Toshifumi Tsukahara

Mosaic Expression of Dystrophin in Symptomatic Carriers of Duchenne's Muscular Dystrophy

A deficiency of the protein dystrophin is known to be the cause of Duchennes muscular dystrophy. To examine the expression of dystrophin in symptomatic female carriers of this X-linked recessive disorder, we performed immunohistochemical studies on muscle-biopsy specimens from three such carriers, using an antiserum raised against a synthetic peptide fragment of dystrophin. In all three carriers, most individual muscle fibers reacted either strongly or not at all to the antiserum for dystrophin; only 2 to 8 percent of fibers showed partial immunostaining. This mosaic staining pattern was present on both cross-sectional and longitudinal muscle specimens. Although the mosaic pattern was seen in all fiber types, more than 80 percent of type 2B and 2C fibers from two of the carriers did not react with the antiserum. Similar studies in nine normal subjects showed consistently strong staining of all muscle fibers. No muscle fibers from 31 patients with Duchennes muscular dystrophy reacted with the antiserum. We conclude that symptomatic carriers of Duchennes muscular dystrophy can be identified by a distinct mosaic pattern in the immunohistochemical staining of the surface membrane of skeletal-muscle specimens. This finding may have practical implications for genetic counseling, although it remains to be shown whether the same staining pattern will be found in muscle specimens from asymptomatic carriers of Duchennes muscular dystrophy.

Biosyntheses and processing of lysosomal cysteine proteinases in rat macrophages

The intracellular processing and release of three lysosomal cysteine proteinases, cathepsin B, H and L, by rat peritoneal macrophages were investigated by pulse‐chase experiments. Newly synthesized procathepsins B (39 kDa), H(41 kDa) and L (39 kDa) after 15 min labeling were processed to the mature, single‐chain enzymes within 1 h. The single‐chain forms of cathepsin B, H and L were further processed to two‐chain forms at different rates: conversion of cathepsin L to the two‐chain form was rapid, whereas the conversions cathepsin B and H took at least 6 h. Macrophages released 30% of the procathepsins B and L, and 10% of the procathepsin H.

Identification of a putative amyloid A4-generating enzyme as a prolyl endopeptidase

The A4 amyloid peptide is deposited in Alzheimers disease inside neurons as neurofibrillary tangles or extracellularly as vascular amyloid. The A4 peptide is cleaved off by an unidentified proteinase from a larger precursor protein (APP), which resembles a cell surface receptor. The proteinase, which cleaves off the membrane‐spanning domain of APP, may be important in amyloid formation. To evaluate this, a model peptide substrate, succinyl‐ isoleucyl‐alanine‐methylcoumarinamide, which is homologous to the C‐terminal portion of A4 peptide, was synthesized to screen the putative A4‐generating proteinase. On chromatographie purification, it was found that two proteinases are involved in the hydrolysis of the peptide, the major one being identified as a prolyl endopeptidase. This evidence may facilitate elucidation of the mechanism of amyloid deposition in Alzheimers disease.

Nuclear accumulation of expanded PABP2 gene product in oculopharyngeal muscular dystrophy.

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is an adult‐onset disease caused by (GCG) repeat expansions in exon 1 of the poly(A) binding protein 2 gene (PABP2). To elucidate the molecular mechanism underlying the disease, we raised an antiserum against a synthetic peptide fragment predicted from PABP2 cDNA. The peptide corresponded to amino acids 271–291 where a cluster of posttranslational arginine methylation occurs. We examined the subcellular localization of PABP2 in muscle specimens from five patients with OPMD, 14 patients with various neuromuscular disorders, and three normal controls. All Japanese patients with OPMD have been shown to have expanded (GCG)8, 9, or 11 mutations in PABP2, as well as intranuclear tubulofilamentous inclusions (ITFI) of 8.5 nm. None of 50 separate Japanese control individuals were shown to have expanded (GCG) repeat in PABP2. Positive immunoreaction for polyclonal PABP2 was confined to the intranuclear aggregates of muscle fibers exclusively in patients with OPMD. Frequency of the nuclei positive for PABP2 (2%) was similar to that of ITFI detected by electron microscopy (2.5%). There was no apparent relationship between the frequency of PABP2‐positive intranuclear aggregates and the severity of muscle fiber damage. In contrast, nuclear immunoreaction was not detected in any samples from normal controls or from other neuromuscular diseases. These results suggest the presence of molecular modification of the product of expanded (GCG) repeat in PABP2, since the synthetic antigen peptide may not recognize a highly dimethylated cluster of arginine residues of the native PABP2, but may recognize the mutated form. Nuclear accumulation of expanded PABP2 product implies a causative role for ITFI.

Cloning and characterization of two neural-salient serine/arginine-rich (NSSR) proteins involved in the regulation of alternative splicing in neurones.

In neurones, alternative splicing regulates the functions of many gene products. However, the molecular basis of neural‐specific splicing, and how splicing regulation is modulated in different neurones remains to be determined.

CDNA microarray analysis of gene expression in fibroblasts of patients with x-linked Emery–Dreifuss muscular dystrophy

To clarify the molecular nature of the pathogenesis in X‐linked Emery–Dreifuss muscular dystrophy (EDMD), we monitored the expression of 2400 genes in control and EDMD fibroblasts by using complementary DNA (cDNA) microarray techniques. A total of 60 genes whose expression was altered in EDMD fibroblasts when compared with control fibroblasts were identified. Twenty‐eight genes whose expression was altered with the emerin deficiency were rescued by infection with a recombinant adenovirus expressing emerin. The altered expression in five genes, including the lamin A/C gene, was confirmed by reverse transcription–polymerase chain reaction. Our preliminary results suggest a correlation between disease similarity and gene expression. We conclude that the cDNA microarray is a very efficient tool to clarify genetic and pathological features of diseases.

Wortmannin enhances activation of CPP32 (Caspase-3) induced by TNF or anti-Fas.

CPP32/apopain (Caspase-3), a protease of the Ced-3/ICE family, is a central mediator in the apoptosis induced by TNF or anti-Fas. In this study we demonstrate that wortmannin, an inhibitor of PI-3K, enhances the activation of CPP32 (Caspase-3) and DNA fragmentation in TNF-treated U937 cells and anti-Fas-treated Jurkat cells. Caspase-3-like activity, Ac-DEVD-MCA cleavage activity, is enhanced by wortmannin in the range of the concentration (1–100 nM) specifically inhibiting PI-3K. LY294002, another PI-3K inhibitor, also enhances Caspase-3-like activity, but inhibitors for myosin light chain kinase and calmodulin dependent kinase do not have any effect on the Caspase-3-like activity. Wortmannin (1–100 nM) enhances the processing of Caspase-3 (32K) into active form (17K) in TNF- or anti-Fas-treated cells, but not in untreated cells. These observations suggest that inhibition of PI-3K induces the activation of processing enzyme of Caspase-3 or increases the susceptibility of Caspase-3 to the processing enzyme. PI-3K seems to protect the cells from apoptosis by suppressing the activation of Caspase-3.

Alternative splicing of Mef2c promoted by Fox-1 during neural differentiation in P19 cells.

Mef2c protein is one of the MADS‐box type transcription factors involved in muscular differentiation and synaptic formation. Previously, it has been reported that the Mef2c gene is responsible for three alternative splicing regulations. Here, we investigated the alternative splicing variants of Mef2c during neural differentiation of P19 cells and during cardio muscular differentiation of P19 clone 6 (P19CL6). We detected that two Mef2c mRNA isoforms, using exon α1 with and without the γ region at exon 10, are mainly produced in immature P19 cells. Remarkably, Mef2c isoforms containing exon β specifically appeared in the neural cell stage. Because most transcripts contain exon β in the neural cell stage and in the brain, this suggests that the alternative splicing of exon β is highly regulated. Among known regulators, Fox‐1 was specifically expressed in the neural cell stage in correlation with Mef2c exon β. Fox‐1 promoted exon β inclusion in transfection experiments using Mef2cβ minigene. Moreover, we found that the promotion required RNA‐binding activity of Fox‐1 and GCAUG sequence located in adjacent intron of exon β. Taken together, our results suggest that Fox‐1, expressed specifically in the neural cell stage, promoted Mef2c exon β inclusion via the GCAUG.

CHANGES IN PRE-MRNA SPLICING FACTORS DURING NEURAL DIFFERENTIATION IN P19 EMBRYONAL CARCINOMA CELLS

Alternative RNA splicing can be regulated in a highly cell- and tissue-specific or developmentally specific manner. In neurons, the functions of many gene products, such as those of trk genes are regulated by alternative splicing. In this paper the mechanism of neural-specific RNA splicing is investigated using trk genes as models. First, we confirm the splicing patterns of trk transcripts during neural differentiation of P19 embryonal carcinoma (EC) cells. The full-length form of trk B was expressed in the neuronal state. In contrast, both the full-length and truncated forms of trk C were expressed constitutively in all differentiation states. However, two alternatively spliced forms with either 42- or 117-nucleotide insertions in the tyrosine kinase domain were detected only in the neuronal state. Thus, the expression of functional trk B and C was found to be regulated by alternative splicing during neural differentiation. To examine the molecular basis of neural-specific splicing, and how splicing regulation is modulated in different neurons. The expression of a number of general splicing factors was studied. The mRNA levels of the splicing factors ASF/SF2, U2AF SF3a, p54nrb and PTB was found to decrease rapidly during differentiation. In contrast, Nova, an RNA-binding protein was expressed in the neuronal state. We also found that the levels of two SR proteins, members of a family of splicing factors, increased in the neuronal state. These results suggest that the stoichiometric balance among some splicing factors, including SR proteins, may be associated with the alternative splicing of trk transcripts during differentiation.

Upregulations of Gata4 and oxytocin receptor are important in cardiomyocyte differentiation processes of P19CL6 cells.

Oxytocin induces P19 cells to differentiate into cardiomyocytes possibly through the oxytocin/oxytocin receptor system. We added oxytocin to the growth medium of P19CL6, a subline of P19, but they did not differentiate into cardiomyocytes as indicated by RT‐PCR and Western blotting results. During the cardiac commitment time of P19CL6 cells, the mRNA expression levels of the oxytocin receptor were upregulated by the addition of oxytocin as well as DMSO, but an upregulation of Gata4 expression levels was only observed for the cells induced by DMSO. The in silico analysis of the upstream sequence of the oxytocin receptor predicted putative binding sites for Gata4 and Nkx2.5. These results suggest that upregulations of the oxytocin receptor and Gata4 are important for cardiomyocyte differentiation processes. J. Cell. Biochem. 100: 629–641, 2007.