Biomedical subjects

E Fujimoto

Publications and source records attributed to E Fujimoto.

At least 19 recordsLinked to original sources

PubMed2001

A novel mechanism associated with idiopathic ventricular fibrillation (IVF) mutations R1232W and T1620M in human cardiac sodium channels.

Two mutations associated with idiopathic ventricular fibrillation (IVF) are localized within extracellular loops between segments DIIIS1-S2 (R1232W) and DIVS3-S4 (T1620M) of the human cardiac sodium channel (hNav1.5) alpha-subunit. We studied wild-type hNav1.5 channels and hNav1.5 channels with the R1232W/T1620M double mutation expressed in Xenopus oocytes using the cell-attached macropatch technique. We demonstrate that these mutations destabilize the fast-inactivated state (described with a two-state first-order reaction model) by decreasing reaction valence, accelerating recovery, and slowing the onset of fast inactivation, collectively resulting in delayed decay of macroscopic currents. R1232W/T1620M mutations in hNav1.5 channels also significantly increase steady-state channel availability, indicating that mutated channels occupy the slow inactivated state less than hNav1.5 channels. Under the stress of repetitive depolarizing pulses, R1232W/T1620M channels demonstrate less use-dependent current reduction compared to wild-type channels. We propose that increased channel availability coupled with destabilized fast inactivation contributes to the pathological effect of R1232W/T1620M mutations, and leads to increased excitability of cardiac tissue in vivo.

Animals↗

PubMed2001

A single residue differentiates between human cardiac and skeletal muscle Na+ channel slow inactivation.

Slow inactivation determines the availability of voltage-gated sodium channels during prolonged depolarization. Slow inactivation in hNa(V)1.4 channels occurs with a higher probability than hNa(V)1.5 sodium channels; however, the precise molecular mechanism for this difference remains unclear. Using the macropatch technique we show that the DII S5-S6 p-region uniquely confers the probability of slow inactivation from parental hNa(V)1.5 and hNa(V)1.4 channels into chimerical constructs expressed in Xenopus oocytes. Site-directed mutagenesis was used to test whether a specific region within DII S5-S6 controls the probability of slow inactivation. We found that substituting V754 in hNa(V)1.4 with isoleucine from the corresponding position (891) in hNa(V)1.5 produced steady-state slow inactivation statistically indistinguishable from that in wild-type hNa(V)1.5 channels, whereas other mutations have little or no effect on slow inactivation. This result indicates that residues V754 in hNa(V)1.4 and I891in hNa(V)1.5 are unique in determining the probability of slow inactivation characteristic of these isoforms. Exchanging S5-S6 linkers between hNa(V)1.4 and hNa(V)1.5 channels had no consistent effect on the voltage-dependent slow time inactivation constants [tau(V)]. This suggests that the molecular structures regulating rates of entry into and exit from the slow inactivated state are different from those controlling the steady-state probability and reside outside the p-regions.

Amino Acid Sequence↗

PubMed2000

Pretibial cyst formation after anterior cruciate ligament reconstruction using auto hamstring grafts: two case reports in a prospective study of 89 cases.

Eighty-nine cases after anterior cruciate ligaments (ACL) reconstruction were followed prospectively with magnetic resonance imaging (MRI). The patients were examined using axial and sagittal MRI at least twice during the postoperative evaluation of reconstructed ACL. Two cases of pretibial cyst formation were observed. At the time of cyst formation, neither patient had any subjective or objective evidence of knee instability. The cyst of one case communicated with the intra-articular. The minimum follow-up period after the surgical excision was 9 months, with no evidence of recurrence. We might speculate that the critical period for cyst formation in both patients occurred at less than 12 months after their ACL reconstruction. We concluded that the cyst formation was most likely due to incomplete graft tendon incorporation within the osseous tunnel.

Adolescent↗

PubMed2000

The delay in recovery from fast inactivation in skeletal muscle sodium channels is deactivation.

1. Using macropatch techniques, we tested the assumption that deactivation underlies the observed delay in the onset to recovery from fast inactivation by comparing open-state deactivation to recovery delay for rat skeletal muscle mutations R1441C and R1441P. 2. Deactivation kinetics from the open state were determined from the exponential decay of tail currents. R1441C and R1441P prolonged open-state deactivation, with the greatest effect produced by R1441P. 3. Delays in the onset to recovery from fast inactivation for R1441P and for R1441C were abbreviated compared to those for rSkM1. Recovery delay was longer in R1441P than R1441C at voltages more negative than -110 mV. Recovery from inactivation exhibited a voltage dependence which, unlike delay, saturated at depolarized voltages. Recovery rate constants were increased to a similar extent for R1441C and R1441P at -150 to -120 mV compared to rSkM1. 4. These results indicate that the delay in the onset to recovery from fast inactivation in skeletal muscle sodium channels is due to deactivation. Lessening of charge immobilization for R1441C and R1441P may contribute to observed biophysical defects underlying the hyperexcitability of muscle fibers containing paramyotonia congenita mutations. The second stage of recovery from fast inactivation may be affected differentially by these mutations.

Animals↗

PubMed1999

Differential effects of homologous S4 mutations in human skeletal muscle sodium channels on deactivation gating from open and inactivated states.

1. The outermost charged amino acid of S4 segments in the alpha subunit of human skeletal muscle sodium channels was mutated to cysteine in domains I (R219C), II (R669C), III (K1126C), and IV (R1448C). Double mutations in DIS4 and DIVS4 (R219C/R1448C), DIIS4 and DIVS4 (R669C/R1448C), and DIIIS4 and DIVS4 (K1126C/R1448C) were introduced in other constructs. Macropatch recordings of mutant and wild-type (hSkM1-wt) skeletal muscle sodium channels expressed in Xenopus oocytes were used to measure deactivation kinetics from open or fast inactivated states. 2. Conductance (voltage) curves (G (V)) derived from current (voltage) (I (V)) relations indicated a right-shifted G (V) relationship for R669C and for R669C/R1448C, but not for other mutations. The apparent valency was decreased for all mutations. Time-to-peak activation at -20 mV was increased for R1448C and for double mutations. 3. Deactivation kinetics from the open state were determined from the monoexponential decay of tail currents. Outermost charge-to-cysteine mutations in the S4 segments of domains III and IV slowed deactivation, with the greatest effect produced by R1448C. The deactivation rate constant was slowed to a greater extent for the DIII/DIV double mutation than that calculated from additive effects of single mutations in each of these two domains. Mutation in DIIS4 accelerated deactivation from the open state, whereas mutation in DIS4 had little effect. 4. Delays in the onset to recovery from fast inactivation were determined to assess deactivation kinetics from the inactivated state. Delay times for R219C and R669C were not significantly different from those for hSkM1-wt. Recovery delay was increased for K1126C, and was accelerated for R1448C. 5. Homologous charge mutations of S4 segments produced domain-specific effects on deactivation gating from the open and from the fast inactivated state. These results are consistent with the hypothesis that translocations of S4 segments in each domain during deactivation are not identical and independent processes. Non-identical effects of these mutations raise several possibilities regarding deactivation gating; translocation of DIVS4 may constitute the rate-limiting step in deactivation from the open state, DIVS4 may be part of the immobilizable charge, and S4 translocations underlying deactivation in human skeletal muscle sodium channel may exhibit co-operativity.

Algorithms↗

PubMed1999

Characterization of a novel Ras-binding protein Ce-FLI-1 comprising leucine-rich repeats and gelsolin-like domains.

Ras proteins are conserved from yeasts to mammals and implicated in regulation of the actin cytoskeleton. The flightless-1 (fli-1) gene of Drosophila melanogaster and its homologs in Caenorhabditis elegans and humans encode proteins (FLI-1) comprising a fusion of a leucine-rich repeats (LRRs) domain and a gelsolin-like domain. This LRRs domain is highly homologous to those of three proteins involved in Ras-mediated signaling; Saccharomyces cerevisiae adenylyl cyclase, C. elegans SUR-8, and mammalian RSP-1. Here we report that the LRRs domain of C. elegans FLI-1 (Ce-FLI-1) associates directly with Ras (Kd = 11 nM) and, when overexpressed, suppresses the heat shock sensitive phenotype of yeast cells bearing the activated RAS2 gene (RAS2(Val-19)). Further, the gelsolin-like domain of Ce-FLI-1 is shown to possess a Ca2+-independent G-actin-binding activity as well as F-actin-binding and -severing activities. FLI-1 may be involved in regulation of the actin cytoskeleton through Ras.

Actins↗

PubMed1999

Beneficial effect of basic fibroblast growth factor on the repair of full-thickness defects in rabbit articular cartilage.

The effects of exogenous basic fibroblast growth factor (bFGF) on the repair of full-thickness cartilage defects were examined. Four-millimeter diameter, cylindrical defects were made in rabbit articular cartilage and were filled with human recombinant bFGF. The addition of bFGF to the defect induced the formation of a thick cartilage layer composed of chondrocytes and a metachromatic-stained matrix after 6 weeks. The score of the bFGF-treated tissue, as evaluated by a semiquantitative histological scale, was significantly higher than that of the untreated tissue. At 24 weeks, the cartilage-like matrix that contained the proteoglycans and type II collagen was thicker in the bFGF-treated tissue than in the untreated tissue. Immunohistochemical analysis of the tissues at 6-12 weeks with an anti-bFGF monoclonal antibody suggested that a single application of bFGF increased the number of differentiating chondrocytes that synthesized bFGF at a high level. In contrast, immunostaining of the tissues at 6-12 weeks with a monoclonal antibody against proliferating cell nuclear antigen showed that the number of proliferating cells in the bFGF-treated tissue was fewer than in the untreated tissue. These findings suggest that administration of bFGF into cartilagenous defects promotes the differentiation of chondrocytes and their matrix synthesis, and that this growth factor is useful for improving cartilage repair.

Animals↗

PubMed1999

Appearance of anterior cruciate ligament autografts in their tibial bone tunnels on oblique axial MRI.

The objective of this study was to observe the changing appearance of human anterior cruciate ligament (ACL) grafts in their tibial bone tunnels by MRI using oblique axial images. One-hundred and eight knees in 75 patients were studied by MRI at 1-33 months after arthroscopic ACL reconstructions using double-looped, autogenous semitendinosus and/or gracilis tendons. Knees with poor stability were excluded from this study. The examinations were performed at 0.2T with spin echo proton density and T2-weighted oblique axial images. Appearances of grafts were mainly described on spin echo proton density images based upon time after surgery. The grafts appeared as homogeneous, low signal intensity areas in the bone tunnels at 1 month after the surgery. Ring-shaped low signal intensity areas were observed along the wall of the bone tunnels in the 2- to 3-month group. In many grafts from this group, each tendinous bundle appeared as a low signal area separated by a high signal intensity area. In all cases in the 4- to 6-month group, the thickness of the ring-shaped low signal intensity area had increased, whereas the thickness of the high signal intensity area had decreased. In almost all of the cases, the interior of the bone tunnel gradually became a homologous low signal intensity region by 7 to 12 months after the surgery. According to these results, it is suggested that the maturation of the tendon-bone interface was completed from 6 to 12 months after the ACL reconstruction.

Adolescent↗

PubMed1999

Voltage sensors in domains III and IV, but not I and II, are immobilized by Na+ channel fast inactivation.

Using site-directed fluorescent labeling, we examined conformational changes in the S4 segment of each domain of the human skeletal muscle sodium channel (hSkM1). The fluorescence signals from S4 segments in domains I and II follow activation and are unaffected as fast inactivation settles. In contrast, the fluorescence signals from S4 segments in domains III and IV show kinetic components during activation and deactivation that correlate with fast inactivation and charge immobilization. These results indicate that in hSkM1, the S4 segments in domains III and IV are responsible for voltage-sensitive conformational changes linked to fast inactivation and are immobilized by fast inactivation, while the S4 segments in domains I and II are unaffected by fast inactivation.

Animals↗

PubMed1999

Effect of cerebrospinal fluid shunting on experimental syringomyelia: magnetic resonance imaging and histological findings.

The histological changes associated with syringomyelia after reduction of the syrinx size were investigated after cerebrospinal fluid shunting in experimental syringomyelia in the rabbit. Five weeks after syringomyelia was induced by the injection of kaolin into the cisterna magna in Japanese white rabbits, ventriculosubgaleal shunting or syringoepidural shunting were performed. After 1 week magnetic resonance (MR) imaging and histological examination were then carried out. Five of 11 shunted animals showed postoperative reduction of syrinx size on MR imaging. Grossly, some specimens showed cavity collapse and parenchymal healing, and others showed a small residual syrinx in the dorsal horn. The most dramatic histological changes occurred in the gray matter. Specimens with syrinx collapse showed rarefaction and tearing of the gray matter, with mild glial reaction. The edematous gray matter showed both degeneration and regeneration, with neuronal processes surrounded by edema fluid. Reactive astrocytes were observed mainly at the margin of the residual syrinx. Some astrocytic processes invested the extraaxonal space and gray matter lacked supportive tissue. Greater reduction of the syrinx after shunting operation was correlated with more regeneration and less degeneration, and the white matter was edematous and histological changes were milder. Syrinx shrinkage occurred after shunting in this experimental model of syringomyelia. The selective vulnerability of gray matter even after shunting may explain discrepancies between imaging findings and clinical features in this disease. The study supports the potential benefit from early treatment, considering the associated morphological findings of regeneration.

Animals↗

PubMed1999

Evaluation of the internal thoracic arterial graft patency by the transthoracic Doppler method under continuous intravenous infusion of adenosine triphosphate disodium.

Usefulness of the Doppler method under continuous infusion of adenosine triphosphate disodium (ATP) for improvement of accuracy in the diagnosis of the left internal thoracic arterial graft (LITA) patency was examined using transthoracic ultrasonic echocardiography. 1) Influence of ATP on the Doppler velocity in a graft was examined in 7 patients with good LITA grafts using physiological saline as the control. In the ATP group, 80 mg of ATP was dissolved in 20 ml physiological saline and continuously infused at 0.14 mg/kg/min. In the saline group, an equal volume of physiological saline was administered and the blood flow velocity in the LITA was recorded continuously by the transthoracic Doppler method from the supraclavicular fossa approach. Results; ATP administration increased the blood flow velocity in the LITA and the rate of increase was 48.3% for systolic peak velocity, 111% for diastolic peak velocity, 64.4% for systolic time velocity integral and 99% for diastolic time velocity integral indicating particularly high rates of increase in diastolic components. The diastolic/systolic peak velocity ratio or diastolic fraction did not increase significantly. In the saline group, none of the parameters showed a change. 2) Angiographic findings of the LITA were compared with the measurement values of the diastolic components by the Doppler method to examine usefulness of diastolic component measurement with ATP infusion for diagnosis of LITA patency. Subjects were 19 patients with good LITA (group A) and 8 patients with bad LITA (group B). Results; while there were significant differences in the mean baseline diastolic peak velocity, mean diastolic time velocity integral and mean diastolic fraction between the groups, overlapping was seen in individual cases. However, the inter-group differences were more distinct by ATP infusion and the borderline values were 30 cm/sec for diastolic peak velocity and 10 for diastolic time velocity integral. 3) Reliability of the diagnosis for LITA patency by measuring the diastolic components using the Doppler method with ATP infusion was examined and compared with the angiographic findings as the gold standard. Subjects were 27 patients and the diagnostic criteria for good LITA were set at 30 cm/sec for diastolic peak velocity and 10 for diastolic time velocity integral. Results; sensitivity and specificity of the Doppler method with ATP infusion were 100% for diagnosis of LITA patency by measuring the diastolic components. Conclusion, in diagnosis of LITA patency by the transthoracic ultrasonic cardiography, diagnostic accuracy was improved by measuring the diastolic parameters under continuous infusion of ATP.

Adenosine Triphosphate↗

PubMed1998

A defect in skeletal muscle sodium channel deactivation exacerbates hyperexcitability in human paramyotonia congenita.

1. Paramyotonia congenita (PC) is a human hereditary disorder wherein missense mutations in the skeletal muscle sodium channel lead to cold-exacerbated muscle hyperexcitability. The most common site for PC mutations is the outermost arginine of domain i.v. segment 4 (human R1448, rat R1441). 2. We examined the rat homologues of two PC mutants with changes at this site: R1441P and R1441C. The R-->P mutation leads to the most clinically severe form of the disease. Since PC has so far been attributed to defects in fast inactivation, we expected the R-->P substitution to have a more dramatic effect on fast inactivation than R-->C. Both mutants (R1441P and R1441C), however, had identical rates and voltage dependence of fast inactivation and activation. 3. R1441P and R1441C also had slowed deactivation, compared with wild-type, raising the possibility that slowed deactivation, in combination with defective fast inactivation, might be a contributing cause of paramyotonia congenita. Furthermore, deactivation was slower in R1441P than in R1441C, suggesting that the worse phenotype of the human R-->P mutation is due to a greater effect on deactivation, and supporting our hypothesis that slowed sodium channel deactivation contributes to paramyotonia congenita. 4. We show that the downstroke of the muscle action potential produced a sodium tail current, and thus slowed deactivation opposes repolarization and therefore leads to hyperexcitability. Hyperexcitability due to slowed deactivation, which has previously been overlooked, also predicts the temperature sensitivity of PC, which has otherwise not been adequately explained.

Action Potentials↗

PubMed1998

Effects of prelesioned peripheral nerve graft on nerve regeneration in the rat spinal cord.

OBJECTIVE: The aim of this study was to examine the effects of prelesioned peripheral nerve grafts on central nerve regeneration compared with the freshly transected peripheral nerve grafts in the dorsal funiculus of the rat spinal cord. METHODS: The experimental paradigm consisted of ligating the common peroneal nerve at the midthigh level for 7 days, while the adjacent tibial nerve was left intact. Numerous Schwann cells appeared accompanying regenerating axons in the proximal stump of the ligated nerve. The proximal stumps of the ligated (prelesioned) common peroneal nerve and the intact (untreated) tibial nerve were excised as one tissue block and autografted into the dorsal funiculi of the upper cervical cord. The graft was placed so that the prelesioned common peroneal nerve was positioned on the left dorsal funiculus and the untreated tibial nerve was positioned to the right of the midsagittal plane. Nerve regeneration was examined by light and transmission electron microscopy 1 to 16 weeks after grafting, comparing the effectiveness of prelesioned and untreated nerve grafts. RESULTS: Numerous regenerating axons were observed in the caudal border of both grafts 1 to 2 weeks after grafting. Astrocyte proliferation was suppressed in the prelesioned grafts compared to the untreated grafts. Four to 16 weeks later, the number of regenerating axons was approximately 10-fold as large in the prelesioned grafts as in the untreated grafts. The regenerating axons were myelinated by Schwann cells. Astrocytic glial scar formation was inconspicuous in the prelesioned grafts, whereas it was prominent in the untreated grafts. Schwann cells were contiguous with astrocytes along regenerating axons, forming a continuous conduit from the central to peripheral nerve microenvironments for the outgrowth of regenerating axons. CONCLUSION: The prelesioned peripheral nerve graft is more effective than the untreated graft in suppressing astrocytic scar formation and in supporting the outgrowth of regenerating axons in the dorsal funiculus of rat spinal cord.

Animals↗

PubMed1997

Remyelination in the rat dorsal funiculus following demyelination by laser irradiation.

Excimer laser (KrF excimer laser, 248 nm wavelength) was used to damage cellular components in the dorsal funiculus at the lumbar level (L2) of the rat spinal cord. An open lesion was not found at the irradiation site on the spinal cord. However, the cytological examination revealed that cellular components were damaged to the depth of 200-500 microm from the pial surface. The characteristic feature was that at the border of the lesion, many axons remained naked but intact after their myelin sheaths had been completely disintegrated. Such naked axons were subsequently remyelinated by mature or immature glial cells. Mature oligodendrocytes, while retaining their cytoplasmic processes connected with the myelin sheaths of unaffected axons, extended new cytoplasmic processes on nearby naked axons and made new myelin sheaths around them. In contrast, 7 days after the irradiation, numerous immature glial cells appeared in association with naked axons, and some of them were differentiated into oligodendrocytes forming thin myelin sheaths on naked axons. These findings suggest that demyelinated axons can cause the proliferation and probably dedifferentiation of the oligodendrocyte lineage. The use of lasers provides a unique experimental model of demyelination and remyelination in the central nervous system of adult mammals.

Animals↗

PubMed1997

Basic fibroblast growth factor promotes extension of regenerating axons of peripheral nerve. In vivo experiments using a Schwann cell basal lamina tube model.

Schwann cell basal lamina tubes serve as attractive conduits for regeneration of peripheral nerve axons. In the present study, by using basal lamina tubes prepared by in situ freeze-treatment of rat saphenous nerve, the effects of exogenously applied basic fibroblast growth factor (bFGF) on peripheral nerve regeneration was examined 2 and 5 days after bFGF administration. Regenerating axons were observed by light and electron microscopy using PGP9.5-immunohistochemistry for specific staining of axons. In addition, the localizations of bFGF and its receptor (FGF receptor-1) were examined by immunohistochemistry using anti-bFGF antibody and anti-FGF receptor-1 antibody, respectively. Regenerating axons extended further in the bFGF-administered segment than in the bFGF-untreated control segment. Electron microscopy showed that regenerating axons grew out unaccompanied by Schwann cells. Findings concerning angiogenesis and Schwann cell migration were very similar between the bFGF treated and control nerve segment. bFGF-immunoreactivity was not detected in the control nerve segment. In contrast, bFGF-immunoreactivity was detected on the basal lamina tubes as well as on the plasmalemma of regenerating axons facing the basal lamina in the bFGF treated nerve segment up to 5 days after administration, suggesting that exogenous bFGF can be retained in the basal lamina for several days after administration. FGF receptor was detected on the plasma membrane of regenerating axons where they abutted the basal lamina. These results indicate that bFGF could promote the extension of early regenerating axons by directly influencing the axons, but not via Schwann cells or angiogenesis.

Animals↗

PubMed1995

Prevalence of fatty liver in Japanese children and relationship to obesity. An epidemiological ultrasonographic survey.

The prevalence of fatty liver in children is unknown and its relationship to obesity is poorly defined. The present study of 810 northern Japanese children (4-12 years old) determined the prevalence of fatty liver in the pediatric population and its relationship to obesity. Diagnosis of fatty liver was based on established real-time ultrasonographic criteria. The overall prevalence of fatty liver was 2.6% and was higher for boys (3.4%) than for girls (1.8%), although not statistically significant (P = 0.15). Fatty liver was found in children as young as 6 years of age. There was no significant association between the prevalence of fatty liver and height (physical growth). There was a strong positive correlation between fatty liver prevalence and established obesity indices: Rohrer's Index--chi 2 linear trend = 59.2, P < 0.0001; body mass index--chi 2 linear trend = 91.6, P < 0.0001; and age-gender-adjusted Japanese standard index of weight for height--chi 2 linear trend = 93.2, P < 0.0001. However, direct measurement of abdominal subcutaneous fat thickness by ultrasonography was the best predictor of fatty liver: chi 2 linear trend = 159, P < 0.0001. These results indicate that fatty liver may develop very early in life, and there is a direct relationship between degree of obesity and fatty liver in children.

Body Height↗

PubMed1994

A physical map and candidate genes in the BRCA1 region on chromosome 17q12-21.

We have constructed a physical map of a 4 cM region on chromosome 17q12-21 that contains the hereditary breast and ovarian cancer gene BRCA1. The map comprises a contig of 137 overlapping yeast artificial chromosomes and P1 clones, onto which we have placed 112 PCR markers. We have localized more than 20 genes on this map, ten of which had not been mapped to the region previously, and have isolated 30 cDNA clones representing partial sequences of as yet unidentified genes. Two genes that lie within a narrow region defined by meiotic breakpoints in BRCA1 patients have been sequenced in breast cancer patients without revealing any deleterious mutations. These new reagents should facilitate the identification of BRCA1.

Autoantigens↗

PubMed1994

Genetic mapping of the BRCA1 region on chromosome 17q21.

Chromosome 17q21 harbors a gene (BRCA1) associated with a hereditary form of breast cancer. As a step toward identification of this gene itself we developed a number of simple-sequence-repeat (SSR) markers for chromosome 17 and constructed a high-resolution genetic map of a 40-cM region around 17q21. As part of this effort we captured genotypes from five of the markers by using an ABI sequencing instrument and stored them in a locally developed database, as a step toward automated genotyping. In addition, YACs that physically link some of the SSR markers were identified. The results provided by this study should facilitate physical mapping of the BRCA1 region and isolation of the BRCA1 gene.

Base Sequence↗