Myosin heavy chain expression in zebrafish and slow muscle composition.

In the zebrafish embryo, two distinct classes of muscle fibers have been described in the forming myotome that arise from topographically separable precursor populations. Based entirely on cross-reactivity with antibodies raised against mammalian and chick myosin heavy chain isoforms slow twitch muscle has been shown to arise exclusively from "adaxial" myoblasts, which migrate from their origin flanking the notochord to form a single layer of subcutaneous differentiated muscle cells. The remainder of the myotome differentiates behind this migration as muscle fibers recognized by anti-fast myosin heavy chain (MyHC) antibodies. To identify unambiguous molecular markers of cell fate in the myotome, we have characterized genes encoding zebrafish fast and slow MyHC. Using phylogenetic and expression analysis, we demonstrate that these genes are definitive molecular markers of slow and fast twitch fates. We also demonstrate that zebrafish embryonic slow twitch muscle co-expresses both slow and fast twitch MyHC isoforms, a property that they share with primary fibers of the amniote myotome.

Ozone and particulate matter air pollution in Wisconsin: trends and estimates of health effects.

OBJECTIVES: Ozone and particulate matter 10 microns in diameter or less (PM10) are associated with increased risk of morbidity and mortality. This paper will report ozone and PM10 trends in Wisconsin and provide estimates of potentially susceptible populations and adverse health outcomes associated with current levels of these pollutants. METHODS: Ozone and PM10 monitoring data is provided by Department of Natural Resources (DNR) air monitors. Exposure-response modeling and data, provided by the United States Environmental Protection Agency (US EPA), was used to estimate hospital admissions associated with ozone and PM10 pollution. RESULTS: Days with high ozone concentrations have become less frequent and the annual PM10 levels have decreased over the 1990s. However, the potentially susceptible population has increased, the number of children who could experience a lung function decrement due to ozone is between 12,978 and 38,653, and high PM10 concentrations are estimated to have resulted in 43 hospital admissions. CONCLUSION: Despite decreasing ozone and PM10 levels, the burden of respiratory and cardiovascular disease affected by these pollutants remains significant.

Competence to stand trial: a neuropsychological inquiry.

How might the deconstruction of the legal theory of competence be related to modern neuropsychological models of cognition? To address this question, we examined retrospectively the relationship between clinical judgments of competence of defendants committed to a maximum security psychiatric facility and their neuropsychological test scores on measures of intelligence, memory, attention, academics, and executive function. In addition, based on both neuropsychological and legal theory, we examined whether subtypes of memory, namely episodic and semantic, and intelligence, specifically social intelligence, would have special relevance to these clinical judgements of competence. Results indicated that in relation to the defendants recommended as incompetent to stand trial (IST), defendants recommended as competent to stand trial (CST) scored significantly higher on summary indexes of psychometric intelligence, attention, and memory, especially verbal memory, but not significantly higher on tests of academics or executive function. Moreover, CST defendants scored significantly higher than did IST defendants on selective tests of episodic memory and social intelligence, but not on measures of semantic memory. Partial correlation also revealed a significant relationship between the likelihood of an IST recommendation and lower scores on tests of episodic memory and social intelligence, but not on measures of semantic memory. These findings illustrate the theoretical import of neuropsychological methods and concepts to the burgeoning nomological net known as competence.

The relationship between perlecan and dystroglycan and its implication in the formation of the neuromuscular junction.

Perlecan is a major heparan-sulfate proteoglycan (HSPG) within the basement membrane surrounding skeletal muscle fibers. The C-terminus of its core protein contains three globular domain modules which are also found in laminin and agrin, two proteins that bind to dystroglycan (DG, cranin) on the muscle surface with these modules. In this study, we examined whether perlecan can also bind to DG and is involved in signaling the formation of the neuromuscular junction (NMJ). By labeling cultured muscle cells with a polyclonal anti-perlecan antibody, this protein is found both within the extracellular matrix in a fibrillar network and at the cell surface in a punctate pattern. In Xenopus muscle cells, the cell-surface perlecan is precisely colocalized with DG. Both perlecan and DG are clustered at ACh receptor clusters induced by spinal neurons or by beads coated with HB-GAM, a heparin-binding growth factor. Blot overlay assays have shown that perlecan binds alpha-DG in a calcium and heparin-sensitive manner. Furthermore, perlecan is present in muscle lysate immunoprecipitated with an anti-DG antibody. Immunolabeling also showed colocalization between HB-GAM and perlecan and between HB-GAM and DG. These data suggest that perlecan is anchored to muscle surface via DG-dystrophin complex. Since DG is also a site of agrin binding, the neural agrin secreted by motoneurons during NMJ formation may compete with the pre-existing perlecan for cell surface binding. This competition may result in the presentation of perlecan-bound growth factors such as HB-GAM to effect synaptic induction.

Effects of lead on rat kidney and liver: GST expression and oxidative stress.

The effect of acute exposure to lead acetate on the expression of glutathione S-transferase (GST) subunits and the levels of reduced and oxidized glutathione (GSH) and malondialdehyde (MDA) in rat kidney and liver was determined. The purpose of this study was to determine if GSH depletion and/or oxidative stress were responsible for changes in the expression of some or all GSTs that followed lead exposure. In kidney, all GST subunits increased following injection of lead. The level of kidney GSH was not changed at either 0.5 or 1 h after lead exposure, but increased 3, 6, 12 and 24 h after a single injection of lead. MDA levels (a marker of lipid peroxidation) did not change in kidney following lead injection. Immunohistochemical markers of oxidative stress and nitric oxide production were also unchanged by lead administration. Therefore. we conclude that the increases in GST levels in kidney following lead exposure were not dependent on oxidative stress. In liver, lead injection caused GSH depletion (61% of control 12 h after lead treatment) and increased MDA production (2.5-fold increase 6 h after lead exposure), while GSTA1, GSTA2, GSTM1 and GSTM2 did not increase. Analysis of the effects of lead on GST mRNA and GST cellular localization were performed by Northern blot and immunohistochemical techniques. Immunoperoxidase light microscopy and immunogold electron microscopy revealed that the increase in kidney GSTM1 and GSTP1 occurred in nuclei, cytoplasm and microvilli of proximal tubules. Northern blot analysis of GSTA2 and GSTP1 mRNAs showed that their increase following lead exposure was inhibited by actinomycin D, suggesting transcriptional induction. This study demonstrates that acute lead exposure causes dramatic changes in the subcellular distribution and expression of rat kidney GSTs, and that these changes are not a result of oxidative stress.

Glutathione S-transferases: two-dimensional electrophoretic protein markers of lead exposure.

Glutathione S-transferases (GST) are a family of detoxification isoenzymes that catalyze the conjugation of xenobiotics and their metabolites with reduced glutathione. Lead exposure in rats is known to induce GST isoenzymes in the liver and kidney. These changes in expression have potential use as biomarkers of lead exposure. Because two-dimensional electrophoresis (2-DE) enables one to analyze both protein abundance changes and chemical changes in protein structure, 2-DE was used to determine the effect of in vivo lead exposure on GST isoform expression in rat kidney cytosols. Male Sprague-Dawley rats were exposed to inorganic lead, and proteins were separated by conventional ISO-DALT and NEPHGE-DALT techniques and blotted for immunological identification. Lead exposure caused detectable inductions in both GSTP1 and GSTM1 and quantifiable charge modification in GSTP1. These preliminary data confirm the utility of 2-D electrophoretic GST analysis as indicative of lead exposure and toxicity and support its use for further elaboration of lead's effects on renal protein expression.

Effects of triethyl lead administration on the expression of glutathione S-transferase isoenzymes and quinone reductase in rat kidney and liver.

The effects of triethyl lead chloride (TEL) on the expression of two detoxication enzyme families, glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase (QR) were determined in rat liver and kidney. Fischer 344 rats were given one intraperitoneal (i.p.) injection of TEL. GST activity, GST isoenzyme levels, mRNA levels of alpha class GST isoenzymes Ya1, Ya2, and Yc1 and activity of QR were determined. Treatment of rats with TEL caused a significant increase in GST activity in kidney. In kidney, the levels of all GST subunits were significantly elevated; the largest increase was a 3.2-fold increase in GST Yb1. The levels of GST Ya1, Ya2, and Yc1 mRNA also increased after injection of TEL. In liver, TEL injection resulted in decreased GST activity and lower levels of hepatic GSTs Yb2, Yb3, Ya1, and Ya2. The largest decrease was a 40% reduction of GST Ya1. In contrast, the level of liver GST Yc1 increased from day 4 through day 14 after injection of 10 mg/kg TEL and Yp was increased 1.4-fold 4 days after injection of 12 mg/kg TEL. The levels of liver mRNAs coding for alpha class GSTs Ya1, Ya2, and Yc1 were reduced 12 h after injection of TEL. The mRNA levels of GST Ya1 and Ya2 returned to basal level while Yc1 message increased to a level higher than controls 24 h after TEL injection. The increase in Yc1 protein between days 4 and 14 is consistent with the increase in the corresponding mRNA. The activity of QR was elevated 1.5-fold in kidney and 2.7-fold in liver 14 days after the injection of TEL. This report demonstrates that administration of organic lead significantly affects GST expression and QR activity in a tissue-specific and isoenzyme-specific manner. These results indicate that GST expression and QR activity are not co-regulated.

Increased levels of glutathione S transferases and appearance of novel alpha class isoenzymes in kidneys of mice exposed to mercuric chloride. I. Biochemical and immunohistochemical studies.

Glutathione S transferases (GST) are a family of enzymes that detoxify electrophilic xenobiotics. This enzyme family was examined in kidneys of mice exposed to mercuric chloride, a known nephrotoxin, because GST have been shown to protect cells against toxicant-induced damage and may serve as biomarkers for toxicant exposure. The purpose of this study was to determine the effect of mercuric chloride on GST activity, isoenzyme levels, and cellular localization in the kidney of Swiss Webster mice. The cellular localizations of alpha, mu, and pi class GST in the kidneys of control and mercuric chloride treated mice were studied immunohistochemically. The GST isoenzyme levels were measured by high-performance liquid chromatography. The mice treated with mercuric chloride had (1) increased amounts of GSTA1/A2 protein in kidney homogenates as compared with controls when analyzed by chromatography and electrophoresis; (2) two new isoforms of the alpha isoenzyme in kidney as demonstrated by Western blot, polyacrylamide gel electrophoresis, and high-performance liquid chromatography, and (3) increased reactivity between antibodies, against GSTA1/A2 or GSTM1 isoenzymes, and cells in the proximal and distal renal tubules as shown by immunohistochemical techniques. The authors conclude that the GSTA1/A2 may protect those cells in the proximal and distal tubules of the renal cortex from toxicant effects of mercuric chloride. This would be one general mechanism for cell protection against a wide variety of toxicants including heavy metals and halogenated aromatics.

Phase II enzyme expression in rat liver in response to the antiestrogen tamoxifen.

The genotoxicity and carcinogenicity of tamoxifen have been attributed to metabolic activation of tamoxifen to an electrophile. Phase II enzymes are known to be involved in the metabolism of the drug and possibly in the formation or elimination of the active metabolite. To determine the effects of tamoxifen on phase II enzyme expression, the drug was administered to F344 rats, and hepatic glutathione S-transferase (GST), UDP-glucuronosyltransferase (UGT), and sulfotransferase (ST) expression was evaluated. Some of the tamoxifen-induced effects, including dramatic suppression of selected GST enzymes and activity, were observed at a dose in rats that is directly equivalent, on a mg/kg b.w. basis, to the doses used for breast cancer treatment. Most of the observed responses are not consistent with the previously described phenobarbital-like properties of tamoxifen and could be the result of the partial agonist activity of tamoxifen at the estrogen receptor. Northern blot analysis was performed with isozyme-specific oligonucleotide probes for rat GST, ST, and UGT. In addition, GST subunit protein levels were assayed by high-performance liquid chromatography. In females, tamoxifen treatment resulted in a 60% suppression of GST Ya1 mRNA and protein levels and a 40% suppression of GST Ya2 levels. In males, tamoxifen treatment suppressed GST Ya1 expression approximately 60%, and GST Ya2 expression was suppressed at low doses but induced above control at high doses. Male GST Yc1 was induced approximately 80% over control. The expression of all other major forms of rat hepatic GST subunit protein, including GST Yb1, Yb2, Yb3, Yp, and Yl, was unaffected by tamoxifen treatment. GST conjugation activity toward delta 5-androstene-3,17-dione, a GST Ya1- and Ya2-specific substrate, was suppressed approximately 40% in both sexes, consistent with our protein and mRNA data. Total GST activity, as measured by the rate of chlorodinitrobenzene conjugation, was not changed. Tamoxifen also produced a dose-dependent increase in UGT2B1 mRNA, a phenobarbital-inducible enzyme; mRNA levels reached 210 and 420% of control in females and males, respectively. In addition, mRNA levels for ST2A2, a female-specific ST gene, were suppressed 50% in females and induced 120% over control in males. mRNA expression for all other forms of rat liver UGT and ST isozymes that were tested was not significantly affected by tamoxifen treatment. Overall, these results demonstrate that tamoxifen has significant effects on hepatic phase II enzyme expression that may have implications for the carcinogenicity and/or therapeutic activity of the drug.

Cellular localization of glutathione S-transferases in retinas of control and lead-treated rats.

PURPOSE: The glutathione S-transferases (GSTs) constitute a family of cytosolic isoenzymes that are involved in the detoxication of electrophilic xenobiotics. The purpose of this investigation was to determine the concentration and cellular distribution of the various classes of cytosolic GSTs in the retina of control and triethyl lead-treated rats and thereby reveal mechanisms by which the cells are protected from damage by lead and other toxicants. METHODS: The regional and cellular distribution of cytosolic GSTs in rat retina of control and lead-treated animals was studied by immunohistochemistry. Enzyme activity was determined by a spectrophotometric assay. The GST subunit distribution of the entire retina of control and lead-treated animals was determined and quantified by reverse-phase high-performance liquid chromatography (HPLC). RESULTS: Polyclonal antibodies against mu-class Yb1 and Yb2 GSTs were primarily and strongly reactive with Müller cells and their processes. Anti-Yb1 also reacted with photoreceptor outer segments. Antibodies against two alpha-class GSTs (Ya and Yk) were strongly reactive with Müller cells and their cell processes. Antibodies against Yp and Yc GSTs were reactive with amacrine cells and their processes, and anti-Yp antibodies were reactive against retinal ganglion cells. Treatment of rats with triethyl lead caused diminished reactions of the antibodies against Yb1 and Yp GSTs and increased reactions of anti-Ya with its retinal targets, whereas the total GST activity did not change significantly. CONCLUSIONS: The positive reaction between the amacrine neuronal cells of retina and the anti-Yp and anti-Yc class antisera broadens the class of neurons that contains GST enzymes protective against toxicant insult. It also has been shown that Müller cells are strongly immuno-positive for Yb1 and Yb2 GST. Because these phagocytic cells are in contact with the vitreous fluid and proximate to pigmented epithelial layer of the eye, these GSTs may protect the cells from toxicants accumulated from this fluid.

Full-length agrin isoform activities and binding site distributions on cultured Xenopus muscle cells.

Agrin is a large multidomain protein involved in the induction of postsynaptic differentiation of the neuromuscular junction. As a step toward further understanding the mechanisms by which agrin induces the aggregation of acetylcholine receptors (AChRs), we have characterized the activity of purified, full-length chick agrin isoforms on Xenopus muscle cells. Incubation with agrin isoforms led to the formation of numerous small AChR clusters primarily on the ventral surface of cells with differing efficacies: Y4Z8 > Y4Z11 = Y4Z19, with Y4B0 being ineffective. Agrin activity appeared to be tyrosine phosphorylation-dependent as the kinase inhibitor tyrphostin RG50864 (80 microM) completely abolished the effect. Initial binding sites for all agrin isoforms were evenly distributed in a punctate manner on the muscle cell surface. After a 14-h incubation, the active isoforms induced AChR clustering, and agrin was enriched at these sites of clustering. Agrin binding to the cell surface and induction of AChR clustering were Ca(2+)-dependent, as previously shown in other systems. This is the first quantitative characterization of agrin's effects using Xenopus cell culture, providing a basis for further elucidating agrin's role in synaptogenesis using this elegant system.

The role of an agrin-growth factor interaction in ACh receptor clustering.

The clustering of acetylcholine receptors (AChRs) at the neuromuscular junction is mediated in part by the heparan-sulfate proteoglycan agrin. However, our previous studies have also suggested the role of heparin-binding growth-associated molecular (HB-GAM) in AChR clustering. Here the role of an agrin-HB-GAM interaction in this process was examined using cultured Xenopus muscle cells. Agrin-coated beads further treated with HB-GAM were highly effective in AChR cluster induction. Protein overlay assays showed specific binding of HB-GAM to agrin. In addition, agrin-enriched neuritic tracks bound HB-GAM in a manner that showed a high degree of colocalization between the neural agrin and the applied factor. Finally, the introduction of exogenous HB-GAM together with soluble agrin resulted in the appearance of AChR clusters on the dorsal surface of cells in an agrin isoform-dependent manner; a dramatic change from the characteristic ventral AChR clustering seen in response to agrin alone. These results suggest that agrin may mediate AChR clustering by interacting with muscle-bound heparin-binding growth factors such as HB-GAM.

The role of heparin-binding growth-associated molecule (HB-GAM) in the postsynaptic induction in cultured muscle cells.

The heparan sulfate proteoglycan (HSPGs) is a components of the extracellular matrix of skeletal muscle that is concentrated at the neuromuscular junction (NMJ). Recent studies have suggested that HSPG, together with its bound peptide growth factors, plays important roles in autocrine or paracrine types of regulation of cell growth and differentiation. Heparin-binding growth-associated molecule (HB-GAM; also known as pleiotrophin, or p18) is a newly discovered HSPG-bound factor that is expressed at high levels in the developing CNS and PNS. In this study, we examined the role of this factor in NMJ development by examining its relationship to the formation of ACh receptor (AChR) clusters. Using an antibody against recombinant rat brain HB-GAM, we found that this protein is present prominently on the surface of cultured Xenopus myotomal muscle cells by immunocytochemistry. It is associated with HSPGs as evidenced by the fact that heparin and heparinase treatment greatly diminished the antibody labeling. HB-GAM is concentrated at preexisting AChR hot spots as well as at those induced by polystyrene beads. In addition, this molecule is also concentrated at AChR clusters induced by spinal cord neurons in nerve-muscle cocultures. To assess its function in synaptic induction, we applied recombinant HB-GAM-coated beads to cultured muscle cells to effect its focal presentation. Over 70% of these beads induced the formation of AChR clusters as shown by fluorescent alpha-bungarotoxin labeling. Furthermore, bath application of HB-GAM inhibited the nerve-induced formation of AChR clusters. Thus, HB-GAM is an endogenous muscle-derived factor that may be a component of the molecular mechanism in postsynaptic induction.

Effects of lead administration on developing rat kidney. I. Glutathione S-transferase isoenzymes.

The effects of acute and chronic exposure to lead on glutathione S-transferase (GST) isoforms were determined in developing kidney in the rat. The ontogeny of glutathione S-transferase isoforms was characterized as were the effects of depletion of dietary calcium on glutathione S-transferase isoform profiles in control and lead-treated rats. In the acute exposure experiments, rats of 14 and 50 days of age received three daily injections of lead acetate (114 mg/kg) and in the chronic exposure studies, rats received lead acetate at doses ranging from 50 to 500 ppm in their drinking water. Lead acetate administration in these chronic studies began 1 day after conception. Acute and chronic lead exposure had similar effects, causing increases in all but one glutathione S-transferase isoform (Yb3); these increases were markedly exacerbated by dietary calcium depletion. In all lead paradigms, GST subunits Yb1 and Yp showed the largest increases--greater than 25-fold in rats fed a low-calcium diet. GST subunit Yb3 showed small increases in the 14-day acute lead and the 4 week low-calcium animals and did not increase in other groups. Lead-related increases in GSTs were partially reversed by transferring animals previously receiving lead to lead-free water for a 4-week period. Kidneys of rats fed the low-calcium diet did not have detectable GST Yk, but in rats on this low-calcium diet that received 500 ppm lead; this GST isoform was found at levels comparable to those in control rats fed lab chow.

Concentration of pp125 focal adhesion kinase (FAK) at the myotendinous junction.

Focal adhesion kinase is a recently characterized tyrosine kinase that is concentrated at focal contacts in cultured cells. It is thought to play an important role in the regulation of the integrin-based signal transduction mechanism involved in the assembly of this membrane specialization. In this study, we examined the immunocytochemical distribution of focal adhesion kinase in Xenopus skeletal muscle and its role in the formation of two sarcolemmal specializations, the myotendinous junction and the neuromuscular junction, using a monoclonal antibody (2A7) against this protein. Immunoprecipitation of Xenopus embryonic tissues with this antibody demonstrated a single band at a relative molecular mass of 116 kDa. A distinct concentration of immunolabeling for focal adhesion kinase was observed at the myotendinous junction of muscle fibers in vivo. At this site, the labeling for this protein is correlated with an accumulation of phosphotyrosine immunolabeling. Focal adhesion kinase was not concentrated at the neuromuscular junction in muscle cells either in vivo or in vitro. However, it was localized at spontaneously formed acetylcholine receptor clusters in cultured Xenopus myotomal muscle cells, although its distribution was not exactly congruent with that of the receptors. In these cells, the accumulation focal adhesion kinase was induced by polystyrene microbeads. In addition, beads also induce the formation of acetylcholine receptor clusters and myotendinous junction-like specializations. By following the appearance of the focal adhesion kinase relative to the formation of these sarcolemmal specializations at bead-muscle contacts in cultured muscle cells, we conclude that the accumulation of this protein was in pace with the development of the myotendinous junction, but occurred well after the clustering of acetylcholine receptors. These results suggest that focal adhesion kinase may be involved in the development and/or maintenance of the myotendinous junction through an integrin-based signaling system. Although it can accumulate at acetylcholine receptor clusters formed in culture, it does not appear to be involved in the development of the neuromuscular junction.