Low in situ expression of antioxidative enzymes in rat cerebellar granular cells susceptible to methylmercury.

Methylmercury (MeHg), an environmental neurotoxicant, induces site-specific toxicity in the brain. Although oxidative stress has been demonstrated with MeHg toxicity, the site-specific toxicity is not completely understood. Among the cerebellar neurons, cerebellar granule cells (CGCs) appear vulnerable to MeHg, whereas Purkinje cells and molecular layer neurons are resistant. Here, we use a MeHg-intoxicated rat model to investigate these cerebellar neurons for the different causes of susceptibility to MeHg. Rats were exposed to 20 ppm MeHg for 4 weeks and subsequently exhibited neuropathological changes in the cerebellum that were similar to those observed in humans. We first isolated the three cerebellar neuron types using a microdissection system and then performed real-time PCR analyses for antioxidative enzymes. We observed that expression of manganese-superoxide dismutase (Mn-SOD), glutathione peroxidase 1 (GPx1), and thioredoxin reductase 1 (TRxR1) was significantly higher in Purkinje cells and molecular layer neurons than in CGCs. Finally, we performed immunohistochemical analyses on the cerebellum. Immunohistochemistry showed increased expression of Mn-SOD, GPx1, and TRxR1 in Purkinje cells and molecular layer neurons, which was coincident with the mRNA expression patterns. Considering Mn-SOD, GPx1, and TRxR1 are critical for protecting cells against MeHg intoxication, the results indicate that low expression of these antioxidative enzymes increases CGCs vulnerability to MeHg toxicity.

Effects of Pathogen Density, Soil Moisture, and Soil pH on Biological Control of Clubroot in Chinese Cabbage by Heteroconium chaetospira.

The effects of soil moisture and pH, and pathogen resting spore density, on the effectiveness of the biological control of clubroot by the fungal endophyte Heteroconium chaetospira was evaluated in greenhouse and field experiments. Conditions favoring disease development included low pH (5.5) and high soil moisture content (80%), with significant reductions in the disease being observed at a higher pH (6.3 and 7.2) and lower soil moisture content (40 and 60%). In greenhouse tests, H. chaetospira effectively controlled clubroot (reducing the disease by 90 to 100%) at pathogen resting spore densities of 104 and 105 spores/g of soil at all soil pHs tested (5.5, 6.3, and 7.2). However, when the resting spore density was 106 spores/g of soil, plants were severely diseased, regardless of treatment, and H. chaetospira had no effect on disease. At a soil moisture content of 40%, disease occurrence was low, regardless of pathogen spore density, but disease was significantly lower in H. chaetospira-treated plants at pathogen spore density of 105 spores/g of soil. At 60% soil moisture content, H. chaetospira significantly could affect at pathogen spore densities of 104 and 105 but not 104/g of soil. At 80% soil moisture content, there was no effect of H. chaetospira at pathogen density. In situ, the soil moisture contents were constantly adjusted to relatively low to moderate (pF 2.2 to 2.4 and pF 2.0 to 2.2) and high (pF 1.6 to 1.8). Other environmental conditions, such as resting spore density and soil pH, were maintained at constant levels. Control plants (not treated with H. chaetospira) showed uniformly high disease levels and proportions of diseased plants across all three moisture treatments (disease index = 72 to 80, proportion of diseased plants 85 to 97%). In the field, H. chaetospira-treated plants at low soil moisture (pF 2.2 to 2.4, plot 1) had 68% disease reduction compared with untreated controls and 49% reduction at moderate moisture pF (pF 2.0 to 2.2, plot 2). There was no effect on disease by H. chaetospira at high soil moisture (pF 1.6 to 1.8, plot 3). Based on our results, H. chaetospira is an effective biocontrol agent against clubroot in Chinese cabbage at a low to moderate soil moisture range and a pathogen resting spore density of 105 (or lower resting spores per gram of soil in situ.

Control of Verticillium Yellows in Chinese Cabbage by the Dark Septate Endophytic Fungus LtVB3.

ABSTRACT Three hundred forty-nine fungal endophytes were obtained from a total of 1,214 root segments of eggplant, melon, barley, and Chinese cabbage grown as bait plants in a mixed soil made up of samples from different forest soils in Alberta and British Columbia, Canada. Three of the 349 isolates, when inoculated in axenically reared Chinese cabbage seedlings grown in petri dishes, almost completely suppressed the effects of a postinoculated and virulent strain of Verticillium longisporum. Two isolates effective against the pathogen were Phialocephala fortinii, which had been obtained from the roots of eggplant and Chinese cabbage. The third isolate was a dark septate endophytic (DSE) fungus obtained from barley roots. Hyphae of P. fortinii grew along the surface of the root and formed microsclerotia on or in the epidermal layer. Hyphae of the DSE fungus heavily colonized root cells of the cortex. Seedlings grown for 1 week in the presence of the endophytes were then challenged with the Verticillium pathogen. In DSE-treated roots, some of cell walls in the epidermal and cortical layers showed cell wall appositions and thickenings, which appeared to limit the ingress of the pathogen into adjacent cells. Such marked host reactions were not observed in the root cells colonized by P. fortinii. Chinese cabbage preinoculated with the above endophytes and, for comparison, a previously reported disease-suppressive fungal endophyte, Heteroconium chaetospira, were transplanted into the field and disease symptoms were assessed. The DSE could most effectively inhibit the development of Verticillium yellows, with reductions in the percentages of external and internal disease symptoms of 84 and 88%, respectively. The protective values against the disease are extremely high compared with those of other isolates. Most of the DSE-treated plants in the plots achieved marketable quality.

Coexpression of the CUG-binding protein reduces DM protein kinase expression in COS cells.

Myotonic dystrophy (DM) is the most common form of adult onset muscular dystrophy. Patients have a large CTG repeat expansion in the 3' untranslated region of the DMPK gene, which encodes DM protein kinase. RNA trans-dominant models, which hypothesize that the expanded CUG trinucleotide repeat on DMPK mRNA sequesters a factor or disrupts the RNA metabolism of the DMPK mRNA itself and other mRNAs in a trans dominant manner, have been proposed. A candidate for the sequestered factor, termed CUG-binding protein (CUG-BP), exists in several alternatively spliced isoforms. We found a human isoform with a twelve base insertion (deduced amino acids Leu-Tyr-Leu-Gln) and an isoform with a three base insertion (deduced amino acid Ala) insertion. In order to elucidate the effects of CUG-BP on DMPK expression, we introduced CUG-BP and DMPK cDNA transiently into COS-7 cells. Cotransfection of CUG-BP did not significantly affect the expression of either wild type or mutant DMPK at the mRNA level. On the other hand, cotransfection of CUG-BP significantly affected the expression of both the wild type and mutant DMPKs at the protein level. This reduction was remarkable when the mutant DMPK construct was used.

In vivo protection of a water-soluble derivative of vitamin E, Trolox, against methylmercury-intoxication in the rat.

Methylmercury (MeHg) is a well-known neurotoxicant. MeHg-intoxication causes a disturbance in mitochondrial energy metabolism in skeletal muscle and apoptosis in cerebellum. We report the first in vivo effectiveness of antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carhoxylic acid), a water soluble vitamin E analog, against the MeHg-induced cellular responses. Treatment with Trolox (6-hydroxy-2.5,7,8-tetramethylchroman-2-carboxylic acid) clearly protects MeHg-treated rat skeletal muscle against the decrease in mitochondrial electron transport system enzyme activities despite the retention of MeHg. Tdt-mediated dUTP nick-end-labeling method clarified that Trolox is effective for protecting cerebellum from MeHg-induced apoptosis. These data indicate that MeHg-mediated oxidative stress plays an important role in the in vivo pathological process of MeHg intoxication. Trolox may prevent some of clinical manifestations of MeHg-intoxication in humans.

Ataxia caused by mutations in the alpha-tocopherol transfer protein gene.

A 48 year old woman with ataxia with vitamin E deficiency is described. Gene analysis identified two point mutations in exon 1 of the alpha-tocopherol transfer protein (alpha-TTP) gene, one missense mutation and an upstream initiation codon mutation in the 5'-untranslated region (Kozak sequence). The latter mutation is the first one identified in the translation regulatory region. This mutation decreased the level of alpha-TTP protein expression. The clinical features included uncommon urinary disturbance and deafness and relatively rare retinitis pigmentosa. Supplementary therapy increased her serum vitamin E concentration to the normal range with mild improvement of the deep senses.

Differential signaling pathways following oxidative stress in mutant myotonin protein kinase cDNA-transfected C2C12 cell lines.

We investigated the response to oxidative stress in a model system established in C2C12 cells stably transfected with myotonin protein kinase (MtPK) cDNAs having 5, 46, 60, or 160 CTG repeats. The transformants showed CTG repeat number-dependent susceptibility to oxidative stress. Mutant MtPK cDNA transformants containing 160 CTG repeats showed apoptotic cell death by the exposure to an oxidant, a very low level of methylmercury. The addition of the antioxidant Trolox protected transformants against apoptosis. Oxidative stress activated the extracellular signal-regulated kinases (ERKs) pathway leading to cell survival in wild-type MtPK cDNA transformants, whereas mutant MtPK cDNA transformants having 160 CTG repeats were defective in the induction of the ERK pathway, although the activation of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) was strong and sustained. These results suggest that the susceptibility to oxidative stress in mutant MtPK cDNA transformants involves differential signaling pathways evoked following oxidative stress.

Overexpression of myotonic dystrophy protein kinase in C2C12 myogenic culture involved in the expression of ferritin heavy chain and interleukin-1alpha mRNAs.

The specific function of myotonic dystrophy protein kinase (DMPK) is still not known. We found that overexpression of human DMPK in C2C12 myogenic culture induces the expression of ferritin heavy chain (FN-H) mRNA using differential display analysis. The quantity of FN-H mRNA was greater in the DMPK transfectant with five CTG triplet repeats in the 3'-untranslated region, while it was lower in the transfectant with 46 CTG repeats, over that of the control clone. We also investigated the quantity of interleukin 1-alpha (IL-1alpha) mRNA in each culture, due to the fact that this cytokine is able to induce FN-H expression, regardless of the concentration of free iron. Quantitative, competitive polymerase chain reaction (PCR) analysis revealed that the quantity of IL1-alpha mRNA is higher in the transfectant with five repeats, compared to the quantity of mRNA in the control clone; however, it is markedly lower in the clone with 46 repeats. These results suggest that overexpression of DMPK in C2C12 cultures may up-regulate IL-1alpha expression, resulting in the induction of FN-H expression. However, a large number of CTG repeats in the 3'-untranslated region of the DMPK gene may affect the pathway of IL-1alpha transcription, thereby resulting in decreased expression of FN-H.

Expanded CTG repeats in myotonin protein kinase increase susceptibility to oxidative stress.

The effect of oxidative stress on myogenic cells with expanded CTG repeats in the myotonin protein kinase (MtPK) gene was investigated using MtPK cDNA-transformants in order to investigate the disease process underlying myotonic dystrophy. We employed methylmercury as a model for reagents that produce reactive oxygen species (ROS). Mutant MtPK cDNA transformants containing 46 CTG repeats treated with 1 microM methylmercury for 24 h underwent cell death showing the characteristics of apoptosis. In contrast, methylmercury-induced cytotoxicity was weaker in wild type MtPK cDNA transformants. Antioxidants such as N-acetyl-L-cysteine and trolox suppressed methylmercury-induced apoptosis, indicating that the intracellular generation of ROS plays an important role. These studies suggest that expanded CTG repeats in MtPK increase the susceptibility of cells to oxidative stress.

Developmental regulation of myotonic dystrophy protein kinase in human muscle cells in vitro.

From our previous studies, myotonic dystrophy protein kinase: gene product of myotonic dystrophy is localized at the terminal cisternae of sarcoplasmic reticulum of human adult muscle. Now we have studied the developmental expression of myotonic dystrophy protein kinase in aneurally cultured human muscles and contracting cross-striated muscles innervated with fetal rat spinal cord using a semi-quantitative reverse transcription-polymerase chain reaction method for myotonic dystrophy protein kinase messenger RNA expression, Western blot analysis, immunohistochemical examinations by laser scanning confocal microscopy and immunoelectron microscopy. About 65,000 mol. wt myotonic dystrophy protein kinase was detected in aneurally cultured muscles. Myotonic dystrophy protein kinase messenger RNA was expressed in both aneurally and innervated cultured muscles, but in early innervated cultured muscles the message was transiently lower than in aneurally cultured muscles and innervated cultured muscles in long-term co-culture. In aneurally cultured muscles, immature aneurally cultured muscles show a diffuse and irregular distribution of myotonic dystrophy protein kinase in the deeper cytoplasm near the nuclei. Ultrastructurally the immuno-products against myotonic dystrophy protein kinase were observed as dense deposits in parts of the membranes near the mitochondria. In innervated cultured muscles, immunofluorescent microscopy showed myotonic dystrophy protein kinase to be localized regularly in the I bands and A-I junctions. Ultrastructurally myotonic dystrophy protein kinase was localized in branched duct-like membranes in the early stage of innervated cultured muscles and then in small sacs at the I bands and A-I junctions of the sarcolemma in the mature stage. Our present studies strongly suggest that innervation plays an important role in the localization of myotonic dystrophy protein kinase in human skeletal muscle during development. We conclude that the expression of myotonic dystrophy protein kinase during development is under neuronal influence.

The effect of methylmercury on skeletal muscle in the rat: a histopathological study.

Methylmercury (MeHg)-induced neurotoxicity includes skeletal muscle symptoms (extremity weakness and wasting, muscle cramp) in addition to ataxia and disturbances of sensory and visual function. The underlying mechanisms responsible for the skeletal muscle symptoms are still poorly understood. In this study the effects of MeHg exposure on skeletal muscle were investigated in rats receiving orally administered MeHgCl at 5 mg/kg/day for 12 days. MeHg-treated rats gradually lost body weight and showed muscle weakness and wasting. Seven days after the last MeHg dose, MeHg levels in the skeletal muscle were as high as those in liver, kidney, or cerebrum. The obvious histopathological finding in skeletal muscle was a decrease in mitochondrial enzyme activity. These changes were more prominent in mitochondria-rich soleus muscle than in extensor digitorum longus muscle. Our findings confirm that MeHg exposure disturbs mitochondrial energy metabolism in skeletal muscle.

Expanded CTG repeats in myotonin protein kinase suppresses myogenic differentiation.

A full-length mutant human myotonin protein kinase (MtPK) cDNA having a 46 expanded CTG-repeat size or a wild type containing 5 CTG repeats was stably transfected into mouse C2C12 cell line in order to explore the effects of the expansion mutation of trinucleotide repeats in the 3' untranslated region on developing myogenic cells. Each established clone expressed a human 70 kDa MtPK protein without proteolytic processing. Differentiation experiments indicated that stable mutant MtPK cDNA-transformants suppressed myogenic differentiation, whereas wild-type transformants exhibited almost normal myogenesis. The disturbance of the expression of neuronal nitric oxide synthase, a mediator of myoblast fusion, suggests that signal transduction system might be involved in the muscle manifestations of mutant MtPK.

[Lysosomal glycogen storage disease without acid maltase deficiency].

Lysosomal glycogen storage disease without acid maltase deficiency is characterized by the triad of clinical manifestations (hypertrophic cardiomyopathy), mental retardation, and mild myopathy), morphologic findings (glycogen storage, glycogenosomes, and autophagic vacuoles), and normal glycolytic enzyme activities. Though most of the patients suffering from the triad were males, family studies often revealed female patients with only cardiomyopathy. So far 27 cases have been reported. The cardiac involvement is progressive and fatal and as severe in females as in males. Many patients of both sexes die in their youth, unexpectedly, because of cardiac failure. The specific biochemical defect causing this disease remains unknown. From abnormal lectin staining patterns on the membrane and preclinical morphologic changes in biopsied skeletal muscle, membranous abnormality is suspected in this disease.

[Myotonin protein kinase].

The mutation underlying myotonic dystrophy is the expansion of polymorphic CTG repeat in the 3'-noncoding region of the myotonin protein kinase (MtPK) gene mapping to chromosome 19q13.3. A full-length cDNA of human MtPK was cloned and expressed in COS-1 cells. MtPK is recovered from the COS cell extract as a 70 kDa protein, which coincides with the size deduced from the predicted amino acid sequence. Biochemical characteristics of MtPK expressed in COS cells and its expression are investigated.

Up-regulation of dystrophin mRNA by exposure to dibutyryl cAMP in the C2C12 muscle cell line.

The effect of dibutyryl cAMP on dystrophin mRNA expression was investigated in C2C12 myogenic cells by a semi-quantitative RT-PCR method. The dystrophin mRNA level was enhanced by the addition of low concentrations of dibutyryl cAMP for 16h in the growth medium. The relative amounts of dystrophin/G3PDH mRNA at 10(-4) M dibutyryl cAMP were about 2.6-fold higher compared to control. We examined MLC1a mRNA expression in order to know whether the induction of dystrophin mRNA is due to the facilitation of myogenic differentiation by dibutyryl cAMP. MLC1a mRNA expression at 10(-4) M dibutyryl cAMP was similar to that of control. Thus we conclude that the dibutyryl cAMP enhancement of dystrophin mRNA expression is not due to a secondary event involving the formation of differentiated myotubes. The induction of CREB mRNA by dibutyryl cAMP suggests that the up-regulation of dystrophin mRNA expression might occur via transcriptional activation.

Morphologic findings in biopsied skeletal muscle and cultured fibroblasts from a female patient with Danon's disease (lysosomal glycogen storage disease without acid maltase deficiency).

A family is reported in which three members were affected by cardiomyopathy. Two members died unexpectedly in their second decade. Only a 23-year-old male suffered from the triad of clinical manifestations (cardiomyopathy, mental retardation and vacuolar myopathy). Morphologic findings and biochemical studies of his biopsied skeletal muscle and cultured fibroblasts confirmed lysosomal glycogen storage disease with normal acid maltase that was first described by Danon et al. In this study we demonstrated early morphologic changes, storage of glycogen and abnormal membranous structures in disorganized myofibers in biopsied skeletal muscle from the elder sister, who only showed cardiomyopathy clinically. The aggregation of autophagosomes was prominent in cultured fibroblasts, with an increased glycogen content. The activity of acid alpha-glucosidase was higher than normal. This is a systemic storage disease with different expression in males and females.

Steroid-responsive myalgia in a patient with Becker muscular dystrophy.

We report a male patient with exercise-induced focal myalgia in the leg muscles. Dystrophin immunostaining of a biopsied muscle specimen from the patient showed the absence of or only faint immunoreactivity in 20% of the muscle fibers. The patient was diagnosed as having Becker muscular dystrophy. The myalgia was intractable and did not respond to non-steroidal anti-inflammatory drugs. The patient was placed on prednisone and found to be sensitive to it. Although he had recurrences of the symptom during tapering of the steroid, slower tapering over one year was tolerated. Steroid treatment may be useful for other Becker muscular dystrophy patients with myalgia.

Phenotypic Duchenne muscular dystrophy with C-terminal domain.

We report a patient with X-linked muscular dystrophy who had rapidly progressive muscle weakness and became wheelchair-bound at age 10 years. Clinically, he was diagnosed as having Duchenne muscular dystrophy; however, he was diagnosed as having Becker muscular dystrophy by dystrophin tests using a C-terminal monoclonal antibody. No immunolabelling was observed with a monoclonal antibody against the N-terminal domain. Multiplex polymerase chain reaction analysis revealed the deletion of exons 3-19. The data suggest that the deletion of the N-terminal domain of dystrophin can cause a severe phenotype even when the C-terminus of the protein is well preserved.

[Clinical significance of serum cardiac myosin light chain I in patients with Duchenne muscular dystrophy].

The cardiac myosin light chain I (LCI) is one of the cardiac muscle structural proteins. A sensitive immunoradiometric assay kit for LCI by using LCI monoclonal antibodies is developed. We estimated LCI in the patients with Duchenne muscular dystrophy (DMD) and Kugelberg-Welander disease (KW). The results suggested that LCI has close relationships with the functional disturbances of skeletal muscles, especially disturbances of pulmonary ventilation. Therefore we studied properties and localizations of LCI in the skeletal muscles by Western blotting and immunohistochemical methods. In Western blotting method LCI monoclonal antibodies have a band of 27 KD proteins of skeletal muscles. LCI has also found to be localized in type 1 fibers in frozen sections of biopsied of human skeletal muscles. LCI was measured from 47 patients with DMD and 8 patients with KW. The average serum LCI levels in the patients with DMD were 11.79 ng/dl and its levels in the patients with KW were in the normal range (under 2.5 ng/dl). Among 12 patients receiving negative pressure chest respirator, the levels of LCI were also under 2.5 ng/dl. Serum LCI decreased with increasing age and reduced physical activity. The levels of LCI has obvious positive correlations with CK and myoglobin. These results suggested that the measurements of serum LCI are useful as one of the markers of disease severity and the determination of suitable time of using respirator.