Regulation of splicing by MBNL and CELF family of RNA-binding protein.

Myotonic Dystrophy (DM), the most common form of adult-onset muscular dystrophy, comprises at least 2 subtypes, DM1 and DM2. DM1 is caused by the expansion of a CTG repeat located in the 3' untranslated region of the DM protein kinase (DMPK) gene. Recently, the expansion of a CCTG tetranucleotide repeat located in the first intron of the ZNF9 gene was identified as the mutation responsible for DM2. Since both DM1 and DM2 are caused by the expansion of repetitive sequences, some common factors that interact with these sequences might be involved in the pathogenesis of DM. MBNL1 is a candidate for such factors and is thought to be sequestered by the expanded forms of DM transcripts.

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.

Limited proteolysis of filamin is catalyzed by caspase-3 in U937 and Jurkat cells.

Members of the caspase family have been implicated as key mediators of apoptosis in mammalian cells. However, few of their substrates are known to have physiological significance in the apoptotic process. We focused our screening for caspase substrates on cytoskeletal proteins. We found that an actin binding protein, filamin, was cleaved from 280 kDa to 170, 150, and 120 kDa major N-terminal fragments, and 135, 120, and 110 kDa major C-terminal fragments when apoptosis was induced by etoposide in both the human monoblastic leukemia cell line U937, and the human T lymphoblastic cell line Jurkat. The cleavage of filamin was blocked by a cell permeable inhibitor of caspase-3-like protease, Ac-DEVD-cho, but not by an inhibitor of caspase-1-like protease, Ac-YVAD-cho. These results suggest that filamin is cleaved by a caspase-3-like protease. To examine whether caspase-3 cleaves filamin in vitro, we prepared a recombinant active form of caspase-3 directly using a Pichia pastoris overexpression system. When we applied recombinant active caspase-3 to the cell lysate of U937 and Jurkat cells, filamin was cleaved into the same fragments seen in apoptosis-induced cells in vivo. Platelet filamin was also cleaved directly from 280 kDa to 170, 150, and 120 kDa N-terminal fragments, and the cleavage pattern was the same as observed in apoptotic human cells in vivo. These results suggest that filamin is an in vivo substrate of caspase-3.

Interaction between emerin and nuclear lamins.

Emerin is an inner nuclear membrane protein that is involved in X-linked recessive Emery-Dreifuss muscular dystrophy (X-EDMD). Although the function of this protein is still unknown, we revealed that C-terminus transmembrane domain-truncated emerin (amino acid 1-225) binds to lamin A with higher affinity than lamin C. Screening for the emerin binding protein and immunoprecipitation analysis showed that lamin A binds to emerin specifically. We also used the yeast two-hybrid system to clarify that this interaction requires the top half of the tail domain (amino acid 384-566) of lamin A. Lamin A and lamin C are alternative splicing products of the lamin A/C gene that is responsible for autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD). These results indicate that the emerin-lamin interaction requires the tail domains of lamin A and lamin C. The data also suggest that the lamin A-specific region (amino acids 567-664) plays some indirect role in the difference in emerin-binding capacity between lamin A and lamin C. This is the first report that refers the difference between lamin A and lamin C in the interaction with emerin. These data also suggest that lamin A is important for nuclear membrane integrity.

The VNTR polymorphism of the human dopamine transporter (DAT1) gene affects gene expression.

The human dopamine transporter (DAT1) gene contains a variable number of tandem repeats (VNTR) in its 3'-untranslated region (UTR). The linkage and association between the VNTR polymorphism of DAT1 and various neuropsychiatric disorders have been reported. We have determined the genomic structure of DAT1 genes containing 7-, 9-, 10-, and 11-repeat alleles and examined the effect of VNTR polymorphism in the 3'-UTR region of DAT1 on gene expression using the luciferase reporter system in COS-7 cells. Luciferase expression was significantly higher when the 3'-UTR of the DAT1 gene contained the 10-repeat allele than when it contained the 7- or 9-repeat alleles. This suggests that VNTR polymorphism affects the expression of the dopamine transporter.

The CUG-binding protein binds specifically to UG dinucleotide repeats in a yeast three-hybrid system.

The CUG-binding protein (CUG-BP) has been reported to be involved in the pathogenesis of myotonic dystrophy (DM) through binding to a CUG trinucleotide repeat located in the 3' untranslated region (3'UTR) of the DM protein kinase (DMPK) gene. We found that CUG-BP associates with long CUG trinucleotide repeats ((CUG)(11)(CUG)(12)), but not with short repeats ((CUG)(12)) in a yeast three-hybrid system. On the other hand, CUG-BP+LYLQ, an alternatively spliced isoform of CUG-BP, does not associate with CUG trinucleotide repeats regardless of the repeat length. In addition to these findings, we found that CUG-BP and CUG-BP+LYLQ strongly and specifically associate with UG dinucleotide repeats. Deletion analyse of CUG-BP revealed that the absence of the first or third RNA-binding domain (RBD I and RBD III, respectively) does not affect the interaction between CUG-BP and UG dinucleotide repeats. Loss of the second RNA-binding domain (RBD II) decreases the affinity of CUG-BP for UG dinucleotide repeats by about 40%. Unexpectedly, deletion of the linker domain most severely reduces the interaction, although this region does not contain a known RNA-binding motif. Our results suggest the possibility that both CUG-BP and CUG-BP+LYLQ associate with UG repeat-containing mRNAs and regulate such metabolic properties as mRNA localization, stability, and translation, and provide new insights into the pathogenesis of DM.

Late leaflet fracture and embolization of a Duromedics mitral prosthesis.

A case of leaflet fracture and embolization of a mitral prosthetic valve is described. A 54-year-old man had received mitral valve replacement with an Edwards-Duromedics 29M prosthetic valve, at 10 years ago. Emergency mitral valve replacement was performed because the patient had severe congestive left heart failure with severe acute mitral regurgitation caused by a fracture in one of the mitral valve leaflets. The leaflet, which was fractured into 2 pieces, was removed from the right common iliac artery at 3 months after valve replacement. Visual inspection revealed that the leaflet contained a midline fracture. The fracture originated within a cavitary erosion pit near the major radius of the leaflet. The patient recovered from acute renal failure, requiring hemodialysis for 80 days, and is currently without complaints. We have used a Duromedics mitral valve in 11 patients, from April 1987 to April 1988. No subsequent valve failure has occurred. The diagnosis, treatment and cause of a mechanical valve fracture are discussed.

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.

An expanded CTG trinucleotide repeat causes trans RNA interference: a new hypothesis for the pathogenesis of myotonic dystrophy.

Here we report a novel mechanism for the pathogenesis of myotonic dystrophy (DM). The DMPK mRNA with expanded CTG trinucleotide repeats interacts with other transcripts having expanded CAG repeats. This "trans RNA interference" occurs in vitro only when the number of CTG repeats is over 140 and the number of target CAG repeats exceeds 35. The trans RNA interference can explain all the phenomena previously reported about DM.

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.

Synthesis of long trinucleotide repeats in vitro.

More than a dozen diseases have been associated with the expansion of trinucleotide repeats. Most of these expanding repeats are GC-rich (CGG/CCG or CTG/CAG), but it is difficult to amplify GC-rich repeat DNA from patient samples by the polymerase chain reaction. We invented a pair of methods to synthesize long trinucleotide repeats in vitro by polymerase extension utilizing a thermal cycler. A combination of the two methods, termed the non-template PCR method and SLIP (Synthesis of Long Iterative Polynucleotide) method, produced (CTG/CAG)190 repeat DNA. We expect that these two methods will contribute to studies of all diseases associated with tri-nucleotide repeat expansion, since they can be applied to any type of repeat DNA.

An integrated multimedia medical information network system.

An integrated multimedia medical information network system at Shimane Medical university has been developed to organize medical information generated from each section and provide information services useful for education, research and clinical practice. The report describes the outline of our system. It is designed to serve as a distributed database for electronic medical records and images. We are developing the MML engine that is to be linked to the world wide web (WWW) network system. To the users, this system will present an integrated multimedia representation of the patient records, providing access to both the image and text-based data required for an effective clinical decision making and medical education.

Novel isoform of myotonin protein kinase: gene product of myotonic dystrophy is localized in the sarcoplasmic reticulum of skeletal muscle.

It is quite important to know the exact localization and function of myotonin protein kinase (MtPK), identified as the gene product of myotonic dystrophy, the most prevalent disease with multisystem disorders among muscular dystrophies. To investigate the localization of MtPK, we raised a polyclonal antibody against a synthetic peptide chosen within the deduced sequence of MtPK. This antibody detected both a membrane-bound 70-kd protein and a soluble 55-kd protein on Western blots of human muscles. By using this antibody for immunohistochemical studies of both biopsied human skeletal muscle fibers and mature innervated cultured muscle fibers, we can now demonstrate by confocal laser scanning microscopy that MtPK is localized mainly in the I-band. By immunoelectron microscopy, it was determined that MtPK is a membrane-bound protein localized mainly in the terminal cisternae of the sarcoplasmic reticulum. To our knowledge, this is the first documentation of the ultrastructural localization of MtPK. This finding is quite important for clarifying the pathophysiological basis of myotonic dystrophy, which might be due to a dysregulation of calcium metabolism.

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.

Immunocytochemical localization of a full-length myotonin protein kinase in rat L6 myoblasts.

Expansion mutation of CTG-repeat motifs within myotonin protein kinase (MtPK) gene is responsible for pathological changes in myotonic dystrophy (DM). To explore its pathological role in skeletal muscle, a full-length human MtPK cDNA was transfected into rat L6 myogenic cell line. Recombinantly expressed human MtPK protein in L6 cell line has a predicted molecular mass of 70 kDa. We have raised a polyclonal antibody against a synthetic peptide in the deduced sequence of the C-terminal portion of MtPK. MtPK in L6 cell is localized to perinuclear region, that resembles with sarcoplasmic reticulum. The MtPK-transfected myoblast cells established in this study will allow us to elucidate the molecular pathomechanism of muscle manifestations in DM.

[Two cases of mitral stenosis associated with ball thrombus in the left atrium].

Two patients (55-year-old female and 77-year-old female) were operated on for mitral stenosis associated with left atrial ball thrombus. The first case had the episode of cerebral infarction and second case had syncopal attack. Both of them presented with systemic arterial embolism. After confirmation of the diagnosis by echocardiography, removal of ball thrombi, OMC and MVR were carried out urgently. Considering high risk of "hole in one sudden death" and multiple-episodes of systemic embolization, ball thrombus should be removed as urgent following confirmation of diagnosis.

Aneurysm of a branch of the subclavian artery with multiple arteriovenous fistulae.

A rare case is reported of an 83-year-old woman with an aneurysm of a branch of the subclavian artery with multiple arteriovenous fistulae. The patient was admitted to our hospital with a pulsatile mass in the supraclavicular space and a prominent continuous murmur which radiated to the anterior chest, right forearm and right neck. She first noticed a pulsatile 2 cm mass in 1972, 1 year following a subtotal gastrectomy. At that time, she had intravenous therapy through a right neck vein. In 1993, the mass became larger, and she developed a shunt murmur. Digital subtraction angiography (DSA) demonstrated an aneurysm of the right subclavian artery and an arteriovenous fistula between the right subclavian artery and vein. The right common carotid artery and right subclavian artery arose from the aortic arch separately. The aneurysm arose from a branch of the subclavian artery which may be the costocervical trunk. The 5 x 4 cm aneurysm was resected and the arteriovenous fistula was divided. On postoperative day 5, a new murmur was ausculated. A repeat DSA detected a new fistula between the axillary artery and vein. Reoperation was performed to ligate and divide the fistula. Pathological examination revealed an atherosclerotic aneurysm.

[A case report of surgical management of blunt ruptured right atrium with blunt ruptured liver].

This is a case report of a 23 year old female involved in a car accident that caused blunt trauma to the patient. First of all, she was found to have cardiac tamponade when the abdomen was explored to suture and to put the gaze compression on for hemostasis of the ruptured liver. Then, she was brought to our institution by the ambulance. Upon the exploration of the heart under standby of extracorporal circulation (ECC), small multiple lacerations were found at the junction of the right atrium and superior vena cava. These were sutured directly to close without ECC. On the 2nd postoperative day, she was bought to the OR again to removal of the gaze tamponade from the ruptured liver and to complete hemostasis. The patient was discarded 35 days after admission.

Effect of artificial (CTG) repeat expansion on the expression of myotonin protein kinase (MtPK) in COS-1 cells.

A major challenge in the study of a new genetic entity called triplet-repeat disease is to identify the role of triplet repeats in the pathogenesis of the disease. We have developed a strategy to demonstrate the effect in the 3'-untranslated end of the (CTG) repeats in myotonic dystrophy gene (MtPK) and found that repeat expansion (CTG46) causes a slight decrease in the translation rate of MtPK cDNA which correlates with the finding in patients with myotonic dystrophy of a low amount of MtPK protein in muscle. These results provide an important clue for characterizing the genetic abnormality in other triplet-repeat diseases.