form), although pure sheep liver SDH enzyme did not possess defluorination activity when subsequently investigated. The FSD2 protein was identified from peptide mass data and best matched with the amino acid sequence of mouse and human Zeta 1 of glutathione S-transferase (GSTZ1) and showed a high GSTZ1 specific activity. This study suggests that the major FSD component (FSD1) represents a new and unique dehalogenating or dehydrogenating enzyme present in rat liver cytosol. The minor FSD component (FSD2) is due to the GSTZ1 present in rat liver cytosol. However, it is not yet clear that FSD1 is indeed SDH and FSD2 is indeed GSTZ1, due to sequence homology being less than 60 and 45%, respectively.

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BACKGROUND: Hyperacute rejection in xenotransplantation is caused by activation of complement (C) on endothelium. We have previously shown that purified C-regulators of the pig (CD59 and membrane cofactor protein [MCP]) are efficient regulators of human C (HuC). The aim of this study was to clarify the role of endogenously expressed C-regulatory molecules on pig endothelium in the protection against hyperacute rejection. METHODS: Porcine aortic endothelial cells (PAEC) were harvested and cultured for various passages. PAEC were examined for the expression of endogenous pig CD59 and MCP by flow cytometry. PAEC were assessed for their susceptibility to lysis by HuC. The effect of phorbol 12-myristate 13-acetate and various cytokines on the expression of MCP and CD59 and C-susceptibility was assessed. RESULTS: Primary PAEC showed an initial high level of expression of pig CD59, however, upon culturing, CD59 levels decreased dramatically to about 20% after five passages. In contrast, levels of MCP doubled upon culturing of PAEC to confluency and remained stable during at least five passages. Primary cells and cells in the early passages were more resistant to HuC than cells that were cultured for longer. Blocking the function of CD59 but not of MCP using monoclonal antibody increased the susceptibility to HuC. Purified human CD59 incorporated to a level of expression similar to that of pig CD59 reversed the increased C-susceptibility, suggesting that pig and human CD59 are similarly protective against HuC. Increase of C-resistance and of expression of pig MCP, but not of CD59, was achieved upon incubation with phorbol 12-myristate 13-acetate. Tumor necrosis factor-alpha, interleukin-1beta, interleukin-4, or interferon-gamma had no effect on C-regulator expression or C-susceptibility. CONCLUSIONS: These data demonstrate the importance of using primary PAEC or cells in the first passages of culturing in in vitro models of xenotransplantation and show that pig MCP and, in particular, pig CD59 play an important role in protection of PAEC from HuC.

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OBJECTIVES: Four phospholipase C (PLC) beta isoforms have been described in pig aortic vascular smooth muscle. The aim was to determine if all four PLC beta isoforms are commonly expressed in vascular smooth muscle cells (VSMC) of three species, i.e. pig, human and rat, and if the individual isoforms had distinctive intracellular distributions. METHODS: Vascular smooth muscle cell cultures were derived from explants of porcine and rat aorta and a human renal artery cell line. PLC beta isoform content was resolved using Western blotting. Intracellular location was determined by immunocytochemistry and confocal microscopy. RESULTS: All three species expressed PLCs, beta 1, beta 2, beta 3 and beta 4. In all species, PLC beta 1 demonstrated foci of concentration throughout the cytoplasm; PLC beta 2 demonstrated a punctate pattern that was principally at the cell periphery or was in the Golgi, depending upon the antibody used; PLC beta 3 was also cytoplasmic but showed a different pattern from PLC beta 1 and PLC beta 4 was cytoplasmic, except in pig quiescent cells, where it was associated with filamentous structures at the intersection with the plasma membrane. CONCLUSIONS: VSMCs of three different species express all four PLC beta isoforms. Each isoform has a unique and consistent signature of distribution that is generally common to all species.

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Treatment of acute burn wounds with silver sulfadiazine has raised concern of potential silver toxicity. As the wound heals, a barrier forms between the silver sulfadiazine and the blood, but this membrane is not impenetrable, and so silver absorption is still possible. In this work, we have modeled chemical systems to investigate the transport of silver sulfadiazine and silver chloride through cellulose, chitosan, collagen, and polyethylene membranes into the following media: synthetic serum electrolyte solution (SSES), SSES plus glutathione, and human serum, to simulate some of the chemical processes occurring at a burn wound during healing. Our results clearly indicate that membranes can retard the movement of silver ions, especially those that have silver-binding properties. This suggests that silver absorption at a healing wound will be minimized by entrapment of silver in the growing membrane network, and thus the likelihood of silver toxicity will be reduced.

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Silver sulfadiazine cream has been a standard treatment for burns over the past two decades. Although many studies have described the phenomenon of silver absorption from burn wounds treated with silver sulfadiazine, they failed to examine the chemistry underlying the absorption process: Silver chloride was assumed to form at the burn wound and absorption of silver was believed to be negligible. Here we have developed chemical model systems to investigate the interactions of silver sulfadiazine and silver chloride in direct contact with synthetic serum electrolyte solution (SSES), with SSES plus endogenous ligands or beef blood plasma, and with human serum. The results indicate that silver absorption from an acute burn site can be significant, because human serum is capable of solubilizing silver. This finding is of concern, given the potential for silver toxicity as a direct consequence of applying silver sulfadiazine to extensive burn wounds.

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The major components of an alkaloid-free, flue-cured, tobacco essential oil sample are isolated and identified. This is accomplished by utilizing modern hyphenated analytical methods. The instrumentation developed to accomplish this are an automated multidimensional gas chromatograph/mass spectrometer/flame ionization detector (MDGC/MS/FID) and a multidimensional gas chromatograph/matrix isolation/Fourier transform infrared spectrometer (MDGC/MI/FTIR). A total of 306 compounds is identified in the essential oil, of which 80 are found as tobacco constituents for the first time.

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