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Biomedical subjects

S Ichinose

Publications and source records attributed to S Ichinose.

At least 19 recordsLinked to original sources
PubMed2000

Purification, characterization, and cDNA cloning of a novel acidic endoglycoceramidase from the jellyfish, Cyanea nozakii.

Endoglycoceramidase (EC ) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids. We report here the purification, characterization, and cDNA cloning of a novel endoglycoceramidase from the jellyfish, Cyanea nozakii. The purified enzyme showed a single protein band estimated to be 51 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme showed a pH optimum of 3.0 and was activated by Triton X-100 and Lubrol PX but not by sodium taurodeoxycholate. This enzyme preferentially hydrolyzed gangliosides, especially GT1b and GQ1b, whereas neutral glycosphingolipids were somewhat resistant to hydrolysis by the enzyme. A full-length cDNA encoding the enzyme was cloned by 5'- and 3'-rapid amplification of cDNA ends using a partial amino acid sequence of the purified enzyme. The open reading frame of 1509 nucleotides encoded a polypeptide of 503 amino acids including a signal sequence of 25 residues and six potential N-glycosylation sites. Interestingly, the Asn-Glu-Pro sequence, which is the putative active site of Rhodococcus endoglycoceramidase, was conserved in the deduced amino acid sequences. This is the first report of the cloning of an endoglycoceramidase from a eukaryote.

Amidohydrolases↗
PubMed2000

Novel apoptosis-inducing activity in Bacteroides forsythus: a comparative study with three serotypes of Actinobacillus actinomycetemcomitans.

Bacteroides forsythus, which has been reported to be associated with periodontitis but has not been recognized as a key pathogen, was found to induce cytolytic activity against HL-60 and other human leukemic cells. This cytolytic activity was demonstrated according to three different criteria: (i) loss of both mitochondrial membrane potential and membrane integrity in cells treated with bacterial extracts and then with Rh123 and propidium iodide, respectively, as demonstrated by flow cytometry; (ii) damage to cytoplasmic membrane, as revealed by scanning electron microscopy (SEM); and (iii) DNA ladder formation and activation of caspase-3. These results indicate that B. forsythus produced an apoptosis-inducing factor(s) found to be composed of protein as judged by heat and trypsin sensitivity. In addition to extracts from B. forsythus, the culture supernatant of this bacterium has the ability to induce a cytolytic effect against peripheral white blood cells, especially lymphocytes. For comparison with B. forsythus, the same analyses were applied to two strains with different serotypes of Actinobacillus actinomycetemcomitans, serotypes a (ATCC 43717) and c (ATCC 43719), in addition to previously reported apoptosis-inducing serotype b (ATCC 43718), which was used as a positive control. The strains of A. actinomycetemcomitans serotypes a and b induced apoptosis in HL-60 cells as judged by the above three criteria but to a slightly lesser extent than did B. forsythus, while the serotype c strain produced apoptosis to a negligible extent. Detailed SEM images showed that the A. actinomycetemcomitans serotype a strain induced large-pore formation and the serotype b strain produced small pores with typical blebbing, while B. forsythus induced severe membrane ruffling. Further DNA ladder formation and caspase-3 activation were observed in the serotype a and b strains but not in the serotype c strain. The present paper is the first report of a protein factor(s) from B. forsythus and the A. actinomycetemcomitans serotype a strain which induces apoptotic cell death.

Aggregatibacter actinomycetemcomitans↗
PubMed1999

Molecular cloning, sequencing, and expression of the gene encoding alkaline ceramidase from Pseudomonas aeruginosa. Cloning of a ceramidase homologue from Mycobacterium tuberculosis.

We previously reported the purification and characterization of a novel type of alkaline ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N., Tani, M., Imayama, S., and Ito, M. (1998) J. Biol. Chem. 273, 14368-14373). Here, we report the molecular cloning, sequencing, and expression of the gene encoding the ceramidase of this strain. Specific oligonucleotide primers were synthesized using the peptide sequences of the purified ceramidase obtained by digestion with lysylendopeptidase and used for polymerase chain reaction. DNA fragments thus amplified were used as probes to clone the gene encoding the ceramidase from a genomic library of strain AN17. The open reading frame of 2,010 nucleotides encoded a polypeptide of 670 amino acids including a signal sequence of 24 residues, 64 residues of which matched the amino acid sequence determined for the purified enzyme. The molecular weight of the mature enzyme was estimated to be 70,767 from the deduced amino acid sequence. Expression of the ceramidase gene in Escherichia coli, resulted in production of a soluble enzyme with the identical N-terminal amino acid sequence. Recombinant ceramidase was purified to homogeneity from the lysate of E. coli cells and confirmed to be identical to the Pseudomonas enzyme in its specificity and other enzymatic properties. No significant sequence similarities were found in other known functional proteins including human acid ceramidase. However, we found a sequence homologous to the ceramidase in hypothetical proteins encoded in Mycobacterium tuberculosis, Dictyostelium discoideum, and Arabidopsis thaliana. The homologue of the ceramidase gene was thus cloned from an M. tuberculosis cosmid and expressed in E. coli, and the gene was demonstrated to encode an alkaline ceramidase. This is the first report for the cloning of an alkaline ceramidase.

Acid Ceramidase↗
PubMed1999

Correlations between the apparent diffusion coefficient, water content, and ultrastructure after induction of vasogenic brain edema in cats.

OBJECT: The authors examined the correlation between changes in the apparent diffusion coefficient, regional water content, and tissue ultrastructure after vasogenic brain edema. METHODS: Vasogenic edema was induced in the white matter of six cats by cortical cold lesioning. The trace of diffusion tensor (Trace[D]) obtained from magnetic resonance imaging to measure the orientationally averaged water diffusibility was compared with the corresponding tissue water content determined by gravimetric studies and with ultrastructural water localization. Edema fluid had spread to the subcortical and deep white matter by 4.5 hours postlesioning. The increase in Trace(D) showed a significant linear correlation with the increase in tissue water content, both in the subcortical and deep white matter as follows: y = 45.5x - 2367 (r = 0.94) and y = 37.0x - 1769 (r = 0.93), respectively, where x is the water content (gram water/gram tissue) and y the Trace(D) (x 10(-6) mm2/second). On histological examination, nerve fibers were found to be dissociated in the white matter and the extracellular space was markedly enlarged with protein-rich fluid. No noticeable hydropic swelling of the cellular components was observed. CONCLUSIONS: A linear correlation was observed between increases in Trace(D) and increases in extracellular water volume in in vivo vasogenic brain edema. A similar correlation between the subcortical and deep white matter showing different arrangements of nerve fibers (parallel compared with intermingled, respectively) indicated that measurement of Trace(D) is a suitable parameter for the evaluation of vasogenic brain edema.

Animals↗
PubMed1998

Mitochondrial encephalomyopathy showing prominent microvacuolation and necrosis of intestinal smooth muscle cells: a case diagnosed by rectal biopsy.

A 40-year-old woman who developed intestinal dysmobility was found, at rectal biopsy, to have marked microvacuolation of mucosal muscle layer cells, which corresponded to increased accumulation of abnormal mitochondria. Skeletal muscle biopsy specimens showed ragged-red fibers, vessels strongly reactive for succinic dehydrogenase, and focal deficiency of cytochrome c oxidase. Autopsy performed at the age of 50 revealed prominent accumulation of abnormal mitochondria in the intestinal smooth muscle cells with a mottled distribution of focal necrosis, multiple small cerebral infarcts with diffuse neuronal loss, and rarefaction of the perivascular white matter. Mitochondrial DNA analysis showed a point mutation at position 3243. This case, showing features of both mitochondrial neurogastrointestinal encephalomyopathy and mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), indicates that routine intestinal biopsy can detect mitochondrial encephalomyopathy with gastrointestinal involvement. The main intestinal changes were extensive accumulation of abnormal mitochondria in the leiomyocytes and scattered focal necrosis.

Female↗
PubMed1998

Observation of ultrastructural changes in cultured retinal pigment epithelium following exposure to blue light.

BACKGROUND: The retina can be damaged by light even when levels of energy are well below the threshold for thermal damage, and the experimental damage of the retinal pigment epithelium (RPE) may be induced more easily by blue light than by longer wavelengths of visible light. The present study demonstrates the ultrastructural damage produced by exposure to blue light in cultured RPE. METHODS: Long-Evans rats were enucleated 8-10 days after birth for primary culture. One week after seeding, the monolayer culture of RPE cells was exposed to a cool blue light (wavelength = 440 +/- 10 nm) for 36 h (12 h/day, 3 days) at 2.0 mW/cm2. Transmission electron microscopy was used to compare the exposed RPE with the control. The entire experiment was repeated 3 times independently. RESULTS: The cytoplasm of the exposed RPE exhibited degenerative changes, such as large whorls of membrane, lamellar whorls and whorled inclusions. CONCLUSION: The RPE cells can be damaged directly by blue light after excluding the possible influence of phagosomes. This primary culture of RPE can also serve as an in vitro model for the study of light damage to the RPE.

Animals↗
PubMed1997

An Alu-linked repetitive sequence corresponding to 280 amino acids is expressed in a novel bovine protein, but not in its human homologue.

A novel protein harboring a 280-amino acid region from an Alu-linked repetitive sequence (bovine Alu-like dimer-driven family) was isolated from a bovine brain S-100 fraction using monoclonal antibodies against a rat GTPase-activating protein that shares the same epitope. The protein has an apparent molecular mass of 97 kDa (p97). Western blot analysis using extracts prepared from various tissues showed p97 to be predominantly detected in brain and moderately in liver and lung. From sequence analysis of the cDNA encoding p97, it was found that the 840-base pair sequence homologous to a part of the bovine Alu-like dimer-driven family, which has never been shown to be expressed, occurs in the middle of the protein coding region. The protein also contains a pair of intramolecular repeats composed of 40 highly hydrophilic amino acids at the C terminus. Human cDNA homologous to p97 was cloned, and its nucleotide sequence demonstrates that the 840-base pair repetitive sequence and one of the intramolecular repeats are missing. We named p97 bovine BCNT after Bucentaur. These results show that bovine BCNT is a unique molecule and suggest that an analysis of the relationship between bovine bcnt and its human homologue may help further the understanding of gene organization and evolution.

Amino Acid Sequence↗
PubMed1996

Binding of human minichromosome maintenance proteins with histone H3.

Minichromosome maintenance (MCM) proteins play essential roles in eukaryotic DNA replication, but their biochemical properties remain to be determined. We detected in HeLa cell extracts six proteins, CDC47, CDC46/MCM5, Cdc21, P1/MCM3, Mis5, and BM28/MCM2, by their binding to a specific antibody and by partial sequencing. The human homologs of the MCM2 (BM28), Mis5, Cdc21, and CDC47 proteins were tightly bound to a histone-Sepharose column and purified to near homogeneity, whereas the P1/MCM3 and CDC46/MCM5 proteins passed through. Among the four core histones, the human BM28/MCM2, Mis5, Cdc21, and CDC47 proteins had high affinity for histone H3. Immunoprecipitation with anti-Cdc21 antibody revealed that these four MCM proteins form complexes. These results are consistent with the findings that MCM proteins bind with chromatin in vivo.

Amino Acid Sequence↗
PubMed1996

Experimental study on nerve regeneration through the basement membrane tubes of the nerve, muscle, and artery.

We evaluated neurotization after transplantation with lyophilized nerves, muscles, and arteries, and examined the possibility of practical application of long bridging grafts. Grafts of 10 mm and 25 mm of lyophilized nerves, muscles, and arteries harvested from Fisher rats were transplanted to the sciatic nerves of recipient Lewis rats. The histological changes undergone by short grafts were observed at weekly intervals. The sham-operated and isograft groups were used to compare the results of long grafts. In both the nerve and muscle-graft group, regenerated axons grew out through the residual basement membrane tube. But in the muscle graft group, phagocytosis of myofibril debris took longer than that of degenerated axons. No statistical differences were found between results of TSI, induced EMG, and quantitative analysis of myelinated axons in the nerve and muscle graft groups. No neurotization was noted in the long artery graft. In long grafts, laminin found on the basement membrane may not be sufficient to accelerate neurotization, and arteries should not be used for tubulization.

Animals↗
PubMed1995

[A morphological study of interactions of Candida albicans and Streptococcus mutans].

Denture stomatitis is caused mainly by infection of Candida albicans and Streptococcus mutans, the most predominant members of the bacterial flora on denture surfaces. The purpose of this study was to investigate the adhesion in vitro of C. albicans and of S. mutans to acrylic surfaces and their interactions. Concentrations of C. albicans and of S. mutans (mutational phase I and III in which activities of glucosyltransferases are higher and lower respectively) were of high levels (> or = 5.0 x 107 colony-forming-units per ml; CFU/ml). The colonies in brain-heart-infusion (BHI) broth added 5% sucrose incubated at 37 degrees C for 18 and 72 hours. Then the samples were dried and evaporated, and examined by S-700 and S-4100 SEM (HITACHI). It was found that S. mutans (mutational phase I) synthesized water-insoluble glucans concomitantly with the adhesion of both S. mutans and C. albicans to the acrylic surfaces. Also, adhesion of C. albicans, mediated by cell-to-cell interactions with S. mutans (especially mutational phase III), to the surfaces was observed.

Acrylic Resins↗
PubMed1994

Is the swelling in brain edema isotropic or anisotropic?

A study was conducted to examine whether swelling of the brain due to vasogenic-type and cytotoxic-type edema is isotropic or anisotropic. Vasogenic edema was induced by cryogenic injury in cats, and coronal sections of the brain were examined at 4-5 h after injury. The swelling of the edematous white matter longitudinal to and transverse to the subcortical neuronal fibers was 2.3% and 91.1%, respectively. Ischemic edema was examined using cortical tissue specimens of cat brain subjected to either middle cerebral artery occlusion for 3 h or immersion in saline after decapitation for 3 h. The swelling parallel to the left-right axis, caudo-rostral axis and antero-posterior axis was 9.6%, 10.1% and 8.5%, respectively. Neuroglial cell swelling was prominent in the ischemic cortex. Thus swelling of the white matter in vasogenic-type edema was anisotropic, whereas that of gray matter in cytotoxic-type (ischemic) edema was isotropic. This observed difference in the biomechanical properties of brain tissue should be taken into account when the etiology of edema-mediated tissue injury, such as herniation, secondary bleeding or ischemia is investigated.

Animals↗
PubMed1991

[A three-dimensional computerized reconstruction of the rectum, anus and surrounding muscles of the rat fetus at the 20th gestation day].

Using fetuses of Wistar/I rats on the 20th gestation day. We designed a three-dimensional computerized reconstruction of the rectum, anus and the surrounding muscles. The tissues were fixed in Bouin's solution sagittally sectioned serially were stained with hematoxylin-eosin. Light microscopic pictures at 40 x magnification were subjected to the analysis using a three-dimensional image processing system, consisting of a drum-scanner, a general purpose computer (Micro VAX II), and a color image processor. The results showed that this system clearly reconstructed the three-dimensional image of the rectum, anus and the surrounding muscles and suggested the presence of the puborectal muscle sling.

Anal Canal↗
PubMed1978

Unusual mechanism in the initiation of the paroxysmal supraventricular tachycardia in a patient with WPW syndrome.

A heretofore unreported unusual mechanism in the initiation of the paroxysmal supraventricular tachycardia (PSVT) in a patient with WPW (Wolff-Parkinson-White) syndrome was observed using His bundle recordings. The patient initially had some degree of AV conduction disturbance at the level of the AV node. A premature atrial impulse initially activated the ventricle exclusively through the accessory pathway and the same impulse re-excited the ventricle via the AV nodal-His axis after finishing the pure pre-excitation with marked prolongation of the AH and the HV intervals. After finishing this double ventricular response it traversed to the atrium to produce the PSVT.

Atrioventricular Node↗