Oxidants, antioxidants and alcohol: implications for skeletal and cardiac muscle.

The chronic form of alcoholic skeletal myopathy is characterized by selective atrophy of Type II fibers and affects up to two thirds of all alcohol misusers. Plasma selenium and alpha-tocopherol are reduced in myopathic alcoholics compared to alcoholic patients without myopathy. Plasma carnosinase is also reduced in myopathic alcoholics, implicating a mechanism related to reduced intramuscular carnosine, an imidazole dipeptide with putative antioxidant properties. Together with the observation that alcoholic patients have increased indices of lipid peroxidation, there is evidence suggestive of free radical (i.e., unpaired electrons or reactive oxygen species) mediated damage in the pathogenesis of alcohol-induced muscle disease. Protein synthesis is a multi-step process that encompasses amino acid transport, signal transduction, translation and transcription. Any defect in one or more of the innumerable components of each process will have an impact on protein synthesis, as determined by radiolabelling of constituent proteins. Both acute and chronic alcohol exposure are associated with a reduction in skeletal muscle protein synthesis. Paradoxically, alcohol-feeding studies in rats have shown that the imidazole dipeptide concentrations are increased in myopathic muscles though alpha-tocopherol contents are not significantly altered. In acutely dosed rats, where protein synthesis is reduced, protein carbonyl concentrations (an index of oxidative damage to muscle) also decline slightly or are unaltered, contrary to the expected increase. Alcoholic cardiomyopathy can ensue from heavy consumption of alcohol over a long period of time. The clinical features include poor myocardial contractility with reduced left ventricular ejection volume, raised tissue enzymes, dilation of the left ventricle, raised auto- antibodies and defects in mitochondrial function. Whilst oxidant damage occurs in experimental models, however this issues remains to be confirmed in the clinical setting. In the rat, circulating troponin-T release increases in the presence of ethanol, a mechanism ascribed to free radical mediated damage, as it is prevented with the xanthine oxidase inhibitor and beta-blocker, propranolol. However, whilst propranolol prevents the release of troponin-T, it does not prevent the fall in whole cardiac protein synthesis, suggestive of localized ischemic damage due to ethanol.

Comparative effects of acute ethanol dosage on liver and muscle protein metabolism.

Experiments were performed to address some outstanding issues and investigate possible mechanisms relating to the acute comparative effects of ethanol on liver and skeletal muscle protein metabolism. Ethanol (EtOH)-treated rats were injected (i.p.) with a bolus of EtOH (75 mmol/kg body weight) and sacrificed at 20 min, 1-, 2.5-, 6-, and 24-hr time points. Control rats were injected with saline (Con-Sal; 0.15 mmol/L NaCl). All 24-hr ethanol-treated animals were compared with saline-injected rats subjected to controlled feeding (i.e. pair-fed controls for 24 hr EtOH). At 24 hr, there was no measurable alcohol in the plasma, whereas high levels were seen from 20 min to 6 hr (up to 448 mg/dL). Plasma levels of albumin were reduced at initial time points, and activities of aspartate aminotransferase increased, but there was no histological evidence of overt tissue damage either in muscle or liver. Hepatic protein and RNA contents and indices of tissue (C(s) and k(s)) and whole-body (V(s)) protein synthesis were significantly increased in ethanol-dosed rats relative to saline-injected pair-fed controls at 24 hr. In the liver, four of the seven cytoplasmic proteases investigated (alanyl-, arginyl-, and pyroglutamyl-aminopeptidases and proline-endopeptidase) showed significant increases in activity at 24 hr relative to pair-fed controls; four of the six lysosomal proteases showed significant decreases in activity (dipeptidyl-aminopeptidase II and cathepsins B, L, and H). In skeletal muscle, k(s) fell progressively between 1 and 24 hr (-25 to -69%; P < 0.001), but no significant changes in skeletal muscle protease activities were seen at 24 hr. At 24 hr after ethanol dosage in vivo, there were no significant increases in protein carbonyl content in liver or skeletal muscle compared to pair-fed controls (muscle levels actually decreased slightly). However, using either rat or human tissue, both liver and muscle carbonyl increased in vitro in response to superoxide and hydroxyl radicals: muscle was more susceptible to carbonyl formation than liver and both tissues were more sensitive to hydroxyl compared to superoxide radicals. These results show divergent effects of acute ethanol treatment on liver and skeletal muscle protein metabolism, which may not be linked to in vivo free radical-mediated protein damage (as indicated by carbonyl formation), at least in the short term.

Protein synthesis in the heart in vivo, its measurement and patho-physiological alterations.

Changes in cardiac protein composition occur in a variety of patho-physiological situations and are usually accompanied by modifications in protein synthesis. Although adjustments in protein synthesis during starvation may be adaptive, the alterations in protein synthesis seen in response to ethanol ingestion may be pathological and an important step in the genesis of alcoholic heart muscle disease. The alterations in heart muscle in hypertension are initially adaptive but in the long term they are deleterious, and involve both transcription and translation. While adequate methods exist for quantifying the amount of mRNA for contractile and non-contractile proteins, such studies of gene-expression provide no dynamic information on the rate at which tissue proteins are lost or accrued. This can only be determined by measuring the rate of protein turnover, i.e. either protein synthesis or protein breakdown. Techniques for directly determining the rates of protein breakdown are limited or involve surgical procedures. Methods for measuring the rate of protein synthesis are described, and are illustrated by their application to the investigation of starvation and ethanol toxicity. In particular, attention is focused on the fact that reliable rates of protein synthesis are obtained only if the specific radioactivity of the precursor at the site of protein synthesis (aminoacyl-tRNA) is assessed.

Protein metabolism in alcoholism: effects on specific tissues and the whole body.

Ethanol is one of the few nutrients that is profoundly toxic. Alcohol causes both whole-body and tissue-specific changes in protein metabolism. Chronic ethanol missuse increases nitrogen excretion with concomitant loss of lean tissue mass. Even acute doses of alcohol elicit increased nitrogen excretion. The loss of skeletal muscle protein (i.e., chronic alcoholic myopathy) is one of several adverse reactions to alcohol and occurs in up to two-thirds of all ethanol misusers. There are a variety of other diseases and tissue abnormalities that are entirely due to ethanol-induced changes in the amounts of individual proteins or groups of tissue proteins; for example, increased hepatic collagen in cirrhosis, reduction in myosin in cardiomyopathy, and loss of skeletal collagen in osteoporosis. Ethanol induces changes in protein metabolism in probably all organ or tissue systems. Clinical studies in alcoholic patients without overt liver disease show reduced rates of skeletal muscle protein synthesis though whole-body protein turnover does not appear to be significantly affected. Protein turnover studies in alcohol misusers are, however, subject to artifactual misinterpretations due to non-abstinence, dual substance misuse (e.g., cocaine or tobacco), specific nutritional deficiencies, or the presence of overt organ dysfunction. As a consequence, the most reliable data examining the effects of alcohol on protein metabolism is derived from animal studies, where nutritional elements of the dosing regimen can be strictly controlled. These studies indicate that, both chronically and acutely, alcohol causes reductions in skeletal muscle protein synthesis, as well as of skin, bone, and the small intestine. Chronically, animal studies also show increased urinary nitrogen excretion and loss of skeletal muscle protein. With respect to skeletal muscle, the reductions in protein synthesis do not appear to be due to the generation of reactive oxygen species, are not prevented with nitric oxide synthase inhibitors, and may be indirectly mediated by the reactive metabolite acetaldehyde. Changes in skeletal muscle protein metabolism have profound implications for whole body physiology, while protein turnover changes in organs such as the heart (exemplified by complex alterations in protein profiles) have important implications for cardiovascular function and morbidity.

Role relationships: lesbian perspectives.

This questionnaire study of 140 lesbians, 70 couples who have lived together for one or more years, explores equality and role relationships. We predicted that lesbians would achieve partnerships characterized by equality and freedom from traditional butch-femme role playing. Financial sharing and decision-making were found to be characterized by high degrees of equality. Household responsibilities tended to be performed individually, but no role-playing was evident. Sexual intimacy items indicated less perceived equality than other areas. And overall, some partners were viewed as unequal. The variables of age, income, education, occupation, assets, and years living together did not explain the inequality observed.

Power-sharing in lesbian partnerships.

This study of 70 lesbian couples explored whether partners who characterized certain aspects of their relationships as equal in power-sharing were similar in age, income, education, and financial assets, while those who viewed their power-sharing as unequal would be dissimilar on these social status variables, and, second, whether or not most lesbian couples considered their relationships as egalitarian. Power-sharing was assessed through a number of questionnaire items, and snowball sampling was utilized. Three types of couples emerged: (a) equal, (b) unequal but in agreement about who had more power, and (c) couples with differing perceptions about power-sharing. The findings indicated that power-sharing arrangements could not be explained by age, income, education, and asset differences between partners. Egalitarianism was the ideal in most relationships, but had not been achieved to the same degree in each of the areas investigated.

In vivo rates of skeletal muscle protein synthesis in rats are decreased by acute ethanol treatment but are not ameliorated by supplemental alpha-tocopherol.

Some studies have shown that reductions in tissue protein synthesis, under a variety of cytotoxic conditions, are ameliorated by alpha-tocopherol (ATC) supplementation. We have also shown evidence of increased oxidative stress and reduced protein synthesis rates in alcohol-exposed muscle. Serum levels of ATC fall and rates of muscle protein synthesis are reduced in patients with alcoholic myopathy. We therefore tested the hypothesis that treatment with ATC could ameliorate the ethanol-induced changes in muscle protein synthesis, a contributory event in the pathogenesis of alcoholic muscle disease. Studies were carried out on gastrocnemius (Type II fiber-predominant and usually considered representative of the musculature as a whole), soleus (Type I fiber-predominant) and plantaris (Type II fiber-predominant) muscles. For comparative purposes, we also investigated the liver. Young male Wistar rats (90 g body weight) were injected intraperitoneally (i.p.) daily with ATC (30 mg/kg body weight) in Intralipid fat emulsion (0.1 mL/100 g body, i.p.) for 5 d. Controls were similarly injected with the Intralipid vehicle alone. After ATC supplementation, rats were given ethanol (75 mmol/kg body weight, i.p., 2.5 h) or saline (0.15 mol/L NaCl, i. p.). Fractional rates of tissue protein synthesis (i.e., the percentage of the tissue protein pool renewed each day, k(s), %/d) and RNA activities [i.e., the amount of protein synthesis each day per unit RNA, k(RNA), mg protein/d/mg RNA)] were then measured. Supplementation increased ATC concentrations in plasma, gastrocnemius and liver. There was no effect of ATC supplementation alone on k(s) in any of the tissues. ATC supplementation in the absence of alcohol increased k(RNA) in the plantaris muscle. In nonsupplemented groups, acute ethanol treatment reduced skeletal muscle (soleus, plantaris and gastrocnemius) k(s). Hepatic k(s) was not altered by ethanol, although ATC concentrations in this tissue increased due to ethanol. However, none of the changes in muscle k(s) or k(RNA) due to ethanol were significantly affected by ATC supplementation. In conclusion, ATC supplementation does not appear beneficial in ameliorating acute alcohol toxicity in skeletal muscle as defined by reductions in protein synthesis.

Skeletal muscle ribonuclease activities in chronically ethanol-treated rats.

Alcoholic myopathy occurs in up to two thirds of alcohol misusers and is characterized by selective atrophy of type II (anaerobic, fast-twitch) fibers; type I (aerobic, slow twitch) fibers are relatively unaffected. Both clinical and animal studies have indicated that skeletal muscle RNA content is reduced in response to ethanol exposure, and contributes to impaired protein synthesis. We hypothesized that the reduction in muscle RNA may be due to raised ribonuclease (RNase) activities that enhance RNA catabolism. To test this hypothesis, we measured the total tissue and plasma RNase activities as well as the activities of general (RNase A) and specific or "restriction" RNases (T1L, T2L) in ethanol-treated rats. Chronically treated rats were fed a nutritionally complete liquid diet with 35% of calories as ethanol. Weight-matched controls were pair-fed with isocaloric glucose. Rats were killed at time-points up to 6 weeks. For comparative purposes, the effect of acute (24 hr) starvation was also analyzed in a second group of rats relative to a group of control rats allowed free access to food and water over 24 hr. Results showed that the type II fiber-predominant plantaris muscle exhibited a significant increase in total RNase, RNase A and RNase T1L activities (increases ranged from +59% to +196%; P-values between 0.025 and 0.01) concomitant with large falls in RNA and protein content. In contrast, none of the RNase activities measured in the type I fiber-predominant soleus muscles were significantly affected; compositional changes were also smaller in the soleus. This effect was independent of reduced nutrition. In conclusion, the raised total RNase, RNase A and RNase T1L activities may contribute to the type II fiber-specific reduction in total RNA in chronically ethanol-treated rats. In turn, this may contribute to the alterations in cellular protein metabolism seen under these treatments.

Protein and mRNA levels of the myosin heavy chain isoforms Ibeta, IIa, IIx and IIb in type I and type II fibre-predominant rat skeletal muscles in response to chronic alcohol feeding.

Alcoholic myopathy occurs in between one and two-thirds of all alcohol misusers and is thus one of the most prevalent muscle disorders (2000 cases per 100,000 population). It is characterised by myalgia, muscle weakness and loss of lean tissue mass. Histological features include a reduction in the diameter of Type II muscle fibres, particularly the IIb fibre subset. In contrast, Type I fibres are relatively protected. It is possible that the myopathy is due to perturbations in myosin protein and mRNA expression. To test this hypothesis, we fed rats a liquid diet containing 35% of calories as ethanol. Control rats were pair-fed identical amounts of the same diet in which ethanol was replaced by isocaloric glucose. At the end of 6 weeks, total myofibrillary proteins and myosin heavy chain (MyoHC) Ibeta, IIa, IIx and IIb protein and mRNA were analysed in the plantaris (Type II fibre-predominant) and soleus (Type I fibre-predominant) muscles. The data showed that there were significant reductions in the total myofibrillary protein content in the plantaris of ethanol fed rats compared to pair-fed controls (P < 0.05). These changes in the plantaris were accompanied by reductions in total myosin (P < 0.025), as a consequence of specific reductions in the Ibeta, (P < 0.01), IIx (P < 0.05) and IIb (P < 0.05) protein isoforms. The mRNA levels of Ibeta were significantly reduced in the plantaris (P < 0.05). However, mRNA levels of IIa, IIx and IIb in the plantaris were not significantly affected by alcohol feeding. Other changes in the plantaris included significant reductions in desmin (P < 0.01), actin (P < 0.025), and troponin-I (P < 0.05) compared to pair-fed controls. In the soleus, the only significant changes related to a fall in Ibeta mRNA levels and a decline in troponin-C content. We conclude that in the rat, alcoholic myopathy is a feature of Type II fibre rich muscles and is accompanied by multiple protein changes. The decline in specific myosin protein levels, such as IIx and IIb in the absence of corresponding reductions in their mRNAs, is probably due to altered proteolysis or more likely reductions in translational efficiencies, rather than changes in transcription.

Studies on the time-course of ethanol's acute effects on skeletal muscle protein synthesis: comparison with acute changes in proteolytic activity.

A study of the effects of ethanol on skeletal muscle protein synthesis and protease activities was carried out in young male Wistar rats (150 g) for up to 24 hr after a single intraperitoneal dose of 75 mmol of ethanol/kg of body weight. At 20 min, the mean blood ethanol levels were 448 mg/dl. This level dropped steadily to zero through the following 24 hr. Compared with pair-fed controls, significant reductions in total protein, RNA, and DNA contents were seen only after 24 hr in all skeletal muscles studied: changes were more marked in the muscles containing large proportions of type II fibers. In plantaris muscle, the fractional rate of protein synthesis (ks, %/day) did not fall 20 min after dosage but was reduced after 1 hr by 23% (p < 0.001), and by 63% after 24 hr, compared with control saline-injected rats (p < 0.001). This effect was independent of dietary intake because, compared with the pair-fed group, the 24-hr ethanol-treated rats still showed a 52% decrease in fractional rates of protein synthesis (p < 0.001). Smaller reductions in ks were seen in soleus muscles in response to ethanol at 24 hr (-39%, p < 0.001). The activities of a variety of lysosomal and nonlysosomal proteases in plantaris muscle of 24-hr treated rats were not significantly affected by ethanol. Only alanyl- and tripeptidyl-aminopeptidase activities were reduced significantly (26%, p < 0.05 and 39%, p < 0.01, respectively). These results suggest that the muscle compositional changes seen over acute periods of ethanol toxicity are predominantly associated with impaired synthesis of protein and that the contribution of cellular proteolytic systems may be minimal. The effects of ethanol on skeletal muscle protein metabolism are greater in muscles containing a predominance of type II fibers than in those containing mainly type I fibers. Ethanol's effects on muscle may be influenced by hormonal changes after 24 hr, because protein synthesis is still compromised and free plasma T3 and corticosterone are altered at this time-point.

Identification of anti-idiotypic antibodies in the sera of ryegrass-allergic and nonallergic individuals.

Anti-idiotypic (Id) antibodies (Abs) are generated in the humoral response to antigen (Ag). Some of these anti-Id Abs are capable of binding to the combining site or paratope of Abs that bind Ag. These Ab2 can competitively inhibit the binding of Ab1 to Ag may trigger an Ab1 response similar to the response induced by Ag. To determine if specific Ab2 that inhibit the binding of IgE Abs to ryegrass (RG)-pollen allergens are present in the sera of RG-allergic (RGA) individuals before the initiation of allergen-specific hyposensitization with RG-pollen extract, we studied sera from five RGA and four nonallergic (NA) subjects in an IgE anti-RG RAST-inhibition assay. Ab2-enriched serum fractions were prepared from these study subjects by exhaustive mixed grass-pollen affinity chromatography to remove IgE and IgG anti-RG Ab1 from whole serum aliquots containing Ab1. Unabsorbed, twice-concentrated sera were diluted to one-time concentrated sera with equal volumes of either Ab2-enriched sera without Ab1 or borate-buffered saline absorbed by mixed grass-pollen affinity chromatography. IgE anti-RG Ab1 was determined by a standard RG RAST assay. We have detected Ab2 in the sera of the RGA patients, which inhibit the binding of autologous and allogeneic IgE anti-RG to RG Ags in solid phase. Parallel RG RAST assays with sera from NA subjects demonstrated no significant inhibition. Ab2-enriched sera from some grass-allergic and some NA subjects inhibited IgE anti-RG binding found in some RGA patients' sera. We conclude that anti-Id Abs, Ab2, specific for IgE anti-RG, Ab1, are present in some RGA patients and NA individuals.

Increased frequency of HLA-DR11 in pediatric human immunodeficiency virus-associated parotid gland enlargement.

We sought to determine whether an increased frequency of the HLA-DR11 (formerly DR5) phenotype is found in human immunodeficiency virus (HIV)-infected children with parotid gland enlargement. In HIV-infected adults, parotid gland enlargement may be part of the diffuse infiltrative CD8 lymphocytosis syndrome. An increased frequency of expression of HLA-DR11 has been described in association with diffuse infiltrative CD8 lymphocytosis syndrome. We conducted a case-control study with 26 HIV-infected children, 13 of whom had parotid gland enlargement and 13 of whom did not but who were matched for age, race, and sex with those with parotid gland enlargement. Clinical and laboratory parameters (including HLA-DR11 phenotype) were compared between the two groups. HIV-positive children with parotid gland enlargement showed an increased frequency of HLA-DR11, similar to their adult counterparts with diffuse infiltrative CD8 lymphocytosis syndrome. The HLA-DR11 phenotype may be associated with the development of parotid gland enlargement in HIV-infected children and may be a marker for a more benign outcome of HIV infection.

Expression of the rheumatoid factor cross-reactive idiotype in JRA: association with disease onset subtype, disease activity and disease severity.

We previously reported that approximately one-third of patients with juvenile rheumatoid arthritis (JRA) express high concentrations of antibodies marked by the rheumatoid factor cross reactive idiotype (RCRI) in their sera (6). In order to determine if an expression of RCRI is associated with certain clinical features of the disease, we prospectively studied 49 patients with JRA over a six month period, and determined serum RCRI concentrations by inhibition ELISA. RCRI concentrations correlated significantly with the duration of morning stiffness (r = .3866, p less than .01), and the functional class (p less than .001), but not with the number of active joints. Expression of RCRI was higher in patients with systemic onset disease (p less than .03), compared to patients with pauciarticular or polyarticular disease. In patients studied on more than one occasion, the RCRI expression was relatively constant despite changes in disease activity. A subset of JRA patients with systemic onset disease, higher serum concentrations of the RCRI.

Enriched HLA-DQ3 phenotype and decreased class I major histocompatibility complex antigen expression in recurrent respiratory papillomatosis.

Respiratory papillomas, caused by human papillomaviruses, are benign tumors that recur following removal. We evaluated immune function and major histocompatibility complex (MHC) phenotype and expression in these patients. MHC-independent immune function appeared normal. The frequency of peripheral blood MHC class II phenotypes was highly enriched for DQ3 and DR11, one split of DR5. Class I MHC antigen expression on papilloma tissue was markedly reduced. Together, these phenomena may facilitate papillomavirus evasion of the cellular immune response.