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Biomedical subjects

Y Ohyama

Publications and source records attributed to Y Ohyama.

At least 109 records · Page 6Linked to original sources
PubMed1996

A comparison of glandular involvement between chronic graft-versus-host disease and Sjögren's syndrome.

Patients with chronic graft-versus-host disease (cGVHD) occasionally suffer from symptoms of xerostomia and xerophthalmia, which are also features of Sjógren's syndrome (SS). To identify differences in the glandular involvement between cGVHD and SS, we measured the proportions of infiltrating lymphocyte subsets and the expression of HLA-DR antigen and cell adhesion molecules in labial salivary glands (LSG). In cGVHD, more than 90% of the infiltrating lymphocytes were T cells with a slight predominance of CD8+ over CD4+ cells. In SS, CD4+ cells were predominant, and B cells accounted for 10-30% of the infiltrating lymphocytes. Ductal epithelial cell associated with lymphocytic infiltration expressed HLA-DR antigen in both cGVHD and SS. In SS alone, HLA-DR antigen expression also occurred without associated lymphocytic infiltration. The expression of adhesion molecules on ductal epithelial cells, especially vascular cell adhesion molecule 1, was more intense in SS than in cGVHD, while that on endothelial cell was similar in cGVHD and SS. These data suggest that the pathogenesis of glandular involvement of cGVHD is different from that of SS.

Adolescent↗
PubMed1996

Oral involvement in chronic graft-versus-host disease after allogeneic bone marrow transplantation.

We examined 37 patients who had undergone an allogeneic bone marrow transplantation and compared their oral findings to their systemic involvement with chronic graft-versus-host disease. Among the clinical signs and symptoms in their oral region, only the presence of oral lichenoid lesions had a statistically significant relationship to the diagnosis of chronic graft-versus-host disease. The histologic findings in the labial salivary glands and buccal mucosa closely reflected the status of chronic graft-versus-host disease. Statistically, the presence of diffuse and periductal lymphocytic infiltration in labial salivary glands, subepithelial lymphocytic infiltration and epithelial changes in buccal mucosa also showed a significant relationship to the diagnosis of chronic graft-versus-host disease. The present study suggests that a systematic oral examination, especially pathologic examination of the labial salivary glands and buccal mucosa, is useful in evaluating the status of chronic graft-versus-host disease.

Adolescent↗
PubMed1996

A phylogenetic study of Rubrobacter radiotolerans by sequence analysis of the 16S ribosomal RNA gene.

Partial sequence of the 16S ribosomal RNA gene of an extremely high radiotolerant bacterium, Rubrobacter radiotolerans (reclassified from Arthrobacter radiotolerans by chemical characteristics) was determined by PCR-amplification from a small amount of heat-lysed biomass, followed by direct sequencing of the PCR product. The sequence was aligned with seven species of Arthrobacter and also with representatives of various other bacterial groups. R. radiotolerans was confirmed to be out of the Arthrobacter group justifying the reclassification. Moreover, it has an individual position among the selected representatives being closer to the eubacterial groups.

Arthrobacter↗
PubMed1996

Rate of nitric oxide release in the lung and factors influencing the concentration of exhaled nitric oxide.

The level of nitric oxide (NO) in exhaled air fluctuates in normal individuals depending on the physiological conditions. We evaluated the effects of duration of exhalation and breath-holding on the exhaled concentrations of NO in 16 normal human volunteers. Exhaled gas corresponding to vital capacity was collected in 6-liter Tedlar bags and analyzed by chemiluminescence. The NO concentration in exhaled gas increased significantly in proportion to the duration of exhalation [P = 0.009 +/- 0.011 (SD)] and was increased after breath-holding. There was no significant difference in the exhaled NO concentration among 10-s phases of a 30-s exhalation, as determined from multiple breath collections. The NO released from the airways is presumably unaffected by fluctuation of exhalation speed. The NO release rate, calculated from a single regression analysis between the NO concentration and the duration of exhalation, was 39 +/- 29 pmol/s, a value which was about fourfold greater in nine patients with bronchial asthma.

Adult↗
PubMed1996

HPLC determination of carnitine and acylcarnitines in human plasma by means of fluorescence labeling using 2-(4-hydrazinocarbonylphenyl)-4,5-diphenylimidazole.

Carnitine and acylcarnitines are important substances involved in the oxidation and metabolism of fatty acids. An HPLC method is presented for the quantitative analysis of these compounds. The method is based on the detection of fluorescent derivatives of carnitine and short- and medium-chain acylcarnitines labeled with 2-(4-hydrazinocarbonylphenyl)-4,5-diphenylimidazole (HCPI). The labeling of carnitine and acylcarnitines with HCPI was performed at room temperature for 1 h using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as a condensing reagent. The analytes prepared using cation-exchange cartridges were separated on an octadecylsilyl silica gel (ODS) column by a gradient elution system of acetonitrile/Tris-HCI buffer and determined using a synthetic internal standard. Fluorescence detection was performed at 475 nm with excitation at 340 nm. The detection limits for carnitine, acetyl-, propionyl-, hexanoyl- and octanoylcarnitine ranged from 0.24 to 1.97 nmol per ml human plasma, at a signal-to-noise ratio of 3. The within-day and between-day precision of the assay for carnitine and acylcarnitines in plasma samples had relative standard deviations (RSDs) lower than 10.3%. The concentration of free carnitine, acylcarnitines and total carnitine in human plasma could be successfully determined by the proposed method.

Adult↗
PubMed1996

Three-dimensional analysis of nystagmus induced by neck vibration.

The role of dorsal neck proprioceptive inputs to vestibular compensation was investigated in 11 patients with unilateral vestibular dysfunction. Subjects had neither spontaneous nystagmus nor disequilibrium, indicating that they were well compensated. Vibratory stimulation to the dorsal neck produced marked nystagmus. All subjects showed horizontal component directed towards the contralateral side of the lesion. Vertical and torsional components of the nystagmus were exhibited by 10 and 7 subjects, respectively. These results indicate that the neck vibration caused a discompensation in vestibularly well compensated subjects after unilateral dysfunction. In addition, the influence of neck afferent on the vestibulo-ocular reflex pathways, not only of the horizontal, but also the vertical systems, is discussed.

Eye Movements↗
PubMed1995

Solution structure of a human cystatin A variant, cystatin A2-98 M65L, by NMR spectroscopy. A possible role of the interactions between the N- and C-termini to maintain the inhibitory active form of cystatin A.

The solution structure of a human cystatin A variant, cystatin A2-98 M65L, which maintains the full inhibitory activity of the wild-type protein, was determined at pH 3.8 by sD/3D heteronuclear double- and triple-resonance NMR spectroscopy. The structure is based on a total of 1343 experimental restraints, comprising 1139 distance, 154 phi and chi 1 torsion angle restraints, and 50 distance constraints for 25 backbone hydrogen bonds. A total of 15 structures was calculated using the YASAP protocol with X-PLOR, and the atomic rms distribution about the mean coordinate positions for residues 8-93 was 0.55 +/- 0.10 A for the backbone atoms and 1.05 +/- 0.11 A for all heavy atoms. The structure consists of five antiparallel beta-sheets and two short alpha-helices. Comparison with the X-ray structure of cystatin B in the papain complex shows that the conformation of the first binding loop is quite similar to that of cystatin A, with an rms deviation of 0.78 A for the backbone atoms in the 43-53 region (cystatin A numbering). The second binding loop, however, is significantly different in the two structures, with an rms deviation greater than 2 A. There are some other significant differences, especially for the N-terminal and alpha-helix regions. The overall structure of cystatin A is also compared with the recently reported NMR structure of the wild-type cystatin A (stefin A) at pH 5.5 (Martin et al., 1995) and reveals the following features. that differ in our structure from the previous one: (1) the N-terminal segment, which was unstructured in the previous report, folds over in close vicinity to the C-terminus, as revealed by the distinctive NOEs between those segments; (2) two discrete short alpha-helices linked by a type II reverse turn were found, instead of the continuous single alpha-helix with a slight kink shown in the previous structure; (3) the second binding loop, which was not well converged in the previous study at pH 5.5, is determined very well in our structure. The effect of the N-terminal truncation on the cystatin A structure was examined by comparing the 1H-15N HSQC spectrum of cystatin A2-98 with that of the cystatin A5-98 variant, which lacks the anti-papain activity, revealing significant chemical shift differences in the residual N-terminal segment and the first binding loop, together with small shifts in the other parts.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

Human cystatin A is inactivated by engineered truncation. The NH2-terminal region of the cysteine proteinase inhibitor is essential for expression of its inhibitory activity.

A series of NH2-terminal truncated forms of human cysteine proteinase inhibitor, cystatin A, was prepared by genetic engineering using Escherichia coli harboring mutated genes. Each variant of cystatin A was efficiently expressed as a fused protein with porcine adenylate kinase and released by CNBr degradation after exchange of the sole inner Met to Leu. The mutant cystatin A lacking an amino-terminal Met residue (called standard variant starting from Ile2, CystA2-98(M65L) showed the same inhibitory activity as authentic one isolated from human epidermis. Two-residue truncation scarcely influenced the activity, but further truncations deleting Pro3 and beyond conservative Gly4 and Gly5 caused a remarkable decrease of their inhibitory activity. But little effect was observed by a substitution of Pro3 with Leu. The loss of the activity by amino-terminal truncation was compensated slightly by engineered substitution of Gly75 with His on a second loop. In the two-dimensional 15N-1H HSQC NMR spectrum, four-residue truncation was found to cause changes in the chemical shifts of Val47 and Val48, which locate on a first loop and consist of a conservative QVVAG sequence. Furthermore, the truncation led to a change in fluorescence spectroscopic behavior of Trp75, which was introduced as a probe on the second loop. Fluorescence intensity of the Trp of the truncated (5-98) form was more affected by heating than the active standard variant. Conversely, fluorescence of Trp75 in 2-98 form was more quenched by acrylamide than the 5-98 variant. Thus, the amino-terminal region of cystatin A is essential for the expression of its inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Regulation and localization of midkine in rat ovary.

In a previous experiment, it was shown that midkine (MK) was quite abundant in the follicular fluid; the concentration of MK in bovine follicular fluid was estimated to be 125 micrograms/l. To investigate the regulation of MK production in the ovary, we examined the effect of pregnant mare's serum gonadotropin (PMSG) and PMSG-hCG treatment on the expression of MK in rat ovary. The mRNA of midkine (MK) was increased by PMSG injection and decreased by PMSG and hCG injection; the profile of change of mRNA of MK was similar to that of FSH receptor. Using in situ hybridization, we observed that the MK mRNA localized to granulosa cells. These results suggest that the granulosa cells produce MK under the control of gonadotropin.

Animals↗
PubMed1995

Effect of 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) enantiomers, major metabolites of phenytoin, on the occurrence of chronic-gingival hyperplasia: in vivo and in vitro study.

The purpose of this study was to assess the possible role of the (R)- and (S)- enantiomers of the phenytoin metabolite p-HPPH in the pathogenesis of gingival hyperplasia (GH). About 98% of circulating p-HPPH is in the (S)-form. There were significant differences between patients with and without GH in (R)-p-HPPH level (0.055 vs 0.042 microgram.ml-1), both enantiomer/racemate level ratios, and R/S enantiomeric ratio (0.0313 vs 0.0232); an increase in serum (R)-p-HPPH level was observed in patients with GH. In separate experiments, the effect of p-HPPH enantiomers on the proliferation of the normal human dermal fibroblast was studied. The in vitro study showed that (R)-p-HPPH selectively stimulated fibroblast growth. The results suggest that the least abundant metabolite, (R)-p-HPPH, is the most toxic with respect to gingival hyperplasia.

Adult↗
PubMed1995

Microdetermination of the prostaglandin B1 in human plasma by gas chromatography/selected ion monitoring using [18O]prostaglandin B1 as an internal standard.

We devised a simple and effective purification for the microdetermination of prostaglandin B1 (PGB1), a metabolite of PGE1. [18O]PGB1 was synthesized by the repeated base-catalyzed hydrolysis of methyl ester derivatives in H(2)18O, to obtain an internal standard for the gas chromatography/selected ion monitoring (GC/SIM) of PGB1. The methyl ester-methoxime-dimethylisopropylsilyl ether derivative was prepared, then GC/SIM was carried out by monitoring the ion at m/z 448.3 for PGB1 and that at m/z 452.3 for internal standard. A good linear response over the range of 10 pg to 100 ng was demonstrated. We detected PGB1 to a level of about 40 pg/mL in human plasma. This method can be used to determine PGB1 in biological samples.

Adult↗
PubMed1995

T-cell receptor V alpha and V beta gene use by infiltrating T cells in labial glands of patients with Sjögren's syndrome.

Sjögren's syndrome is an autoimmune disease affecting the exocrine glands; it is thought to result from T-cell-mediated damage. In the labial glands of 20 patients with Sjögren's syndrome, infiltrating T cells were immunohistochemically characterized, and T-cell receptor (TCR) gene expression was examined with a method based on polymerase chain reaction. Most of these lymphocytes expressed CD3, CD4, CD45RO, and TCR alpha beta, whereas less than 5% of them expressed CD25 and CD69. The TCR V alpha and V beta genes expressed in peripheral blood mononuclear cells were diverse, whereas the TCR V alpha and V beta repertoires in the labial glands were more restricted but were still heterogeneous. The predominantly used V alpha and V beta families in the labial glands, when compared with those in peripheral blood mononuclear cells, were found to vary in individual patients and also to differ from patient to patient. Thus the T-cell population in the labial glands was polyclonal but showed a more restricted pattern than that seen in peripheral blood mononuclear cells.

Adult↗
PubMed1995

Regulation of vitamin D-responsive gene expression by fluorinated analogs of calcitriol in rat osteoblastic ROB-C26 cells.

We compared the activation of vitamin D-responsive genes by 24,24-difluorocalcitriol [F2-1 alpha,25(OH)2D3] and 26,26,26,27,27,27-hexafluorocalcitriol [F6-1 alpha,25(OH)2D3] with that by calcitriol [1 alpha,25(OH)2D3] in rat osteoblastic ROB-C26 cells. F2-1 alpha,25(OH)2D3 and F6-1 alpha, 25(OH)2D3 were ten times more potent than 1 alpha,25(OH)2D3 in inducing the expression of 1 alpha, 25(OH)2D3-24-hydroxylase (24-OHase) mRNA 6 h after adding vitamin D compounds. The lower affinity of these two fluorinated analogs compared with that of 1 alpha,25(OH)2D3 for vitamin D binding protein in serum (serum DBP) seemed to be partly involved in their increased ability to activate the 24-OHase gene. A time course study revealed that the expression of the 24-OHase and osteopontin mRNAs in the cells incubated with 1 alpha, 25(OH)2D3 and F2-1 alpha,25(OH)2D3 attained maximal levels at 6 h for 24-OHase mRNA and 18 h for osteopontin mRNA, the both decreased thereafter. On the contrary, F6-1 alpha,25(OH)2D3 increased the expression of 24-OHase and osteopontin exponentially until 72 h. While F2-1 alpha,25(OH)2[1 beta-3H]D3 was catabolized quickly by ROB-C26 cells, F6-1 alpha,25(OH)2[1 beta-3H]D3 was slowly and quantitatively converted into putative 26,26,26,27,27,27-hexafluoro-23S-hydroxy[1 beta-3H]calcitriol (F6-1 alpha,23S,25(OH)3[1 beta-3H]D3). This may explain why the time-course profiles of the accumulation of mRNAs for 24-OHase and osteopontin differed in the cells exposed to the fluorinated analogs. In addition to the longer retention, unknown up-regulating mechanisms appeared to be involved in the exponential activation of the 24-OHase and osteopontin genes induced by F6-1 alpha,25(OH)2D3.

Animals↗
PubMed1995

Significance of the highly conserved Gly-4 residue in human cystatin A.

The expression system for human recombinant cystatin A has already been established to be a fusion protein with porcine adenylate kinase in Escherichia coli [Kaji et al. (1990) Biol. Chem. Hoppe-Seyler 371, Suppl., 145-150]. After cyanogen bromide cleavage of the fused protein expressed in E. coli, the cystatin portion could be readily isolated. The inhibitory activity of the obtained variant (Cyst A (2-98)) was found to be almost identical with that of the wild type, and thereafter a mutation was introduced into this variant (Ctst A(2-98)), called the standard variant. To elucidate the role of the Gly-4 residue, which is completely conserved in all cystatin species, this residue was substituted with 17 other amino acids by means of cassette mutagenesis. Thus 17 variants (Cyst A(2-98)[G4X]) obtained were examined as to their inhibitory activity towards papain. As the side chain of the substituted amino acid residue became more bulky, the inhibitory activity of the variant markedly decreased. Variants whose side chains were bulkier than a Val residue showed almost no inhibitory effect towards papain. Consequently, it was deduced that the large side chain of a substituted amino acid may cause steric hindrance, which may be responsible for the decrease in inhibitory activity. Thus, we could conclude that the 4th (Gly) residue on cystatin A must be small, because amino acids which existed on the N-terminal side of this residue could interact with a papain molecule.

Amino Acid Sequence↗
PubMed1995

Quantitative immunoelectron microscopic analysis of the localization and induction of 25-hydroxyvitamin D3 24-hydroxylase in rat kidney.

25-Hydroxyvitamin D3 24(R)-hydroxylase (24-hydroxylase) is involved in the metabolism and regulation of vitamin D3 and is markedly induced by administration of vitamin D3. We detected this enzyme by electron microscopy and an immunogold technique along nephrons of normal and vitamin D3-administered rats. After the rats were administered vitamin D3, 50,000 IU/day for 1 week, they were perfusion-fixed with a paraformaldehyde solution. The fixed kidneys were then removed and embedded in LR White resin. Ultrathin sections were prepared and labeled by the immunogold technique using a mouse anti-rat 25-hydroxyvitamin D3 24-hydroxylase monoclonal antibody. We counted the number of gold particles bound per micron 2 of the mitochondria (particle density) of the tubule epithelial cells along the nephrons. In normal and vitamin D3-administered rats, gold particles were observed in the mitochondria of epithelial cells along the tubules. In normal rats, gold labeling for 24-hydroxylase was statistically significant (p < 0.05), in the S1-S2 segments, the S3 segment of the proximal tubules, and in the distal convoluted tubules. In the rats administered vitamin D3, the particle density increased significantly (p < 0.05) by about 12-fold in the S1-S2 segments of the proximal tubules, whereas it increased less markedly in other parts of the nephron. The marked induction of the S1-S2 segments of the proximal tubules suggests that these segments play an important role in the regulation of vitamin D3 metabolism.

Animals↗