Kinectin, a major kinesin-binding protein on ER.

Previous studies have shown that microtubule-based organelle transport requires a membrane receptor but no kinesin-binding membrane proteins have been isolated. Chick embryo brain microsomes have kinesin bound to their surface, and after detergent solubilization, a matrix with an antibody to the kinesin head domain (SUK-4) (Ingold et al., 1988) bound the solubilized kinesin and retained an equal amount of a microsome protein of 160-kD. Similarly, velocity sedimentation of solubilized membranes showed that kinesin and the 160-kD polypeptide cosedimented at 13S. After alkaline treatment to remove kinesin from the microsomes, the same 160-kD polypeptide doublet bound to a kinesin affinity resin and not to other proteins tested. Biochemical characterization localized this protein to the cytoplasmic face of brain microsomes and indicated that it was an integral membrane protein since it was resistant to alkaline washing. mAbs raised to chick 160-kD protein demonstrated that it was absent in the supernatant and concentrated in the dense microsome fraction. The dense microsome fraction also had the greatest amount of microtubule-dependent motility. With immunofluorescence, the antibodies labeled the ER in chick embryo fibroblasts (similar to the pattern of bound kinesin staining in the same cells) (Hollenbeck, P. J. 1989. J. Cell Biol. 108:2335-2342), astroglia, Schwann cells and dorsal root ganglion cells but staining was much less in the Golgi regions of these cells. Because this protein is a major kinesin-binding protein of motile vesicles and would be expected to bind kinesin to the organelle membrane, we have chosen the name, kinectin, for this protein.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS): novel compound heterozygous mutations in the SACS gene.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disorder first described among French Canadians in Quebec. To date, 24 mutations have been reported in the SACS gene of ARSACS patients. The authors report a clinical and genetic analysis of a Japanese family with ARSACS with novel compound heterozygous mutations in the SACS gene (N161fsX175, L802P). The phenotype is similar to that of previously reported ARSACS patients.

Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis.

Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.

Antibodies to brain proteins in paraneoplastic cerebellar degeneration.

A 68-year-old man had subacute cerebellar degeneration and a non-Hodgkin's lymphoma. Using an immunoblotting method, we found serum antibodies to rat cerebral 250-kd and 110-kd and cerebellar 110-kd acidic cytoplasmic proteins. The antibodies did not react unless the antigens were prepared soon after death with protease inhibitors. Two hundred fifty-kd and 110-kd proteins are minor components of soluble cytoplasmic proteins of the brain. The molecular weights differed from other soluble brain-specific proteins already characterized.

A novel de novo mutation in the desmin gene causes desmin myopathy with toxic aggregates.

OBJECTIVE: To determine the cause and pathogenic mechanisms of a 21-year-old patient's cardioskeletal myopathy. The patient's muscle atrophy and weakness began in distal parts of limbs; cardiac and facial muscles were later involved. BACKGROUND: Desmin myopathy is a skeletal myopathy often associated with cardiomyopathy, caused by mutations in the desmin gene and characterized by desmin accumulation in affected muscle fibers, a leading marker of myofibrillar myopathies. Two kinds of deletions and seven missense mutations in the desmin gene have been identified. METHODS: Clinical examination, electron microscopy of muscle tissue, two-dimensional gel electrophoresis, DNA sequencing, restriction enzyme analysis, and gene transfection were performed. RESULTS: Electron microscopy showed disruption of sarcomeres at Z discs and electron-dense aggregates in biopsied skeletal and heart muscle. Two-dimensional gel electrophoresis of the patient's skeletal muscle proteins showed massive accumulation of desmin. The authors identified a novel desmin mutation, L385P in one allele in the carboxyl end of the rod domain 2B in the patient's leukocytes and skeletal muscle; neither parent had the mutation. Serologic study and DNA markers confirmed the de novo mutation. A peptide harboring desmin rod domains 2A and 2B with L385P tagged with green fluorescent protein induced cytoplasmic aggregates, nuclear DNA condensation, and cell death. CONCLUSIONS: A novel de novo mutation, L385P, causes desmin myopathy. An expression study indicated the toxic effect of the L385P mutation.

Elemental diet modulates the growth of Clostridium difficile in the gut flora.

BACKGROUND AND AIMS: Tube feeding is regarded as a risk factor for Clostridium difficile-associated diarrhoea. Recently, we reported that C. difficile toxin was frequently found in patients receiving an elemental diet. The present study was conducted to clarify whether elemental diets are associated with the growth of C. difficile in the gut flora. METHODS: C. difficile was cultured for 72 h in various concentrations of elemental diet containing 3% thioglycollate, and the growth rate or activity of C. difficile was evaluated by Gram stain or by measuring optical density at 560 nm. Faecal samples from 10 healthy adults were cultured in elemental diet + 3% thioglycollate. RNA was extracted from faeces with glass powder, which can eliminate PCR inhibitors, and mRNA of C. difficile toxin B was measured by reverse transcription PCR. RESULTS: Maximum OD560 value during culture in thioglycollate-containing elemental diet was 2.4 times higher than that in thioglycollate alone (P = 0.0163). Viability of C. difficile was decreased in thioglycollate but not in thioglycollate-containing elemental diet. Toxin B mRNA was detected in five faecal samples (50%) before culture and in all samples after culture. CONCLUSIONS: Our results suggest that an elemental diet can modulate the growth of C. difficile in the gut flora.

Detection and isolation of a 30 kDa abnormal protein in avian dystrophic muscle.

We have studied the protein composition of the pectoralis superficialis muscle of genetically dystrophic (New Hampshire line 413) and normal control (line 412) chickens by one- and two-dimensional gel electrophoresis. A protein, referred to hereafter as the 30 kDa abnormal protein, was specifically detected in the affected muscle. It was purified to homogeneity, and its molecular properties were studied. It is a monomer with a molecular mass of approximately 30 kDa and an isoelectric point of about pI 8.4. We have screened by Western blotting a variety of muscles from line 412 and line 413 chickens for the presence of the 30 kDa protein. While the pattern of total protein is very similar in all cases, the 30 kDa protein was not detected in the pectoralis superficialis muscle of line 412 chickens. However, the immunoreactive bands were detected in the sartorius muscle and the tensor fasciae latae muscle from dystrophic and normal chickens. Interestingly, the immunoreactive bands of normal skeletal muscles are smaller in molecular weight than those of dystrophic skeletal muscles. To determine the early time sequence of the appearance of the abnormal protein, we studied muscles from embryos and post-hatched chickens at various ages. The abnormal protein was detected in dystrophic muscles as early as 15 days ex ovo and occurred throughout development up to six months ex ovo. Although the implication of the dystrophy-associated appearance of the 30 kDa protein in the affected muscle is not clear at present, it would be of particular interest to elucidate the biochemical functions of the 30 kDa protein in the affected muscle (pectoralis superficialis muscle) of genetically dystrophic chicken.

Difference in phosphorylation of neurofilament proteins in motor neurons and spinal ganglion cells.

The composition of the neurofilament proteins (NFPs) in neuronal perikarya was examined by two-dimensional (2-D) gel electrophoresis of isolated perikarya of bovine spinal motor neurons. The extent of phosphorylation of the high molecular weight subunit of NFP (NFP-H) was compared between motor and sensory neuronal perikarya in spinal cord and spinal ganglion by immunocytochemistry with monoclonal antibodies (MAbs) to NFP. Of the 23 MAbs used in this study, one MAb (82E10) was specific to the highly phosphorylated component of NFP-H examined by 2-D immunoblot whereas another MAb (3A8) was specific to NFP-H irrespective of its level of phosphorylation. Immunocytochemically, 82E10 did not stain the perikarya of bovine and rabbit spinal motor neurons but 3A8 stained the perikarya in both animal species. These findings are consistent with 2-D immunoblot of neuronal perikarya of bovine motor neurons isolated in bulk. As for the spinal ganglia, 82E10 stained many, but not all, perikarya of sensory neurons of both animal species. These results indicate that the extent of phosphorylation of NFP-H in the perikarya of most spinal ganglion cells is higher than that of motor neurons. These findings suggest that the rate of phosphorylation of NFP-H in perikarya or the axonal transport of NFP from perikarya to proximal axons is uniform in spinal motor neurons but variable in spinal ganglion cells.

Suppressive effect of E-64c on ischemic degradation of cerebral proteins following occlusion of the middle cerebral artery in rats.

Microtubule-associated protein 2 (MAP2) levels in the left cerebral hemisphere decreased significantly 3 days after occlusion of the left middle cerebral artery in rats to 29 +/- 16.3% of control levels. Since MAP2 is one of the substrates of calpain, E-64c, a synthetic calpain inhibitor, was administered at a dose of 400 mg/kg twice a day for 3 days, with the first dose being given before the production of ischemia. This depletion was significantly inhibited in vivo by E-64c (P less than 0.05) to increase MAP2 levels to 55 +/- 25.7% of control levels. E-64c had no significant effect on the ischemia-induced depletion of myelin-associated glycoprotein. Sham-operated rats were used as controls. Our results suggest that calpain is partially involved in the degradation of MAP2, and that the use of calpain inhibitors can be a useful clinical approach to cerebral ischemia.

Phosphorylation and transport of neurofilament proteins in the rat spinal ganglion.

Neurofilament proteins (NFs) in rat spinal ganglia were labeled with [32P]orthophosphate injected into ganglia and analyzed by two-dimensional autoradiography and immunoblotting. Three polypeptides of NF were labeled irrespective of the extent of phosphorylation. Most of the labeled NFs were transported from cell bodies to proximal axons within 24 h. A major fraction of low phosphorylated NF-H changed to high phosphorylated form in intraganglionic nerve fibers and peripheral nerves adjacent to spinal ganglia. A small fraction of low phosphorylated NF-H appeared earlier than the high phosphorylated form in adjacent peripheral nerves, suggesting that newly synthesized NF-H in cell bodies migrate a long distance before they are extensively phosphorylated and assembled into the cytoskeleton in proximal axons.

Kinectin distribution in chicken nervous system.

Kinectin, a major kinesin receptor on endoplasmic reticulum, was visualized with anti-kinectin monoclonal antibodies (mAbs) in the adult chicken nervous system in comparison with kinesin immunostaining. Anti-kinectin mAbs punctately stained cell bodies and proximal dendrites of motor neurons in spinal cords. Axons of motor neurons were not stained with anti-kinectin mAbs, but stained heavily with anti-kinesin mAbs. This suggest that the kinesin receptor responsible for kinesin-driven anterograde fast axonal transport is different from kinectin. Anti-kinectin mAbs strongly stained neuronal cell bodies in spinal ganglion, nuclei in brainstem, cerebellar nuclei, striatum and cerebral cortex. Small neurons in cerebellar cortex and optic lobe showed relatively weak reaction, suggesting that the amount of kinectin correlates with the size of neuronal cell bodies.

Kinesin accumulation in chick spinal axonal swellings with beta,beta'-iminodipropionitrile (IDPN) intoxication.

Kinesin is a major molecular motor responsible for anterograde axonal transport. Chicks were injected with beta,beta'-iminodipropionitrile (IDPN) to induce axonal swellings in spinal motor neurons and spinal sensory ganglion neurons. Cylindrical swollen axons were found in the anterior horn and anterior funiculus of the spinal cord, anterior root, and spinal ganglia. All of the axonal swellings were heavily stained with two anti-kinesin monoclonal antibodies. The swellings were mildly stained with an anti-cytoplasmic dynein and anti-tubulin antibodies, and weakly stained with an anti-tau antibody. These suggest the isolated disturbance of kinesin transport with neurofilament accumulation in IDPN intoxication.

Massive accumulation of M and H subunits of neurofilament proteins in spinal motor neurons of neurofilament deficient Japanese quail, Quv.

Quiver (Quv) is a non-sense mutation of neurofilament protein L subunit (NF-L) that causes neurofilament deficiency with preserved microtubules in Japanese quail. Anti-NF-M and anti-NF-H mAbs stained cell bodies of motor neurons in Quv embryo spinal cords much more intense than those in control spinal cords. Volume of motor neurons in Quv spinal cords increased to 2.3 times of control motor neurons. Immunoblot of Quv spinal cords revealed a relative increase in non- and hypo-phosphorylated NF-M and NF-H, and a decrease in the total amount of NFs. Quv sciatic nerves showed faintly reacted phosphorylated NF-M and NF-H. These results suggest that deficiency of assembled neurofilament results in decreased axonal transport of NFs and accumulation of NFs in cell bodies of spinal motor neurons.

Detection of heparan sulfate in spinal spheroids of beta, beta'-iminodipropionitrile (IDPN)-treated rats.

Beta,beta'-Iminodipropionitrile (IDPN) is known to produce a massive accumulation of neurofilaments in the proximal portion of axons of spinal anterior horn cells. The spinal cords of 20 Wistar rats treated with IDPN were examined immunohistochemically with a monoclonal antibody against heparan sulfate (HepSS-1). Virtually all axonal swellings were intensely labeled with HepSS-1. This immunoreactivity was almost completely absorbed by the presence of CDSNS-HS (completely desulfated, N-sulfated heparan sulfate). Western blot study revealed that HepSS-1 recognized four distinct bands at the positions of approximately 17 kDa, approximately 20 kDa, approximately 21 kDa, and approximately 25 kDa. The present study suggests that the deposit of heparan sulfate in spheroids is related to the pathomechanism for the formation of axonal swelling.

Phosphorylation of neurofilament proteins and localization of axonal swellings in motor neuron disease.

Seven lumbosacral spinal cords with motor neuron disease were examined immunocytochemically with monoclonal antibodies (MAb) directed against neurofilament proteins (NFP). Each of the 5 MAbs used in this study was monospecific to one of the triplet of NFP. Two of them were specific to the highly phosphorylated form of high molecular weight peptide of NFP (NFP-H). All 5 MAbs stained all axonal swellings examined. In 2 spinal cords examined, some axonal swellings were found in the anterolateral funiculus and some of these were as far as 1000 microns from the grey matter. This localization of axonal swellings suggests that a high degree of phosphorylation of NFP is not the cause of accumulation of NFP in axonal swellings in motor neuron disease.

Kinesin and cytoplasmic dynein in spinal spheroids with motor neuron disease.

Kinesin and cytoplasmic dynein are two major molecular motors responsible for fast axonal transport. As visualized by immunohistochemistry with monoclonal antibodies, both motors were found to be distributed throughout the cell bodies, dendrites and axons of motor neurons in normal human spinal cords. Large axonal swellings, spheroids, in the spinal cords of patients with motor neuron disease showed massive accumulation of kinesin co-localized with highly phosphorylated neurofilaments. Of 114 spheroids in five spinal cords, 87% were stained heavily with the three anti-kinesin antibodies used in this study. Cytoplasmic dynein was scarce or absent in most of the spheroids. These findings suggest that kinesin selectively accumulates in the spheroids of motor neuron axons, causing disturbance of the machinery for anterograde fast axonal transport in motor neuron disease.

Fulminant hepatitis caused by hepatitis C virus during treatment for multiple sclerosis.

A 55-year-old woman was treated at our hospital for multiple sclerosis. Therapy consisted of glucocorticosteroids and cyclosporin. In the 7th week after these drugs were discontinued the patient developed acute liver failure due to fulminant hepatitis (FH) and died. Post-mortem examination showed massive liver necrosis. Serologic examination was negative for hepatitis B virus-related markers. Antihepatitis C virus (anti-HCV) antibody and serum HCV RNA were negative on admission, but HCV RNA appeared concurrently with the onset of FH. Although HCV infection rarely causes FH, it was considered to be the cause of FH in this patient, since there were no other causes of acute liver injury. We suspect that underlying immunologic abnormalities in conjunction with HCV infection may have precipitated the FH.

Pilot study of ofloxacin and interferon-alpha combination therapy for chronic hepatitis C without sustained response to initial interferon administration.

A controlled trial comparing combination therapy with ofloxacin (OFLX) and interferon (IFN) versus IFN monotherapy was conducted in patients with chronic hepatitis C who failed IFN therapy. Twenty patients were assigned randomly to two groups. Equal doses of recombinant IFN alpha-2b were administered to each group for 24 weeks. For the IFN plus OFLX group, OFLX was administered for 12 weeks at a daily dose of 600 mg. Levels of hepatitis C virus RNA declined significantly from the first month after the start of IFN treatment compared with those before administration in both groups. Serum alanine aminotransferase levels were significantly lower in the IFN plus OFLX group at two and six months after the start of treatment than levels in the IFN group. The fraction of subjects whose levels of serum ALT normalized was also higher in the IFN plus OFLX group. Larger clinical trials should be undertaken.

A multicentre randomized controlled trial of recombinant interferon-alpha-2a in the treatment of patients with chronic hepatitis C.

Sixty-one chronic hepatitis C patients were randomly assigned to receive either 6 x 10(6) or 9 x 10(6) U of recombinant interferon-alpha-2a (IFN alpha-2a) six days a week for the first two weeks of treatment, followed in both cases by 6 x 10(6) U three days a week for the next 22 weeks. In the low dose group, 11 patients showed a complete response maintained for at least six months, 12 responded but then relapsed and nine did not respond; the corresponding figures in the high dose group were 10, 15 and five patients, respectively. The differences between groups are not statistically significant. Thus, this study provides no evidence of therapeutic benefit from increasing the initial dose of IFN alpha-2a. In both treatment groups, complete responders had significantly lower pretreatment viral titres than nonresponders and were significantly more likely to be infected by type 2a versus type 1b virus.

Hereditary lipo-muscular atrophy with joint contracture, skin eruptions and hyper-gamma-globulinemia: a new syndrome.

We previously reported two siblings with decreased subcutaneous adipose tissue, muscular atrophy, joint contractures, recurrent skin eruptions, hyper-gamma-globulinemia, and reduced natural killer cell activity. Some of their clinical features are similar to those of partial lipodystrophy, but they are distinct in that muscular atrophy, joint contractures and recurrent skin eruptions are not found in patients with partial lipodystrophy. Thirteen other Japanese patients with similar clinical manifestations have been reported. We propose that such cases should be considered a distinct clinical entity.