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Biomedical subjects

A Ono

Publications and source records attributed to A Ono.

At least 73 records · Page 4Linked to original sources

Nucleotide sequencing analysis of the 146-kilobase segment around the IkBL and MICA genes at the centromeric end of the HLA class I region.

To elucidate the complete gene structure and to identify new genes involved in the development of HLA class I antigen-associated diseases in the class I region of the human major histocompatibility complex on chromosome 6, a YAC clone (745D12) covering the 146-kb segment around the IkBL and MICA loci was isolated from a YAC library constructed from the B-cell line, BOLETH. A physical map of this region was constructed by isolation of overlapping cosmid clones derived from 745D12. Of these, five contiguous cosmids were chosen for DNA sequencing by the shotgun strategy to give a single contig of 146,601 bp from 2.8 kb telomeric of the IkBL gene to exon 6 of MICA. This region was confirmed to contain five known genes, IkBL, BAT1, MICB, P5-1, and HLA-X (class I fragment), from centromere to telomere, and their exon-intron organizations were determined. The 3.8-1 homologue gene (3.8-1-hom) showing 99.7% identity with the 3.8-1 cDNA clone, which was originally isolated using the 3.8-kb EcoRI fragment between the HLA-54/H and the HLA-G genes, was detected between MICA and MICB and was suggested to represent the cognate 3.8-1 genomic sequence from which the cDNA clone was derived. No evidence for the presence of expressed new genes could be obtained in this region by homology and EST searches or coding and exon prediction analyses. One TA microsatellite repeat spanning 2545 bases with as many as 913 repetitions was found on the centromeric side of the MICA gene and was indicated to be a potential hot spot for genetic recombination. The two segments of approximately 35 kb upstream of the MICA and MICB genes showed high sequence homology (about 85%) to each other, suggesting that segmental genome duplication including the MICA and MICB genes must have occurred during the evolution of the human MHC.

Carrier Proteins↗

Characteristics of cardiac hypertrophy in the juvenile visceral steatosis mouse with systemic carnitine deficiency.

The juvenile visceral steatosis (JVS) mouse exhibits hereditary systemic carnitine deficiency and develops cardiac hypertrophy. The aim of this study was to clarify the characteristics of cardiac hypertrophy in the JVS mouse. Total carnitine content in IVS mouse heart was about 10% of that of control mouse heart at 4 and 8 weeks of age. The heart weight/body weight ratio was bigger in JVS mice than that in control mice at 2 weeks of age, and this difference in ratio increased with age. The wall areas of both ventricles and septum in JVS mice were larger than those of the control mice at 2 and 8 weeks. The myocyte diameter in both ventricular walls and septum in JVS mice was longer than that of the control mice. On electron microscopy, the percent of mitochondria in the myocyte was 66% in JVS mice, and 37% in control mice. The percent of lipid fraction in JVS mice was six-fold higher than that in control mice. Total content of adenine nucleotides in JVS mouse heart was about 60% of that in control mouse heart. Adenylate energy charge in JVS mouse heart was 63 and 45% of that in the control mouse heart at 4 and 8 weeks, respectively. Overall, the cardiac enlargement observed in this animal model could be accounted for by a proportional increase in the myocyte diameter in the ventricles and septum, accompanied by an increase in mitochondria. Furthermore, this cellular growth is associated with decreases in the levels of ATP and ADP, and adenylate energy charge.

Adenosine Diphosphate↗

DNA duplex dynamics: NMR relaxation studies of a decamer with uniformly 13C-labeled purine nucleotides.

Dynamics in a DNA decamer duplex, d(CATTTGCATC). d(GATGCAAATG), were investigated via a detailed 13C NMR relaxation study. Every 2'-deoxyadenosine and 2'-deoxyguanidine was chemically enriched with 15% 13C and 98% 15N isotopes. Six nuclear relaxation parameters [R(13Cz), R(1Hz), R(2(1)Hz13Cz), R(13Cx), R(2(1)Hz13Cx) and steady-state 13C¿1H¿ NOE] were measured at 600 MHz and three were measured at 500 MHz (1H frequency) for the CH spin systems of sugar 1', 3', and 4' as well as base 8 and 2 positions. A dependence of relaxation parameter values on chemical position was clearly observed; however, no sequence-specific variation was readily evident within our experimental error of approximately 5-10%, except for 3' and 5' termini. It was demonstrated that the random 15% 13C enrichment effectively suppressed both scalar and dipolar contributions of the neighboring carbons and protons on the relaxation parameters. To analyze dynamics via all observed relaxation parameters, full spectral density mapping (1992, J. W. Peng and G. Wagner, J. Magn. Reson. 98, 308) and the "model-free" approach (1982, Lipari and Szabo, J. Am. Chem. Soc. 104, 4546) were applied complementarily. A linear correlation between three spectral density values, J(omegaC), J(omegaH - omegaC), and J(omegaH + omegaC) was observed in plots containing all measured values, but not for the other spectral density terms including J(0). These linear correlations reflect the effect of overall motion and similar internal motions for each CH vector in the decamer. The correlations yielded two correlation times, 3-4 ns and 10-200 ps. One value, 3-4 ns, corresponds to the value of 3.3 ns obtained for the overall isotropic tumbling correlation time determined from analysis of 13C T1/T2 ratios. The possibility of overall anisotropic tumbling was examined, but statistical analysis showed no advantage over the assumption of simple isotropic tumbling. Lack of correlations entailing J(0) implies that a relatively slow chemical exchange contributes to yielding of effective Jeff(0) values. Based on spectral density mapping and the T1/T2 ratio analysis, three basic assumptions were initially employed (and subsequently justified) for the model-free calculation: isotropic overall tumbling, one internal motion, and the presence of chemical exchange terms. Except for terminal residues, the order parameter S2 and the corresponding fast internal motion correlation time were determined to be about 0.8 +/- 0.1 and 20 +/- 20 ps, respectively, for the various CH vectors. Only a few differences were observed between or within sugars and bases. The internal motion is very fast (ps-ns time scale) and its amplitude restricted; e.g., assuming a simple wobble-in-a-cone model, the internal motion is restricted to an angular amplitude of +/-22. 5 degrees for each of the 1', 3', 4', 2, and 8 positions in the purine nucleotides in the entire duplex.

Adenosine↗

Intradural extension of mucocele complicating frontoethmoid sinus osteoma: case report.

BACKGROUND: Osteomas of the paranasal sinus are often asymptomatic and are sometimes complicated by mucoceles, but intradural extension of such a mucocele has rarely been reported. CASE DESCRIPTION: This 67-year-old man with complaint of headache was diagnosed as having an intradural extension of a mucocele complicating an osteoma of the frontoethmoid sinus. A right frontobasal craniotomy was performed and a mucocele in the frontal sinus extending into the frontal lobe through two dural defects and the osteoma was removed completely. The patient was successfully treated without recurrence. CONCLUSION: The importance of radical surgery for such lesions and the relationship between osteomas and mucoceles are discussed.

Aged↗

Development of glycosylated human interleukin-1alpha, neoglyco IL-1alpha, coupled with D-galactose monosaccharide: biological activities in vivo.

In our previous study, a galactose monosaccharide with C9 spacer was chemically coupled to recombinant human interleukin 1alpha (rhIL-1alpha) in order to study the effect of glycosylation on its activities, and to develop IL-1 with less deleterious effects. The glycosylated IL-la exhibited reduced activities in vitro by 10 to 10000-fold depending upon different aspects of activities addressed. The affinity to type I and II IL-1 receptors were also reduced. In this study we examined a variety of IL-1 activities in vivo, including upregulation of serum levels of IL-6, alpha1-acid glycoprotein, NOx, corticosterone, downregulation of serum level of glucose, and recovery of peripheral white blood cells (WBCs) from myelosuppression in 5-fluorouracil-treated mice. In contrast to the biological activities in vitro, these activities in vivo were uniformly reduced by only about 10 to 20-fold compared to untreated IL-1alpha.

Animals↗

Provirus load in patients with human T-cell leukemia virus type 1 uveitis correlates with precedent Graves' disease and disease activities.

We previously demonstrated the increased provirus load in the peripheral blood of patients with human T-cell leukemia virus type 1 (HTLV-1) uveitis (HU). To delineate the relevance of the increased provirus load to clinical and immunologic parameters, we studied the correlation between them. Seventy-nine HU patients (24 male and 55 female) were included in the study, with their informed consent. Plasma samples and genomic DNA of the peripheral blood mononuclear cells were isolated and the provirus load was estimated by semi-quantitative polymerase chain reaction of the gag region sequence. Serum levels of anti-HTLV-1 antibodies and soluble IL-2R were determined by electrochemiluminescence immuno assay and by ELISA, respectively. Disease activities were assessed and graded 0 to 4 according to the evaluation system. Recurrence of the disease during the follow-up period was diagnosed ophthalmologically. The provirus load was significantly higher in the HU patients after Graves' disease (GD) than in those without GD (P<0.05). It correlated with disease activities assessed in terms of vitreous inflammation and interval to recurrence (both P<0.05). In the HU patients without GD, it correlated with the serum levels of soluble IL-2 receptor (P<0.01), and nearly with those of HTLV-1 antibody (P=0.063). These correlations were not found in the HU patients after GD under methimazole treatment. The results suggested a direct involvement of HTLV-1-infected cells in the pathogenesis of uveitis, and raise the possibility that hyperthyroidism may contribute to the clonal expansion of HTLV-1-infected cells.

Graves Disease↗

Role of matrix in an early postentry step in the human immunodeficiency virus type 1 life cycle.

The matrix protein of human immunodeficiency virus type 1 (HIV-1) has been reported to play a crucial role in the targeting of the Gag polyprotein precursor to the plasma membrane and in the incorporation of viral envelope glycoproteins into budding virions. In this report, we present evidence that mutation of a highly conserved Leu at matrix amino acid 20 blocks or markedly delays virus replication in a range of cell types, including T-cell lines, primary human peripheral blood mononuclear cells, and monocyte-derived macrophages. These mutations do not impair virus assembly and release, RNA encapsidation, or envelope glycoprotein incorporation into virions but rather cause significant defects in an early step in the virus life cycle, as measured by single-cycle infectivity assays and the analysis of viral DNA synthesis early postinfection. This infectivity defect is independent of the type of envelope glycoprotein carried on mutant virions; similar results are obtained in pseudotyping experiments using wild-type or truncated HIV-1 envelope glycoproteins, the amphotropic murine leukemia virus envelope, or the vesicular stomatitis G protein. Intriguingly, matrix residue 20 mutations also increase the apparent binding of Gag to membrane, accelerate the kinetics of Gag processing, and induce defects in endogenous reverse transcriptase activity without affecting virion density or morphology. These results help elucidate the function of matrix in HIV-1 replication.

Amino Acid Sequence↗

Mutation/polymorphism scanning of glucose-6-phosphatase gene promoter in noninsulin-dependent diabetes mellitus patients.

Glucose-6-phosphatase (G6Pase) catalyzes the rate-limiting step of gluconeogenesis, and hepatic G6Pase activity is increased in diabetes. We have cloned and analyzed the human G6Pase gene promoter region and identified putative regulatory sequences for insulin, cAMP, glucocorticoid, and hepatocyte nuclear factors. The promoter region of the G6Pase gene was analyzed in 154 noninsulin-dependent diabetes mellitus patients and 90 control subjects by PCR-single strand conformation polymorphism and direct sequencing methods. Polymorphisms were not found in any subjects. The results suggested that in noninsulin-dependent diabetic patients, the major cause of the hepatic glucose overproduction was not attributed to dysregulation of the G6Pase gene due to mutation/polymorphism of its promoter region.

Adolescent↗

[Estimated prevalence rate of HTLV-I uveitis in Chikugo].

To estimate the prevalence rate of human T-cell lymphotropic virus type 1 (HTLV-I) uveitis, an epidemiological survey was carried out in the Chikugo District of Fukuoka Prefecture between 1 September and 31 October 1995. The survey was done by sending questionnaires on uveitis patients to all ophthalmological institutes in the district and measuring the serum antibody to HTLV-I. The recovery rate of the survey was 39 of 48 institutes (81.3%). A total of 357 patients with uveitis were reported in the survey, of whom 317 (88.8%) were tested for their seropositivity to HTLV-I. Among them, 18 patients were counted as having HTLV-I uveitis (HU) on 1 October 1995. Based on these data together with the age- and sex-specific population of HTLV-I carriers in the Chikugo District, it was estimated that the crude prevalence rates of HU per 100,000 HTLV-I carrier population were 58.6 in males, 152.0 in females, and 112.2 in both sexes. The prevalence rate of HU was slightly higher than that of HTLV-I associated myelopathy.

Adult↗

Proteoglycans in the nucleus pulposus of canine intervertebral discs after chondroitinase ABC treatment.

Experimental chemonucleolysis of the canine intervertebral disc with chondroitinase ABC and chymopapain was compared during a 52-week period. Roentgenograms and magnetic resonance imaging were used to examine changes in disc space and water content, respectively. Disc space narrowing and reductions in disc water content after chondroitinase ABC treatment were less than that after chymopapain. High-performance liquid chromatography was performed to measure changes in proteoglycans. Similarly to chymopapain, chondroitinase ABC degrades proteoglycans in the nucleus pulposus and decreases their quantity. However, large differences in the molecular weight and acidity of the resynthesized proteoglycans and in the chain length of the resynthesized glycosaminoglycans were observed between the two enzymes. The difference in disc space narrowing and the changes in disc water content between the two enzymes might result from differences in the characteristics of the resynthesized proteoglycans.

Acids↗

NMR with 13C, 15N-doubly-labeled DNA: the Antennapedia homeodomain complex with a 14-mer DNA duplex.

Nearly complete 1H, 13C and 15N NMR assignments have been obtained for a doubly labeled 14-base pair DNA duplex in solution both in the free state and complexed with the uniformly 15N-labeled Antennapedia homeodomain. The DNA was either fully 13C, 15N-labeled or contained uniformly 13C, 15N-labeled nucleotides only at those positions which form the protein-DNA interface in the previously determined NMR solution structure of the Antennapedia homeodomain-DNA complex. The resonance assignments were obtained in three steps: (i) identification of the deoxyribose spin systems via scalar couplings using 2D and 3D HCCH-COSY and soft-relayed HCCH-COSY; (ii) sequential assignment of the nucleotides via 1H-1H NOEs observed in 3D 13C-resolved NOESY; and (iii) assignment of the imino and amino groups via 1H-1H NOEs and 15N-1H correlation spectroscopy. The assignment of the duplex in the 17 kDa protein-DNA complex was greatly facilitated by the fact that 1H signals of the protein were filtered out in 13C-resolved spectroscopy and by the excellent carbon chemical shift dispersion of the DNA duplex. Comparison of corresponding 13C chemical shifts of the free and the protein-bound DNA indicates conformational changes in the DNA upon complex formation.

Antennapedia Homeodomain Protein↗

Nucleotide sequence analysis of the HLA class I region spanning the 237-kb segment around the HLA-B and -C genes.

To elucidate the detailed gene organization of the human leukocyte antigen (HLA) class I region on chromosome 6, seven contiguous cosmid genomic clones covering the 237-kb segment around the HLA-B and -C loci were subjected to DNA sequencing by the shotgun strategy to give a single contig of 236,822 bp from the MICA gene (58.2 kb centromeric of HLA-B) to 90.8 kb telomeric of HLA-C. This region was confirmed to contain four known genes, MICA, HLA-17, HLA-B, and HLA-C, from centromere to telomere. Further, a new member of the P5 multicopy genes was found to be about 1.3 kb upstream of the HLA-17 gene and designated P5.8. Five novel genes designated NOB1-5 were identified by RT-PCR and Northern blot hybridization. In addition, two pseudogenes, dihydrofolate reductase pseudogene (DHFRP) and ribosomal protein L3 homologous gene (RPL3-Hom), were also found in the vicinity of the HLA-B and -C genes, respectively. The two segments (about 40 kb) downstream of the HLA-B and HLA-C genes showed high sequence homology to each other, suggesting that segmental genome duplication including the major histocompatibility complex (MHC) class I gene must have occurred during the evolution of the MHC.

Blotting, Northern↗

NMR analysis of the hydrogen bonding interactions of the RNA-binding domains of the Drosophila sex-lethal protein with target RNA fragments with site-specific [3-15N]uridine substitutions.

It has been reported that a 183 residue fragment, consisting of the two RNA-binding domains (RBD1- RBD2) of the Drosophila melanogster Sex-lethal (Sxl) protein, strongly binds an oligonucleotide of the target RNA sequence (5'-GUUUUUUUUC-3') that regulates alternative splicing, and forms four or five hydrogen bonds with the imino groups of the RNA. In the present study, we used site-directed mutagenesis to improve the solubility of the didomain fragment of Sxl, and confirmed that this mutant fragment forms hydrogen bonds with the target RNA in the same manner as that of the wild-type fragment. The mutant fragment was shown to bind the cognate RNA sequences GUUUUUUUUC and AUUUUUUUUC more tightly than UUUUUUUUC. By using a [3-15N]uridine phosphoramidite, we synthesized a series of15N-labeled target RNAs, in which one of the uridine residues was specifically replaced by [3-15N]uridine. By observing the imino1H-15N coupling of the labeled uridine residue, we assigned all four of the hydrogen-bonded imino protons to U1, U2, U5 and U6, respectively, of the target RNA. The imino protons of U2 and U6 exhibited nuclear Overhauser effects with aliphatic protons of the protein. All these results indicate that the A/G, U1, U2, U5 and U6 residues in the target sequence of (G/A)UUUUUUUU are specifically recognized by the two RNA-binding domains of the Sxl protein.

Animals↗

Characterization of human immunodeficiency virus type 1 matrix revertants: effects on virus assembly, Gag processing, and Env incorporation into virions.

The matrix protein of human immunodeficiency virus type 1 (HIV-1) has been postulated to serve a variety of functions in the virus life cycle. Previously, we introduced a large number of mutations into the HIV-1 matrix and determined the effects on virus replication. These studies identified domains involved in virus assembly and release and envelope glycoprotein incorporation into virions. Here we describe the identification and characterization of viral revertants containing second-site changes in the matrix which compensate for the effects of four of the original mutations on matrix function. Specifically, mutations at matrix residues 4 and 6 severely impaired virus assembly and release; substitutions at residues 4 and 6 reversed the phenotype of the amino acid 4 change while second-site mutations at matrix positions 10, 69, and 97 partially or fully reversed the phenotype of the amino acid 6 substitution. A mutation at matrix residue 62 reversed the effect of a position 34 change which blocks envelope glycoprotein incorporation into virions, and substitutions at residues 27 and 51 reversed the phenotype of a position 86 mutation which redirects virus assembly to the cytoplasm. In addition to determining the effects of the compensatory changes in the context of the original mutations, we also introduced and analyzed the second-site changes alone in the context of the wild-type molecular clone. The data presented here define potential intermolecular and intramolecular interactions which occur in the matrix during the virus life cycle and have implications for our understanding of the relationship between matrix structure and function.

Amino Acid Sequence↗

Mitochondrial abnormalities in a murine model of primary carnitine deficiency. Systemic pathology and trial of replacement therapy.

Mitochondrial abnormalities and effectiveness of replacement therapy were examined in a murine model of systemic carnitine deficiency, namely the juvenile visceral steatosis (JVS) mouse. Homozygous JVS mice revealed severe lipid deposition and abnormal mitochondria in liver, heart, skeletal muscle, and kidney, but there was no pathological change in the nervous system, though they showed cerebral signs. There were numerous ragged-red fibers in muscles, but enzyme activities of the respiratory chain were intact. Histograms of oxidative and nonoxidative muscle fibers showed an increase in small and oxidative muscle fibers in 4-week-old JVS mice, but this difference no longer existed in 8-week- or 1-year-old JVS mice. On the contrary, Mn-superoxide dismutase immunostaining of muscle showed a focal increase in every age of JVS mice. With L-carnitine treatment, JVS mice could survive for a year, but to some extent, there were the same pathological changes as those seen in untreated mice.

Animals↗

Differential central modulation of the baroreflex by salt loading in normotensive and spontaneously hypertensive rats.

In salt-sensitive hypertensive animal models and human subjects compared with their salt-resistant counterparts, sympathetic activity is abnormally enhanced during a high salt diet. We examined whether salt loading differentially modulates the arterial baroreceptor reflex (ABR), a major control mechanism of arterial pressure and sympathetic vasomotor activity, in young normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Six-week-old WKY and SHR were fed a normal (0.66%) or high (8.00%) salt diet for 4 weeks. After the diet regimen, baseline levels of mean arterial pressure (MAP), renal sympathetic nerve activity (RSNA), and the overall and central properties of the ABR were compared among the four groups of rats under halothane anesthesia. In WKY, a high salt diet did not affect baseline arterial pressure and RSNA but potentiated the ABR, as evidenced by an increase in the maximal slope of MAP-RSNA and MAP-heart rate relationships. In SHR, by contrast, salt loading accelerated hypertension and sympathetic overactivity and impaired the ABR. Salt-induced modulation of the ABR was associated with that of the central property, since reflex inhibition of RSNA by stimulation of the aortic depressor nerve was augmented in WKY and attenuated in SHR. These results suggest that differential modulation of the central mechanism subserving the baroreflex control of sympathetic activity at least partly accounts for the difference in salt sensitivity between WKY and SHR.

Animals↗

Reactions between hydroxyl-radical-induced 7,8-dihydro-8-oxo-2'-deoxyguanosine precursor and the spin trap alpha-phenyl-N-tert-butylnitrone.

An N2O-saturated aqueous solution containing 2'-dG and the spin trap agent PBN was examined by ESR and HPLC-ECD methods after X irradiation. ESR examination showed that the ESR spectrum obtained consisted of signals due to the PBN-OH and PBN-H adducts. The signal intensity of PBN-H adducts was larger in the presence of 2'-dG than in the absence of 2'dG, while that of PBN-OH adducts was smaller in the presence of 2'-dG than in the absence of 2'-dG. When the OH-radical-induced 8-oxodG was measured by HPLC-ECD, the yield of 8-oxodG was found to be enhanced about twofold in the presence of PBN. By contrast, usual OH-radical scavengers (DMSO, sodium formate and mannitol) inhibited the formation of 8-oxodG beyond expectation. The enhancement of the yield of PBN-H adducts by 2'-dG and the enhancement of the 8-oxodG formation by PBN were explained by the electron transfer and subsequent proton transfer reactions from OH-radical-induced guanine-N7 radicals to PBN to form 8-oxodG and PBN-H. The present study led us to conclude that PBN reacted with the precursor radical of 8-oxodG to accelerate the formation of 8-oxodG, while OH-radical scavengers reacted with it to diminish the formation of 8-oxodG.

8-Hydroxy-2'-Deoxyguanosine↗

A survey of the values of clinical chemistry parameters obtained for a common rat blood sample in ninety-eight Japanese laboratories.

A control survey was conducted to check the accuracy of automated analyzers used in the evaluation of clinical chemistry parameters in nonclinical toxicology studies. Pooled serum samples from male Sprague-Dawley rats were delivered refrigerated to each facility 98 laboratory facilities throughout Japan within 18 hours after sample preparation and analyzed. Commercially available normal human serum samples from a single lot were also analyzed at the same time. Survey results were divided into three categories. (1) Parameters with small coefficient of variation (CV) values for both rat and human serum samples included protein, glucose, cholesterol (CHO), urea nitrogen (UN), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), and inorganic phosphate (IP). Definition of normal values in rats should be straight forward for these parameters. (2) Parameters with large CV values, but with a relatively good correlation between rat and human values include triglycerides (TG), glutamic oxaloacetic transaminase/aspartate aminotransferase (GOT/AST), glutamic pyruvic transaminase/alanine aminotransferase (GPT/ALT), and alkaline phosphatase (ALP). Measurements based on different principles gave different mean values, and this values contributed to the increase in CV values. Assessment of normal values would require a consideration of the measurement principles. (3) Parameters with large CV values only in rat serum samples included albumin (albumin/globulin ratio: A/G ratio), creatinine (CRE), and total bilirubin(BIL). Reactivity was different in rat albumin (ALB), depending on the reagents used. This difference needs to be corrected with values available by electrophoresis, or adjusted by rat ALB values, because of the lack of an appropriate measurement method. The enzyme method gave low values for rat CRE, which suggests the need for further examination of this method. The BIL values were extremely low in rat samples. It seems to be necessary to select appropriate methods to measure clinical pathology parameters correctly for rats. There was no deviation in values due solely to the mechanical operations of the analytical equipment. Non-standard initial settings of the equipment (equipment originally intended for human samples, but now applied to animal samples) was the main cause of the wide range of analytical values seen.

Albumins↗