Effects of a chronic wasting infection on skeletal muscle size and contractile properties.

To evaluate the effects of chronic infection on skeletal muscle dimensions and contractile properties, we used a hamster model of visceral leishmaniasis, a parasitic infection of the reticuloendothelial system produced by Leishmania donovani (LD). To distinguish between effects of reduced caloric intake and infection per se, we also studied healthy control animals and noninfected animals subjected to caloric restriction. Three muscles were tested in vitro: plantaris, soleus, and diaphragm. Both caloric restriction and LD infection caused loss of body weight and reduced muscle cross-sectional areas and wet weights. The interventions had variable effects on in vitro contractile properties, the most pronounced being reduction in peak tension in response to tetanic stimulation. Tension loss was 35-45%, except for a loss of 65% in plantaris of LD-infected animals. We conclude that chronic LD infection affects skeletal muscles in both indirect and direct ways. 1) Reduced caloric intake due to anorexia decreases muscle size and active tension. Disuse probably enhances this effect in limb muscles. 2) Infection produces profound weakness of inactive fast-twitch muscle by unknown mechanisms.

Visceral leishmaniasis: a model for infection-induced cachexia.

Parasitic infections and malnutrition coexist in many tropical and subtropical areas. Studies of Leishmania donovani and of experimentally infected Syrian hamsters have provided important insights into the complex interrelationships between malnutrition and this parasitic disease. Malnutrition, which adversely affects cell-mediated immunity, is associated with the development of visceral leishmaniasis (kala-azar) in children living in endemic areas. In turn, L. donovani can cause wasting as well as hepatosplenomegaly, fever, and anemia. Syrian hamsters infected with L. donovani develop a disease that is comparable to that of humans with kala-azar. Weight loss in infected hamsters is associated with splenic macrophage secretion of potentially catabolic cytokines as measured by the D10.G4.1 assay for interleukin-1 and the L929 cytotoxicity assay for tumor necrosis factor/cachectin. Although decreased food intake contributes to wasting in infected hamsters, studies of skeletal muscle function indicate that it is not the sole factor. Leishmania donovani-infected hamsters have also been used to study drugs with the potential to prevent or reverse cachexia.

Actin isoform expression, cellular heterogeneity, and contractile function in smooth muscle.

Smooth muscles express four isoforms of actin: two smooth muscle specific and two cytoplasmic isoforms typically associated with the cytoskeleton of nonmuscle cells. The relative amounts of each isoform expressed and the total actin content vary with smooth muscle type, with development, in cell culture, pathologically, and potentially between cells within tissues. Our objective was to determine whether actin isoforms contribute to contractile diversity. Functional diversity may be the result of differences in the kinetics of the cross-bridge interaction with thin filaments consisting of different actin isoforms or of the fraction of cross-bridges developing force in series or in parallel resulting from thin filaments of different lengths. Our hypothesis was that functionally significant differences in actin isoform properties (i.e., myosin interactions or properties affecting thin filament lengths) would require isoform segregation into distinct populations of thin filaments within cellular domains (e.g., cytoskeletal and contractile) or in phenotypically different cells. We tested this hypothesis by determining the smooth muscle alpha- and gamma-actin and cytoplasmic beta-actin isoform composition of native thin filaments isolated from swine stomach using isoform-specific antibodies linked to colloidal gold beads with protein A (the cytoplasmic lambda-isoactin content was below the detection limit). The lengths of individual thin filaments were also estimated from electron micrographs. A statistically uniform population of thin filaments was observed consisting of randomly copolymerized isoactins in each filament with the same isoform proportions as the tissue. The average thin filament length was 1.35 +/- 0.06 (SEM) microns. These results, together with other studies, suggest that actin isoforms are functionally equivalent. The data imply that the high stress-generating and shortening capacities of smooth muscles are not primarily due to long thin filament to thick filament length ratios compared with striated muscles.

Sex differences in response to hepatitis B infection among patients receiving chronic dialysis treatment.

Patients undergoing treatment at a community-based renal dialysis clinic were monitored monthly for hepatitis B surface antigen (HBsAg) and antibody to HBsAg (anti-HBs). Of 160 patients who began treatment HBsAg(-)/anti-HBs(-), 77 subsequently became HBsAg(+). Once HBsAg(+), males were more likely to remain HBsAg(+) indefinitely, whereas females were more likely to convert to HBsAg(-) and develop anti-HBs. This was not due to a sex difference in exposure to hepatitis B virus because only patients who became infected while undergoing treatment were included in the analysis. These data are clear evidence of a sex difference in response to hepatitis B virus, which may partially explain the greater incidence of several chronic liver diseases, including primary hepatocellular carcinoma, in males.

Localization of isoactins in isolated smooth muscle thin filaments by double gold immunolabeling.

Smooth muscle cells contain both smooth muscle-specific and cytoplasmic actin isoforms. We tested the hypothesis that these two classes of isoactins are segregated into separate "contractile" vs. "cytoskeletal" populations of thin filaments. Isolated thin filaments from adult swine stomach were double-labeled with isoactin-specific peptide antibodies with the use of two different size protein A-gold markers to localize the isoactins on individual filaments. The large and small gold beads bound to individual filaments were counted on electron micrographs, and the results were analyzed statistically. Both classes of actin isoforms were present in every filament, and the relative proportions of smooth muscle vs. cytoplasmic actin isoforms in each filament showed no significant deviation from a single random population. Furthermore, the distribution of the isoactins along each filament was also random; no significant clustering of isoforms was observed. Controls containing added skeletal muscle F-actin showed that the techniques used could have detected nonrandom isoform distributions if they had been present.

Smooth muscle myosin subfragment-1 is a kinetic analogue for heavy meromyosin in the extended conformation.

The 10S-->6S (Flexed-->Extended) transition in smooth muscle myosin is related to increased ATPase activity, but there is controversy over whether the analogous 9S-->7S transition in HMM is also associated with ATPase activity. We therefore studied the association of ionic strength, phosphorylation, and ATPase activity for HMM as compared to S1 which has no apparent flexed conformation. In addition, we performed both steady state and single turnover analyses, to control for artifacts due to multiple subfragment populations that might skew steady state results. At low ionic strength where myosin and HMM are in the flexed conformation, HMM had a near zero ATPase activity while S-1 had a high ATPase rate (0.07 s-1). At 400 mM ionic strength, where both myosin and HMM are in the extended conformation, S1 and HMM had the same ATPase rate (0.04 s-1). Phosphorylation did not affect S1 significantly, but shifted the HMM curve to higher rates at lower ionic strengths. Both steady state and single turnover experiments gave the same results, indicating that steady state results were not skewed by multiple subfragment populations. These data indicate that HMM has a conformation-ATPase relation similar to that observed with myosin. Furthermore, these findings suggest that the S1 ATPase rate corresponds to that of HMM in the extended conformation.

Product inhibition of the actomyosin subfragment-1 ATPase in skeletal, cardiac, and smooth muscle.

We studied product inhibition of the actin-activated ATPase of myosin subfragment-1 (S-1) from the three types of muscle tissue: skeletal, cardiac, and smooth. Increasing levels of [MgADP] in the 0-1-mM range caused significant inhibition of the actin-activated MgATPase activity of cardiac and gizzard but not skeletal muscle S-1. When total nucleotide concentration ([ATP] + [ADP]) was kept constant at 1 mM, ATPase activity was inhibited by 50% at an ADP/ATP ratio of 6:1 for cardiac S-1 and 3:1 for gizzard S-1. For skeletal S-1, however, even a 19:1 ratio did not cause 50% inhibition of ATPase activity. The observed effect was not due to changes in pH or inorganic phosphate concentration, nor could it be explained by substrate (ATP) depletion. In the absence of actin, ADP had little or no inhibitory effect on the ATPase activity of S-1, and these observations imply that ADP is competing directly for the ATP binding site of the actin-S1 complexes of cardiac and smooth muscle S-1. ADP has previously been shown to be a weak competitive inhibitor of the ATPase activity in skeletal muscle. The current data imply that ADP is a very effective competitive inhibitor for the actin-activated ATPase activity of cardiac and gizzard S-1 and, therefore, that ADP may be a physiologically important modulator of contractile activity in cardiac and smooth muscle.

LC20 and kinetics of gizzard myosin subfragment-1: digestion with papain vs. S. aureus protease.

Previous reports have shown that papain-digested gizzard subfragment-1 (PAP-S1) has a cleaved regulatory light chain (LC20), and Vmax similar to phosphorylated heavy meromyosin (HMM) (Greene et al., Biochemistry 22:530-535, 1983; Sellers et al., J. Biol. Chem. 257:13880-13883, 1982; Umemoto et al., J. Biol. Chem. 264:1431-1436, 1989], while S. aureus protease-digested S-1 (SAP-S1) has intact LC20, but Vmax closer to that of unphosphorylated HMM [Ikebe and Hartshorne, 1985]. To determine whether intact LC20 inhibits ATPase activity for subfragment-1 (S1), we compared the kinetic properties and structures of unphosphorylated PAP-S1 and SAP-S1. SDS-PAGE showed that SAP-S1 had 68 and 24 KDa heavy chain and 20 and 17 KDa light chain components. PAP-S1 (15 minutes digestion at 20 degrees C) also had 68 and 17 KDa bands, but the single 24 KDa band (24HC) was replaced by a group of 22-24 KDa fragments and LC20 was cleaved to a 16 KDa fragment. At 13 mM ionic strength, both PAP-S1 and SAP-S1 had Vmax similar to phosphorylated HMM (1.1-1.5 s-1). SAP-S1 had the same KATPase as phosphorylated HMM (38 microM actin), but KATPase for PAP-S1 was 3-fold stronger (11 microM actin). Subsequent digestion of SAP-S1 with papain did not significantly change Vmax, but as LC20 and 24HC were cleaved, both KATPase and Kbinding strengthened 3- to 5-fold. Thus, intact LC20 did not inhibit, and cleavage of LC20 did not increase Vmax for S1. Rather, papain cleavage of LC20 and 24HC was associated with strengthened actin binding.

Phosphopeptide occupancy and photoaffinity cross-linking of the v-Src SH2 domain attenuates tyrosine kinase activity.

Phosphorylation of c-Src at carboxyl-terminal Tyr-527 suppresses tyrosine kinase activity and transforming potential, presumably by facilitating the intramolecular interaction of the C terminus of Src with its SH2 domain. In addition, it has been shown previously that occupancy of the c-Src SH2 domain with a phosphopeptide stimulates c-Src kinase catalytic activity. We have performed analogous studies with v-Src, the transforming protein from Rous sarcoma virus, which has extensive homology with c-Src. v-Src lacks an autoregulatory phosphorylation site, and its kinase domain is constitutively active. Phosphopeptides corresponding to the sequences surrounding c-Src Tyr-527 and a Tyr-Glu-Glu-Ile motif from the hamster polyoma virus middle T antigen inhibit tyrosine kinase activity of baculovirus-expressed v-Src 2- and 4-fold, respectively. To determine the mechanism of this regulation, the Tyr-527 phosphopeptide was substituted with the photoactive amino acid p-benzoylphenylalanine at the adjacent positions (N- and C-terminal) to phosphotyrosine. These peptides photoinactivate the v-Src tyrosine kinase 5-fold in a time- and concentration-dependent manner. Furthermore, the peptides cross-link an isolated Src SH2 domain with similar rates and specificity. These data indicate that occupancy of the v-Src SH2 domain induces a conformational change that is transmitted to the kinase domain and attenuates tyrosine kinase activity.

Reciprocal relationships between undernutrition and the parasitic disease visceral leishmaniasis.

Little is known about the interrelationship between undernutrition and parasitic infections in areas of the world where both are prevalent. The associations between undernutrition and visceral leishmaniasis, an important protozoal disease, were assessed in a study of residents of an area in Brazil with endemic leishmaniasis. Mid-arm anthropometry was used to assess fat and muscle area. Children with visceral leishmaniasis came from large families (9.6 +/- 1.1 members vs. 6.8 +/- 0.7 members in neighborhood control families), and patient housemates had fat areas that were 78% (P less than .05) those of age- and sex-matched neighborhood controls. The children with visceral leishmaniasis who were studied four months or less after diagnosis had fat areas that were 66% (P less than .05) those of age- and sex-matched household controls or 41% (P less than .01) those of neighborhood controls and muscle areas that were 81% (P less than .025) those of household controls or 75% (P less than .05) those of of neighborhood controls. It is hypothesized, on the basis of these data and other findings, that undernutrition is associated with the development of clinically apparent visceral leishmaniasis and that the disease itself has a profound effect on nutritional status, resulting in loss of both muscle and fat, effects that possibly are mediated by interleukin-1 and/or other factors produced by Leishmania donovani-infected macrophages.

Host responses to hepatitis B infection in patients in a chronic hemodialysis unit.

Host responses to hepatitis B infection were studied in 222 patients in a chronic hemodialysis unit. From 1970 to 1976, patients were monitored monthly for development of hepatitis B surface antigen (HBsAg), antibody to hepatitis B surface antigen (anti-HBs), and serum transaminase (SGPT) elevations. Five categories of patients were identified as: 1) chronic carriers of HBsAg; 2) transiently HBsAg(+), who developed anti-HBs; 3) HBsAg(-) on admission, who developed anti-HBs without becoming HBsAg(+); 4) anti-HBs(+) on admission; 5) uninfected who remained HBsAg(-) and anti-HBs(-). For a patient who became HBsAg(+) in this clinic, the probability of becoming a chronic carrier was 62-8% and rose to 88.5% if he or she had been HBsAg(+) for five consecutive months. Males were more likely to become chronic carriers, and females were more likely to develop anti-HBs. Neither age, race, nor type of underlying kidney disease was associated with particular host responses to hepatitis B virus. No effect of hepatitis B infection on mortality was detected. Variation in host response to hepatitis B infection among renal dialysis patients may affect the usefulness of hepatitis B hyperimmune globulin and hepatitis B vaccine and be related to the outcome of kidney transplantation.

Association of graft survival with host response to hepatitis B infection in patients with kidney transplants.

We studied the relation of host response to hepatitis B infection before transplantation with survival of kidney grafts in 79 patients receiving 87 transplants. Antibody to hepatitis B surface antigen (anti-HBs) signaled early graft rejection (median survival congruent to two months), whereas hepatitis B surface antigen (HBsAg) signaled delayed rejection (greater than 22 months). Patients with neither HBsAg nor anti-HBs had graft survival times (median congruent to 16 months) similar to the HBsAg carriers but significantly longer than the anti-HBs-positive patients (p less than 0.01). Similar results were observed when patients who received HLA-identical kidneys or had anti-HLA antibodies before transplantation were excluded. The highest probability of graft rejection was in patients with anti-HBs who received kidneys from male donors. The probability that such grafts would survive for four months was less than 20 per cent. HLA-nonidentical kidneys transplanted into patients with anti-HBs have a poor prognosis, whereas such grafts in HBsAg carriers have as good a prognosis as grafts in uninfected recipients.