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The etiology of chicken muscular dystrophy is the synthesis of aberrant WW domain containing E3 ubiquitin-protein ligase 1 (WWP1) protein made by a missense mutation of WWP1 gene. The β-dystroglycan that confers stability to sarcolemma was identified as a substrate of WWP protein, which induces the next molecular collapse. The aberrant WWP1 increases the ubiquitin ligase-mediated ubiquitination following severe degradation of sarcolemmal and cytoplasmic β-dystroglycan, and an erased β-dystroglycan in dystrophic αW fibers will lead to molecular imperfection of the dystrophin-glycoprotein complex (DGC). The DGC is a core protein of costamere that is an essential part of force transduction and protects the muscle fibers from contraction-induced damage. Caveolin-3 (Cav-3) and dystrophin bind competitively to the same site of β-dystroglycan, and excessive Cav-3 on sarcolemma will block the interaction of dystrophin with β-dystroglycan, which is another reason for the disruption of the DGC. It is known that fast-twitch glycolytic fibers are more sensitive and vulnerable to contraction-induced small tears than slow-twitch oxidative fibers under a variety of diseased conditions. Accordingly, the fast glycolytic αW fibers must be easy with rapid damage of sarcolemma corruption seen in chicken muscular dystrophy, but the slow oxidative fibers are able to escape from these damages.

The WW domain containing E3 ubiquitin protein ligase 1 (WWP1) is classified into one of ubiquitin ligases which play an important role in ubiquitin-proteasome pathway. Previously, we identified the WWP1 gene as a candidate gene of chicken muscular dystrophy by linkage analysis and sequence comparison. However, the mechanism causing pathological changes and underlaying gene function remains elucidated. In the present study, we analyzed the WWP1 gene expression in various muscles and tissues of normal chickens, and compared with those from muscular dystrophic chickens. Two mRNA isoforms were detected in all tissues examined and revealed almost equal expression level. The WWP1 expression of dystrophic chickens was decreased in almost all skeletal muscles including unaffected muscles. These data indicate that there might not be a causal relationship between the alteration of WWP1 expression level and the severity of muscular dystrophy.

Aims Glucose transporter 10 (GLUT10), encoded by the SLC2A10 gene, is a member of the class III facilitative glucose transporter family. Mutations in the SLC2A10 gene cause arterial tortuosity syndrome (ATS) in humans. To further study the pathogenesis of the disease, we generated mice carrying GLUT10 mutations. Methods and results Using a gene-driven N -ethyl- N -nitrosourea (ENU)-mutagenesis approach, we generated mice carrying GLUT10 mutations c.383G>A and c.449C>T, which resulted in missense mutations of glycine to glutamic acid (p.G128E) and serine to phenylalanine (p.S150F), respectively. Both mutant strains appeared normal at birth, gained weight appropriately and survived to adulthood (>18 months). Blood and urine glucose were normal. Echocardiogram and electrocardiogram were also normal and brain magnetic resonance angiography revealed normal cerebral arteries without tortuosity, stenosis/dilatation, or aneurysm. Histopathology revealed thickening and irregular vessel wall shape of large and medium size arteries characterized by markedly increased elastic fibres, both in number and size. There was also intima endothelial hypertrophy and deranged elastic fibres that resulted in disruption of internal elastic lamina in the aorta of older mice. Conclusion Abnormal elastogenesis with early elastic fibre proliferation provides a clue to the pathogenesis of arterial tortuosity in human ATS. Availability of this mouse model will allow testing of the relationship between diabetes and its vascular complications, including diabetic retinopathy, nephropathy and peripheral vascular disease.

The gracile axonal dystrophy (gad) mice are known to have a deletion within the gene encoding ubiquitin carboxy-terminal hydrolase-1 (Uch-L1) and show hereditary sensory deterioration and motor paresis. Expression of Uch-L1 is reported to be almost limited to the nervous system and testis. To understand whether Uch-L1, one of the major ubiquitin carboxy-terminal hydrolase (UCH) isozymes in the testis, affects spermatogenesis and other UCH isozymes (Uch-L3, L4 and L5) expression in the testis, we compared the testis between gad, hetero and wild type mice by histological, immunohistochemical analyses and RT-PCR. Histological analysis in 25-week-old gad mice showed shrinking of seminiferous tubules, decreasing total number of cells and enlargement of remaining cells in seminiferous tubules. By immunohistochemistry, a significant decrease (p < 0.05) in the number of proliferating cell nuclear antigen (PCNA) positive cells was observed. Expression of other UCH isozyme mRNAs was not apparently affected by Uch-L1 deficiency in 25-week-old gad mice. This study is the first report on the testis of gad mutant mouse.

Accumulation of unfolded and malfolded proteins causes endoplasmic reticulum (ER) stress, stimulating unfolded protein response (UPR) and c-Jun N-terminal kinase (JNK) activation and activating caspase-12 located on the ER. Little is known about the relationship between the ER stress and polyglutamine [poly(Q)] aggregates. Poly(Q)72 repeats [poly(Q)(72)] induced the stimulation of ER stress signals such as JNK activation, upregulation of Grp78/Bip and caspase-12 activation in C2C5 cells. We prepared antiserum against the cleavage site of mouse caspase-12 at D(318) (anti-m12D318), and showed that poly(Q)(72) with perinuclear aggregates, cytoplasmic inclusions and nuclear inclusions stimulated JNK activation and anti-m12D318 immunoreactivity, but poly(Q)(72) with dispersed aggregates and small nuclear aggregates showed a significantly less effect. Poly(Q)(72) and poly(Q)(11) dispersed in cytoplasm did not. Anti-m12D318-positive cells showed apoptotic features. Unlike anti-m8D387 immunoreactivity, the anti-m12D318 immunoreactivity was not coaggregated with poly(Q). Ac-IETD-fmk (caspase-8 inhibitor) and Ac-DEVD-CHO (caspase-3 inhibitor) did not prevent the anti-m12D318 immunoreactivity induced by poly(Q)(72) aggregates. Anti-m12D318 immunoreactivity was detected in caspase-8(-/-) and caspase-3(-/-) mouse embryonic fibroblasts expressing poly(Q)(72) aggregates. Thus, caspase-12 was activated by poly(Q)(72) aggregates via a pathway independent of caspase-8 and caspase-3 activation, and caspase-12 activation was closely associated with poly(Q) aggregate-mediated cell death. Stimulation of ER stress signals may be involved in the pathogenesis of neurodegenerative disorders with poly(Q) expansion.

The male hypermethylated (MHM) region, located near the middle of the short arm of the Z chromosome of chickens, consists of approximately 210 tandem repeats of a BamHI 2.2-kb sequence unit. Cytosines of the CpG dinucleotides of this region are extensively methylated on the two Z chromosomes in the male but much less methylated on the single Z chromosome in the female. The state of methylation of the MHM region is established after fertilization by about the 1-day embryonic stage. The MHM region is transcribed only in the female from the particular strand into heterogeneous, high molecular-mass, non-coding RNA, which is accumulated at the site of transcription, adjacent to the DMRT1 locus, in the nucleus. The transcriptional silence of the MHM region in the male is most likely caused by the CpG methylation, since treatment of the male embryonic fibroblasts with 5-azacytidine results in hypo-methylation and active transcription of this region. In ZZW triploid chickens, MHM regions are hypomethylated and transcribed on the two Z chromosomes, whereas MHM regions are hypermethylated and transcriptionally inactive on the three Z chromosomes in ZZZ triploid chickens, suggesting a possible role of the W chromosome on the state of the MHM region.

Caveolin-3 is a muscle-specific protein integrated in the caveolae, which are small invaginations of the plasma membrane. Mutations of the caveolin-3 gene, localized at 3p25, have been reported to be involved in the pathogenesis of limb-girdle muscular dystrophy (LGMD1C or caveolinopathy) with mild clinical symptoms, inherited through an autosomal dominant form of genetic transmission. To elucidate the pathogenetic mechanism, we developed caveolin-3-deficient mice for use as animal models of caveolinopathy. Caveolin-3 mRNA and its protein were absent in homozygous mutant mice. In heterozygous mutant mice, both the mRNA and its protein were normal in size, but their amounts were reduced by about half. The density of caveolae in skeletal muscle plasma membrane was roughly proportional to the amount of caveolin-3. In homozygous mutant mice, muscle degeneration was recognized in soleus muscle at 8 weeks of age and in the diaphragm from 8 to 30 weeks, although there was no difference in growth and movement between wild-type and mutant mice. No apparent muscle degeneration was observed in heterozygous mutant mice, indicating that pathological changes caused by caveolin-3 gene disruption were inherited through the recessive form of genetic transmission.

Truncated polypeptides containing expanded polyglutamine (polyQ) stretches tend to form cytoplasmic or nuclear aggregates in cultured cells, leading to cell death. Although it has been shown recently that caspase-8 coaggregates with polyQ and is activated during polyQ-mediated cell death, little is known of the location and timing of caspase-8 activation by nuclear polyQ aggregates. Also, the relationship between nuclear polyQ aggregate-mediated cell death and activation of other caspases is unclear. In P19 embryonal carcinoma (EC) cells, which can be made to differentiate into neuronal cells, polyQ72 repeats preferentially aggregate in the nucleus. Nuclear aggregates of polyQ72 induced P19 EC cell death, with a high frequency of cells exhibiting morphology characteristic of apoptosis (i.e., roundness, cell shrinkage, chromatin condensation) and DNA fragmentation. In the present study, we used antisera that specifically recognized the active forms of caspase-8, -3, and -9 but not their proforms, and showed that only caspase-8 and -3 were activated during the generation of polyQ72 aggregates in P19 EC cell nuclei. Furthermore, we showed that the caspase inhibitor z-VAD-fmk inhibited DNA fragmentation, but only partially inhibited the appearance of apoptotic morphology. Thus, caspase activation, including caspase-8 and -3, is necessary for polyQ-mediated DNA fragmentation but not sufficient for polyQ-mediated cell death in P19 EC cells.

The gracile axonal dystrophy (gad) mouse is an autosomal recessive mutant that shows sensory ataxia at an early stage, followed by motor ataxia at a later stage1. Pathologically, the mutant is characterized by 'dying-back' type axonal degeneration and formation of spheroid bodies in nerve terminals2,3,4,5. Recent pathological observations have associated brain ageing and neurodegenerative diseases with progressive accumulation of ubiquitinated protein conjugates6,7. In gad mice, accumulation of amyloid β-protein and ubiquitin-positive deposits occur retrogradely along the sensory and motor nervous systems8,9. We previously reported that the gad mutation was transmitted by a gene on chromosome 5 (refs 10,11). Here we find that the gad mutation is caused by an in-frame deletion including exons 7 and 8 of Uchl1, encoding the ubiquitin carboxy-terminal hydrolase (UCH) isozyme (Uch-l1) selectively expressed in the nervous system and testis12,13,14,15. The gad allele encodes a truncated Uch-l1 lacking a segment of 42 amino acids containing a catalytic residue16. As Uch-l1 is thought to stimulate protein degradation by generating free monomeric ubiquitin16,17,18, the gad mutation appears to affect protein turnover. Our data suggest that altered function of the ubiquitin system directly causes neurodegeneration. The gad mouse provides a useful model for investigating human neurodegenerative disorders.

Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase (GAA), a glycogen degrading lysosomal enzyme. GAA-deficient (AMD) Japanese quails exhibit progressive myopathy and cannot lift their wings, fly, or right themselves from the supine position (flip test). Six 4-wk-old acid maltase-deficient quails, with the clinical symptoms listed, were intravenously injected with 14 or 4.2 mg/kg of precursor form of recombinant human GAA or buffer alone every 2-3 d for 18 d (seven injections). On day 18, both high dose-treated birds (14 mg/kg) scored positive flip tests and flapped their wings, and one bird flew up more than 100 cm. GAA activity increased in most of the tissues examined. In heart and liver, glycogen levels dropped to normal and histopathology was normal. In pectoralis muscle, morphology was essentially normal, except for increased glycogen granules. In sharp contrast, sham-treated quail muscle had markedly increased glycogen granules, multi-vesicular autophagosomes, and inter- and intrafascicular fatty infiltrations. Low dose-treated birds (4.2 mg/kg) improved less biochemically and histopathologically than high dose birds, indicating a dose-dependent response. Additional experiment with intermediate doses and extended treatment (four birds, 5.7-9 mg/kg for 45 d) halted the progression of the disease. Our data is the first to show that an exogenous protein can target to muscle and produce muscle improvement. These data also suggest enzyme replacement with recombinant human GAA is a promising therapy for human Pompe disease.

A cDNA clone, pZOV3, was isolated from the cDNA library of immature chicken ovaries and its gene was mapped to the middle of the short arm of the Z chromosome. The cDNA sequence suggests that ZOV3 is a novel member of the immunoglobulin superfamily. cDNA clones of homologues of chicken ZOV3 were also obtained from Japanese quail and pigeon. Northern blot hybridization suggests that the high-level expression of the ZOV3 gene is restricted to the gonads: embryonic, immature and mature ovaries, and embryonic and immature testes. Western blot analysis and immunocytological detection using specific polyclonal antibodies against amino- and carboxyl-terminal regions of ZOV3 demonstrate that ZOV3 is a plasma membrane-bound glycoprotein that exists in granulosa cells and islets of cells in the theca externa layer of ovarian follicles. The latter islets coincide with those producing estradiol-17 beta. In male and female embryos, production of ZOV3 is first prominent in medullary and seminiferous codes, respectively, of developing gonads. Then, after hatching, it is shifted to the cortex surrounding the primitive follicles in the ovary or is continued weakly in the primary seminiferous tubules in the testis. Expression of the ZOV3 gene and production of ZOV3 are no longer detectable in the mature testis. ZOV3 is unique among the immunoglobulin superfamily proteins in that it is produced predominantly in gonads. Its possible role in differentiation or maintenance of steroidogenic cells in an ovarian follicle is discussed.

Cloning and sequencing of the peripheral myelin protein-22 cDNA and genomic DNA from newly found Trembler mice revealed an in-frame deletion including exon IV which codes for the second (TM2) and a part of third (TM3) transmembrane domain of peripheral myelin protein-22. This mutation was distinct from those in both other allelic Trembler and Trembler-J mice, which carry point mutations within the putative transmembrane spanning regions of peripheral myelin protein-22. Inheritance was autosomal dominant. The affected mice revealed an abnormal gait, which appeared at 15-20 days of age, followed by motor and sensory ataxia, which remained throughout life. Most of the affected mice could survive more than one year. One of the most notable pathological phenotypes was a giant vacuolar formation in the sciatic nerve of homozygotes. They vary in size within the cytoplasm of Schwann cells, which failed to assemble myelin at any ages studied. Heterozygotes showed normal myelination during the early postnatal stages, followed by a segmental demyelination at an advanced stage. Vacuolar formation was not so frequent as in the homozygotes. These results suggest that the missing of transmembrane spanning region (TM2 and TM3) of peripheral myelin protein-22 may disturb a dual biological function of peripheral myelin protein-22, leading to a dysmyelination of axons and to a vacuolar formation within the cytoplasm of the Schwann cells. The latter phenotype is discussed in conjunction with the disruption of an intracellular transport system and subsequent cell death.

Members of the caveolin family are the main component of caveolae, and caveolin-3 is a muscle-specific protein. Caveolin-3 deficiency induces a muscular dystrophic phenotype, while its overexpression is also harmful to muscle cells. Increased caveolae were observed in chicken muscular dystrophy; however, the underlying mechanism causing the onset remains unclear. Therefore, the current study analyzes the expression of caveolin-3 and other caveola-related proteins in dystrophic chickens. Western blotting and semi-quantitative RT-PCR analysis revealed that (1) caveolin-3 is highly expressed in the damaged muscle of dystrophic chickens (7.12-fold); (2) the amount of caveolin-3 protein is regulated in posttranslational modification, since no significant increase is observed at the mRNA level (1.09-fold); and (3) the expression pattern of other caveola-related proteins is similar to that of caveolin-3. These results suggest that the accumulation of caveolin-3 protein may be associated with the causative process of chicken muscular dystrophy.

In the present studies the VIP receptors on plasma membranes from human pancreatic carcinoma cells (PU-PAN-1) were characterized by radioligand analysis and cross-linking reagents assay. Kd for VIP receptors on plasma membranes was 1.7×10 -10 M. After binding of 125 I-VIP on membranes either bifunctional cross-linking reagent 1 mM disuccinimidyl suberate (DSS) or ethylene glycol bis (succinimidyl succinate) (EGS) was added, then SDS-polyacrylamide gel electrophoresis and autoradiography were performed. When plasma membranes with 125 I-VIP without adding crosslinking agents, no radiolabeled band appeared in the autoradiogram

Chicken muscular dystrophy with abnormal muscle (AM) has been studied for more than 50 years, but the gene responsible for it remains unclear. Our previous studies narrowed down the AM candidate region to approximately 1Mbp of chicken chromosome 2q containing seven genes. In this study, we performed sequence comparison and gene expression analysis to elucidate the responsible gene. One missense mutation was detected in AM candidate genes, while no remarkable alteration of expression patterns was observed. The mutation was identified in WWP1, detected only in dystrophic chickens within several tetrapods. These results suggested WWP1 is responsible for chicken muscular dystrophy.

Using the metabolomics-guided screening coupled to N-ethyl-N-nitrosourea-mediated mutagenesis, we identified mice that exhibited elevated levels of long-chain acylcarnitines. Whole genome homozygosity mapping with 262 SNP markers mapped the disease gene to chromosome 5 where candidate genes Hadha and Hadhb, encoding the mitochondria trifunctional protein (MTP) alpha- and beta-subunits, respectively, are located. Direct sequencing revealed a normal alpha-subunit, but detected a nucleotide T-to-A transversion in exon 14 (c.1210T>A) of beta-subunit (Hadhb) which resulted in a missense mutation of methionine to lysine (M404K). Western blot analysis showed a significant reduction of both the alpha- and beta-subunits, consistent with reduced enzyme activity in both the long-chain 3-hydroxyacyl-CoA dehydrogenase and the long-chain 3-ketoacyl-CoA thiolase activities. These mice had a decreased weight gain and cardiac arrhythmias which manifested from a prolonged PR interval to a complete atrio-ventricular dissociation, and died suddenly between 9 and 16 months of age. Histopathological studies showed multifocal cardiac fibrosis and hepatic steatosis. This mouse model will be useful to further investigate the mechanisms underlying arrhythmogenesis relating to lipotoxic cardiomyopathy and to investigate pathophysiology and treatment strategies for human MTP deficiency.

Infantile neuroaxonal dystrophy (INAD) is a rare autosomal recessive hereditary neurodegenerative disease of humans. So far, no responsible gene has been cloned or mapped to any chromosome. For chromosome mapping and positional cloning of the responsible gene, establishment of an animal model would be useful. Here we describe a new mouse model for INAD, named inad mouse. In this mouse, the phenotype is inherited in an autosomal recessive manner, symptoms occur in the infantile period, and the mouse dies before sexual maturity. Axonal dystrophic change appearing as spheroid bodies in central and peripheral nervous system was observed. These features more closely resembled human INAD than did those of the gad mouse, the traditional mouse model for INAD. Linkage analysis linked the inad gene to mouse Chromosome 1, with the highest LOD score (=128.6) at the D1Mit45 marker, and haplotype study localized the inad gene to a 7.5-Mb region between D1Mit84 and D1Mit25. In this linkage area some 60 genes exist: Mutation of one of these 60 genes is likely responsible for the inad mouse phenotype. Our preliminary mutation analysis in 15 genes examining the nucleotide sequence of exons of these genes did not find any sequence difference between inad mouse and C57BL/6 mouse.

Despite intensive studies of muscular dystrophy of chicken, the responsible gene has not yet been identified. Our recent studies mapped the genetic locus for abnormal muscle (AM) of chicken with muscular dystrophy to chromosome 2q using the Kobe University (KU) resource family, and revealed the chromosome region where the AM gene is located has conserved synteny to human chromosome 8q11-24.3, where the beta-1 syntrophin (SNTB1), syndecan 2 (SDC2) and Gem GTPase (GEM) genes are located. It is reasonable to assume those genes might be candidates for the AM gene. In this study, we cloned and sequenced the chicken SNTB1, SDC2 and GEM genes, and identified sequence polymorphisms between parents of the resource family. The polymorphisms were genotyped to place these genes on the chicken linkage map. The AM gene of chromosome 2q was mapped 130 cM from the distal end, and closely linked to calbindin 1 (CALB1). SNTB1 and SDC2 genes were mapped 88.5 cM distal and 27.6 cM distal from the AM gene, while the GEM gene was mapped 18.5 cM distal from the AM gene and 9.1 cM proximal from SDC2. Orthologues of SNTB1, SDC2 and GEM were syntenic to human chromosome 8q. SNTB1, SDC2 and GEM did not correspond to the AM gene locus, suggesting it is unlikely they are related to chicken muscular dystrophy. However, this result also suggests that the genes located in the proximal region of the CALB1 gene on human chromosome 8q are possible candidates for this disease.

We examined the effects of methylcobalamin (methyl-B12, mecobalamin) on degeneration of motor nerve terminals in the anterior gracile muscle of gracile axonal dystrophy (GAD) mutant mice. GAD mice received orally methyl-B12 (1 mg/kg body wt/day) from the 40th day after birth for 25 days. In the distal endplate zone of the muscle, although most terminals were degenerated in both the untreated and methyl-B12-treated GAD mice, sprouts were more frequently observed in the latter. In the proximal endplate zone, where few degenerated terminals were seen in both groups of the mice, the perimeter of the terminals was increased and the area of the terminals was decreased significantly in the methyl-B12-treated GAD mice. These findings indicate that methyl-B12 promotes regeneration of degenerating nerve terminals in GAD mice.

Caveolin, a 20-24 kDa integral membrane protein, is a principal component of caveolar domains. Caveolin-1 is expressed predominantly in endothelial cells, fibroblasts, and adipocytes, while the expression of caveolin-3 is confined to muscle cells. However, their localization in various muscles has not been well documented. Using double-immunofluorescence labeling and confocal laser microscopy, we examined the localization of caveolins-1 and 3 in adult monkey skeletal, cardiac and uterine smooth muscles and the co-immunolocalization of these caveolins with dystrophin, which is a product of the Duchenne muscular dystrophy gene. In the skeletal muscle tissue, caveolin-3 was localized along the sarcolemma except for the transverse tubules, and co-immunolocalized with dystrophin, whereas caveolin-1 was absent except in the blood vessels of the muscle tissue. In cardiac muscle cells, caveolins-1 and -3 and dystrophin were co-immunolocalized on the sarcolemma and transverse tubules. In uterine smooth muscle cells, caveolin-1, but not caveolin-3, was co-immunolocalized with dystrophin on the sarcolemma.

It has been reported that light exposure to one eye induces functional lateralization, which can be inverted by exposing the opposite eye to the light. However, the anatomical basis of the functional inversion by the light has not been shown. To address this issue, we labeled cells in the dorsolateral anterior thalamus (DLA) using retrograde fluorescent tracers injected into visual Wulst, counted the labeled cell number, and compared the anatomical asymmetry of DLA between the left eye occluded and the right eye occluded chickens. We found that a rostral part of DLA (DLAda) and a lateral/ventral part of DLA differentially projected to the visual cortex ipsilaterally and contralaterally, respectively. These regions showed anatomical asymmetry that was inverted by the light. An antibody against a nicotinic acetylcholine receptor subunit more intensively and widely stained the side of DLA receiving the light stimulation and the cell labeled by the tracers co-localized with the immunoreactive neuropil. These results indicated that the light experience induced the anatomical lateralization of thalamofugal visual pathway.

Altered function of the ubiquitin pathway has been implicated in the etiology of neurodegeneration. For example, gracile axonal dystrophy (gad) mutant mice, which harbor a deletion within the gene encoding ubiquitin C-terminal hydrolase L1 (Uch-L1), display sensory ataxia followed by posterior paralysis and lethality. We previously showed that mice homozygous for a targeted deletion of the related Uch-L3 gene are indistinguishable from wild-type. To assess whether the two hydrolases have redundant function, we generated mice homozygous for both Uch-L1gad and Uch-L3Delta3-7. The double homozygotes weigh 30% less than single homozygotes and display an earlier onset of lethality, possibly due to dysphagia, a progressive loss in the ability to swallow food. This is consistent with histological analysis that revealed axonal degeneration of the nucleus tractus solitarius (NTS) and area postrema (AP) of the medulla. The NTS is essential for central nervous system control of swallowing. The double homozygotes also display a more severe axonal degeneration of the gracile tract of the medulla and spinal cord than had been observed in Uch-L1gad single homozygotes. In addition, degeneration of dorsal root ganglia cell bodies was detected in both the double homozygotes and Uch-L3Delta3-7 single homozygotes. Given that both Uch-L1gad and Uch-L3Delta3-7 single homozygotes display distinct degenerative defects that are exacerbated in the double homozygotes, we conclude that Uch-L1 and Uch-L3 have both separate and overlapping functions in the maintenance of neurons of the gracile tract, NTS and AP. This study is the first to successfully document dysphagia in the mouse and is a potentially valuable resource for understanding human neurodegenerative disorders that cause swallowing defects.

A new neurological mutant has been found in the inbred F344 strain of rats. The mutation is inherited as an autosomal recessive trait and is manifest clinically by a hesitant and wobbling gait with asynergic limbs and slight tremor. These symptoms begin at 16–18 days of age and remain essentially constant thereafter. Histologic examination revealed severe degeneration of the Purkinje cells and symmetrical calcification in these and in their dendritic branches in the cerebellar cortex. Such calcified Purkinje cells were intensely stained with the periodic acid-Schiff (PAS) method. PAS-positive substances in the Purkinje cells and extending diffusely over the lesioned sites in the molecular layer were also evident before calcification took place. We have named this neurological mutant the Cerebellar Calcification (CC) rat with the gene symbol cc. This offers a new animal model for the study of the Purkinje cell degeneration and intracranial calcification.

We show the efficient transduction and expression of the lacZ gene in the skeletal muscle of adult C57BL/10ScSn mice after adenovirus-mediated gene transfer. Of the myofibers in the tibialis anterior muscle 62% were beta-galactosidase positive after injection of the lacZ gene under the control of the chicken beta-actin promoter and the cytomegalovirus enhancer. The transduced gene was preferentially expressed in type IIA and IIX fibers, which were richer in oxidative enzymes than type IIB fibers.

Acid maltase deficiency (AMD) causes a lysosomal glycogenosis inherited as an autosomal recessive trait. The infantile type of AMD (Pompe disease) leads to early death due to severe dysfunction of cardiac and respiratory muscles and no effective therapy is available. Replication-defective adenovirus vectors offer a promising tool for in vivo gene delivery and gene therapy. We constructed a recombinant adenovirus containing the human acid maltase (AM) cDNA downstream of the CAG promoter, composed of modified chicken beta-actin promoter and CMV IE enhancer (AxCANAM). Japanese quail with AMD was used for this study as an animal model for human AMD. When cultured fibroblasts from AMD quail were infected with AxCANAM, AM activity in the cells increased in proportion to the multiplicity of infection (MOI). When AxCANAM (4.5 x 10(8) PFU) was injected into unilateral superficial pectoral muscle of AMD quail, PAS staining showed that glycogenosomes disappeared and stainability of acid phosphatase was reduced in the injected area as compared with the contralateral muscle of the same birds. Biochemically, AM activity increased and glycogen content decreased in the injected muscle. Western blot analysis showed that AMD quail muscle injected with AxCANAM expressed human AM protein processed to active forms. These results suggest that the human AM cDNA transferred by an adenovirus vector was sufficiently expressed, leading to a marked reduction of the glycogen accumulation in the skeletal muscle of AMD quail.

Extracellular levels of the excitatory neurotransmitter glutamate in the nervous system are maintained by transporters that actively remove glutamate from the extracellular space. Homozygous mice deficient in GLT-1, a widely distributed astrocytic glutamate transporter, show lethal spontaneous seizures and increased susceptibility to acute cortical injury. These effects can be attributed to elevated levels of residual glutamate in the brains of these mice.

The GAD mouse is a spontaneous neurological mutant with axonal dystrophy in the gracile tract of the medulla oblongata and spinal cord. The immunoreactivity of amyloid precursor protein (APP-IR) and amyloid beta-protein (A beta P-IR) was examined in the gracile tract and the dorsal root ganglia of normal and GAD mice. The mice were studied at 4, 9, 18, and 32 weeks of age. These periods correspond clinically to the initial, progressive, critical, and terminal stages of the disease, respectively. The APP-IR in both axons and glial cells was already accentuated to a higher level as early as 4 weeks of age in the gracile nucleus of GAD mouse. Similarly there was increase in APP-IR of GAD mouse in the dorsal root ganglia. Almost all of the primary neurons in the dorsal root ganglia at the lumbar cord level of GAD mouse revealed stronger APP-IR than those of normal mouse throughout all stages. The cells showing immunoreactivity for amyloid beta-protein became positive in axons and glial cells in the gracile nucleus by approximately the 9th week, and followed by an increase of A beta P-IR in order of the cervical, thoracic and lumbar spinal cords. These results suggest that the initial feature in GAD mouse is an accumulation of amyloid precursor protein induced by axonal dystrophy which then leads to a deposition of amyloid beta-protein within the cytoplasm of both axons and glial cells in the gracile tract.

We have tested the fusogenic properties of cleaved and uncleaved spike (S) protein of mutine coronavirus (MCV) JHMV variant cl-2 by expressing the S protein by recombinant vaccinia viruses (RVVs). The amino acid sequence of the putative cleavage site of cl-2 S protein, Arg-Arg-Ala-Arg-Arg, was replaced by Arg-Thr-Ala-Leu-Glu by in vitro mutagenesis of cl-2 S gene. The RVVs having cl-2 S gene [RVV t(+)] or mutated cl-2 S gene [RVV t(-)] were tested for their ability to induce fusion as well as cleavability in DBT cells. After inoculation with RVV t(+) onto DBT cells, the fusion formation was first observed at 8 h postinoculation (p.i.) and spread throughout the whole culture by 24 h. In cells infected with RVV t(-), fusion appeared by 2 h and most of cells were fused by 30 h p.i. The S protein and its cleavage products were detected in DBT cells expressing wild type S protein. However, no cleavage products of the S protein were detected in RVV t(-) infected cells producing mutated S protein, even though fusion was clearly visible. These results suggest that the cleavage event of JHMV-S protein of MCV is not a prerequisite for fusion formation, but that it enhances fusion.

By a highly sensitive enzyme immunoassay we measured the level of nerve growth factor (NGF) in the cerebellum and cerebrum of the neurologically mutant mice, weaver, reeler and Purkinje cell degeneration (PCD). A significant decrease in NGF level was observed in both cerebellum and cerebrum of weaver and reeler mutants of either sex. However, there was no such difference between normals and mutants in the case of the PCD mice. These results show that weaver and reeler mice have abnormalities of NGF synthesis and/or degradation not only in the cerebellum but also in the cerebrum.

Coronaviruses are enveloped RNA viruses with single positive stranded genomic RNA of approximately 32 kilobases which encodes at least 3 structural proteins; a nucleocapsid protein (N), a matrix glycoprotein (M) and a spike glycoprotein (S), as well as several nonstructural proteins (1). The S protein forms the projecting spikes or peplomers on the surface of the virus particle (2). This protein is considered to be involved in the attachment of virus to susceptible cells, the induction of cell-to-cell fusion, and the induction of neutralizing antibodies (3,4). It is also believed that the S protein is important for determining the pathogenic potential of murine coronaviruses (5,6). For more detailed structural and functional analyses of S protein, we need to have large quantities of S protein uncontaminated with other viral proteins. For this purpose, we have sought to express the S gene product in a baculovirus expression vector. The Autographa californica nuclear polyhedrosis virus (AcNPV) insect baculovirus vector was chosen because of the high levels of foreign gene expression obtained using the AcNPV polyhedrin promoter (7). Also, translational modifications such as glycosylation, phosphorylation, and cleavage of the signal peptide are known to occur in this system. In this paper, we describe the expression of the MHV strain JHM (JHMV) S protein by recombinant baculovirus in insect cells, characterization of the protein and analysis of epitopes existing on the S protein.

We have studied the effects of dietary vitamin E supplement on the clinical signs and pathological changes in GAD (gracile axonal dystrophy) mice. The control diet contained 2 mg of dl-alpha-tocopheryl acetate (2 I.U.) and vitamin E-supplemented diet contained 58.5 mg of dl-alpha-tocopheryl nicotinate (50 I.U.), per 100 mg of feed. The diet was given to normal (gad/+) and GAD (gad/gad) mice from 21 to 130 days of age. During the feeding, there was no improvement in clinical signs in the GAD mice fed the vitamin E-supplemented diet. The gracile nucleus of the medulla oblongata and the gracile fascicules of the spinal cord were investigated for pathology at 130 days of age, and alpha-tocopherol was also assayed in the serum, liver, brain and spinal cord at that time. There were no pathological differences in the gracile nucleus and fascicules between the GAD mice fed the control and vitamin E-supplemented diet. The alpha-tocopherol levels in the serum and target organs in the control GAD mice were not significantly different from those in control normal mice, showing that GAD mice could absorb and transport alpha-tocopherol. In the supplemented GAD mice, no significant increases in alpha-tocopherol levels were observed in the liver, brain or spinal cord. Particularly, the percentage increase of alpha-tocopherol level in the liver of GAD mice was very low in comparison with that in normal mice, even though the liver can store vitamin E. Thus it may be that the capacity to store vitamin E is lowered in GAD mice. Further studies are needed to investigate in detail the vitamin E metabolism in the mutant mice.

ゴニオメーターステージ(goniometer tilting stage)を電子顕微鏡に装着し,孵卵7,12日の鶏胚錯綜筋(M. complexus)および大腿二頭筋(M. biceps femoris)において,筋細胞の融合(fusion)に伴う,細胞膜や細胞質の微細構造学的変化を検討した.最初に形成される一次筋管と,それに隣接して新たに形成される二次筋管の境界には,複雑に入り組んだ両者の細胞膜がみられる.これらの細胞膜は,胚発生の初期には,部分的に接着帯結合を示したり,また,膜の一部が不明瞭となり,両細胞が細胞質橋で連結しているかのような所見を示す場合が多い.ゴニオメーターステージを用いて,膜構造を三次元的に解析した結果,境界部で相対する二枚の細胞膜の一部が認められなくなる原因は,接触する細胞膜の面が,斜めに薄切されたことにより,観察時に生ずる人工産物のためであり,一次と二次筋管の間では,側面融合は起らないことを確かめることができた.筋細胞の融合像は,主に,筋芽細胞と一次および二次筋管の末端での接触面で観察された.筋芽細胞が,両側で筋管と接している場合は,両側の境界部で,同時に融合が始まっていた.細胞融合は,細胞膜の部分的崩壊や消失で始まり,次いで,境界部へ不規則に配列した筋フィラメントの集合や新生が起る.構造変化を起した細胞膜は,次いで,小型の小胞や細管へと,二次的な膜構造の変化を示すことが暗示された.錯綜筋と大腿二頭筋の間で,細胞融合の様式を比較したが,特に差はみられなかった.

Investigations by using animal models offer a useful tool to study the pathogenesis and cure of several incurable human diseases. The laboratory animal science in this field was established about 20 years ago in Japan. This paper describes the outline of its history and the characteristics of each animal model which has been studied extensively and will also be studied in the future in Japan. Some results on dystrophic chicken and gracile axonal dystrophic (gad) mouse which were recently obtained in our laboratory are discussed.

鶏胚錯綜筋(M. complexus)および大腿二頭筋(M. biceps femoris)において,胚発生に伴 う骨格筋線維の形態分化とその増殖機構を検討した.孵卵7日から14日にかけて形成される筋管(myotube)は,最初に形成される大型の一次筋管と,それに接するか,または周辺へ分離して新たに形成 される二次筋管に分けられ,これらの細胞増殖に2相性があることが明らかとなった.筋を構成する筋 線維の数は,これら筋管の胚発生中の増殖によって増加する.筋横断面に含まれる筋線維数の変化を, 孵卵10日から20日にかけて両筋で比較すると,両筋とも孵卵16日でほぼ筋線維数は最大値に達してい た.しかし,孵卵14日から16日にかけての筋線維数の増加率を比較すると,錯綜筋の増加率は,大腿 二頭筋に比べ,著しく低く,筋線維数は孵卵14日で,すでに最大値に近い値を示した.このような筋線 維総数の変化は,その筋組織内に含まれる筋線維の束,特に,その最も低次の単位である一次筋束内の 筋線維数の変化と一致していた.この筋束内に見られる一次筋管の数は,発生日齢を通じてほぼ一定で あり,しかも両筋の間でも,大きな差が認められない.しかし,一次筋管の細胞内小器管や筋原線維の 形態分化は,錯綜筋で著しく進んでいた.一次筋管の細胞径を両筋の間で比較すると,筋管の数の増加 が最も著しい孵卵10日から13日にかけて,差が認められなかった.一方,孵卵12日から14日にかけ て,二次筋管の総胞径を両筋の間で比較すると,錯綜筋の二次筋管は,大腿二頭筋に比べ,形態分化や 細胞径の増大が顕著であった.錯綜筋で見られる,このような二次筋管の著しい発達は,孵卵16日以後 において,筋線維数が増加しにくい現象と深いかかわりがあった.本報告では,胚発生過程の筋組織に おいて,筋線維の数の増加を制御する要因や,その機構を,二次筋管の発達程度と関連して考察した.