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Biomedical subjects

Y Kitagawa

Publications and source records attributed to Y Kitagawa.

At least 19 recordsLinked to original sources

Site-specific and random fragmentation of Cu,Zn-superoxide dismutase by glycation reaction. Implication of reactive oxygen species.

Site-specific and random fragmentation of human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) was observed following the glycation reaction (the early stage of the Maillard reaction). The fragmentation proceeded in two steps. In the first step, Cu,Zn-SOD was cleaved at a peptide bond between Pro62 and His63, as judged by amino acid analysis and sequencing of fragment peptides, yielding a large (15 kDa) and a small (5 kDa) fragment. In the second step, random fragmentation occurred. The ESR spectrum of the glycated Cu,Zn-SOD suggested that reactive oxygen species was implicated in the both steps of fragmentation. The same fragmentations were seen upon exposure of the enzyme to an H2O2 bolus. Catalase completely blocked both steps of the fragmentation process, whereas EDTA blocked only the second step. Incubation with glucose resulted in a time-dependent release of Cu2+ from the Cu,Zn-SOD molecule. The released Cu2+ then likely participated in a Fenton's type of reaction to produce hydroxyl radical, which may cause the nonspecific fragmentation. Evidence that EDTA abolished only the second step of fragmentation induced by an H2O2 bolus supports this mechanism. This is the first report that a site-specific fragmentation of a protein is caused by reactive oxygen species formed by the Maillard reaction.

Amino Acid Sequence

Molecular analysis of rice dwarf phytoreovirus segment S1: interviral homology of the putative RNA-dependent RNA polymerase between plant- and animal-infecting reoviruses.

We have determined the complete nucleotide sequence of the largest segment S1 of rice dwarf phytoreovirus (RDV), a member of the family Reoviridae. S1 is 4423 nucleotides long with a segment-specific inverted repeat located adjacent to the conserved termini (5'GGCAAA---UGAU3'). A major open reading frame (bases 36 to 4367) on the S1 plus strand, which is preceded by a minicistron (bases 6 to 29), encodes the polypeptide (P1) consisting of 1444 amino acids with a M(r) of 164, 142. The sense-strand transcript derived from the full-length S1 cDNA, the minicistron of which was abolished, directed the synthesis of a polypeptide of 170 kDa in addition to smaller polypeptides in wheat germ extracts, and the 170-kDa product comigrated with the minor core protein in SDS-polyacrylamide gel. Thus, P1 is assumed to be localized in the viral core particle. The consensus sequence element conserved in RNA-dependent RNA polymerase is observed in the P1 amino acid sequence predicted from the nucleotide sequence. Based on the dendrogram established from the sequence alignment around the polymerase module region, and sequence identity within the alignment, P1 of plant-infecting RDV was evolutionarily compared with VP1, lambda 3, and VP1 of three other animal-infecting members of the family, rota-, reo-, and bluetongue viruses. Consequently, RDV S1 was shown to be more closely related to the rotavirus gene segment 1, in terms of molecular evolution, than the animal-infecting members are to one another.

Amino Acid Sequence

[Study of an outbreak of itai-itai disease].

We studied an outbreak of itai-itai disease in the Jinzu River basin, in Toyama, Japan. One hundred and fifty females recognized as itai-itai disease patients till by 1990 were studied for the ages and years of onset and residence in the cadmium-polluted area. 1. Cases were recognized from as early as 1929, increased gradually to the peak of 1955-1959 and rapidly decreased up to the 1970s. 2. It was found that the later the patients was born, the younger the age of onset, though there was no difference of ages of onset between the cases born in the 1910s and the cases born from 1920. 3. The onset of itai-itai disease was most frequently seen at 50-59 years of residence in the cadmium-polluted area. It was found that the later a person started to inhabit the cadmium-polluted area, the shorter the period of residence in the cadmium-polluted area up to onset of itai-itai disease. 4. Comparing the patients who inhabited the cadmium-polluted area from birth and those who had moved there from non-polluted areas, the age of onset was higher in the latter, but there were no significant differences in the period of residence up to onset. From these findings, it appeared that itai-itai disease was not caused by aging, but by cadmium exposure starting from the 1910s.

Cadmium Poisoning

Turner's syndrome associated with Hashimoto's thyroiditis and sarcoidosis: a case report.

A 32-yr-old Japanese female who was suffering from Turner's syndrome associated with Hashimoto's thyroiditis and sarcoidosis is reported. It is known that Hashimoto's thyroiditis is not infrequently complicated with Turner's syndrome on the basis of an abnormal X chromosome. In this case, sarcoidosis occurred 4 months after Hashimoto's thyroiditis. This extremely interesting coincidence of Hashimoto's thyroiditis and sarcoidosis in Turner's syndrome has not been reported to date to our knowledge.

Adult

Prediction of insulin dependent diabetes mellitus in non-obese diabetic mice by the endogeneous tumor necrosis factor-alpha level.

The non-obese diabetic (NOD) mouse is an animal model of human insulin dependent diabetes mellitus (IDDM). In this strain, the serum concentration of tumor necrosis factor-alpha (TNF alpha) after OK432 (a streptococcal preparation) stimulation is much lower than in any other non-diabetic control strain. Female NOD mice which have a higher incidence of diabetes have significantly lower TNF alpha level (6.5 +/- 4 U/ml, mean +/- SEM) than do male NOD mice (21 +/- 5 U/ml) (P < 0.02) which have lower incidence of diabetes. On the basis of these results, we designed a prospective study to evaluate the relationship between the serum TNF alpha concentration and the incidence of diabetes in individual male NOD mice. Mice were studied until 30 weeks of age. During this period four of eight mice with a low TNF alpha level (TNF alpha < or = 1.1 U/ml) became diabetic, whereas none of eighteen mice with a high TNF alpha level (TNF alpha > 1.1 U/ml) developed overt diabetes. These results indicate that by measuring of endogeneous TNF alpha level after stimulation by OK432, one could predict IDDM in male NOD mice.

Aging

[A multicenter, fixed-flexible dose study of terazosin hydrochloride in the treatment of symptomatic benign prostatic hypertrophy].

In this study the multicenter, fixed-flexible dose regimen was taken to evaluate the effective dose range of Terazosin for the treatment of micturition disturbance in benign prostatic hypertrophy (BPH) and to clarify the characteristics of patients who are more responsive to Terazosin therapy. After a 1-week washout (placebo) the first two weeks 1 mg/day of Terazosin was administered, then depending on efficacy of subjective symptoms, Terazosin doses were increased up to 2 mg/day and 4 mg/day at intervals of two weeks. After six weeks the final efficacy and safety were assessed. The subjective symptom improvement rate was 18.5% by 1 mg/day, 55.6% by 2 mg/day and 65.4% by 4 mg/day cumulatively. The objective symptom improvement rate were 13.2% by 1 mg/day, 42.1% by 2 mg/day and 50.0% by 4 mg/day cumulatively. The global improvement rate was 14.5% by 1 mg/day, 50.0% by 2 mg/day and 61.8% by 4 mg/day cumulatively. The patients who had a higher subjective symptom score in the lead-in period were more improved rather than those who had a lower score. In objective symptoms, voided volume, maximum flow rate (MFR), MFR nomogram score and average flow rate improved and the ratio of residual urine volume decreased. There was no relationship between clinical improvement on either subjective or objective symptoms and prostatic weight. Adverse reactions, such as dizziness, vertigo, tinnitus, nausea and blurred vision; were seen in 10 cases. In conclusion Terazosin was effective and well tolerated for the treatment of patients who had micturition disturbance with BPH in the dose range of 2 to 4 mg/day.

Adrenergic alpha-Antagonists

Nonmetric tooth crown traits in the Ami tribe, Taiwan aborigines: comparisons with other east Asian populations.

The frequencies of occurrence of 17 tooth crown traits in the living Ami tribe, which inhabits the east coast of Taiwan, were investigated and compared with other East Asian populations based on Turner's (1987) Mongoloid dental variation theory. Principal coordinate analysis based on Smith's mean measure of divergence using frequencies of the 17 traits suggests that the Ami tribe together with the Yami tribe and the Bunun tribe is included in the sinodont group typical of the Chinese mainland and northeast Asia. In light of these results and the estimated distribution of sinodonty and sundadonty in the past and the present, we speculate that the gene flow from Chinese mainlanders to native sundadonts, who seem to have migrated northward to Taiwan, contributed significantly to the formation of the living Taiwan aboriginal groups, sinodonts. Among the aboriginal tribes of Taiwan, the Ami have characteristics intermediate between those of the Yami and the Bunun. The relative positions of these tribes in East Asian populations suggests that the extent of sinodontification and of genetic isolation is one of the causes of the intertribal variation.

Adolescent

[Rapid detection of mecA gene by nested PCR for diagnosis of methicillin resistance in Staphylococcus aureus].

In recent years, the most common causative organism of hospital infections has been methicillin resistant Staphylococcus aureus (MRSA). The major mechanism of beta-lactam resistance in MRSA is attributed to the production of a specific penicillin binding protein (PBP2'), which is a product of mecA gene, with extremely low binding affinities to beta-lactams. In the present study, we have established a rapid identification method of MRSA by sensitive detection of mecA gene using nested PCR. Nested PCR method amplifying the target DNA in two steps enhanced the efficiency of the second round amplification. By means of this method, mecA gene was successfully detected in clinical samples, such as blood, pus, sputum and feces within 3-4 hrs. Rapid diagnosis of MRSA-bacteremia is particularly important for prevention of sever systemic infection. There are some strains of S. aureus which possess mecA gene in spite of low minimal inhibitory concentration of DMPPC. In these strains expression of mecA gene is induced by contact of beta-lactams and they obtain methicillin resistance. Using nested PCR method, these latent MRSA are rapidly and certainly detectable. This method should be useful for early and effective detection of MRSA hospital infections.

Cross Infection

Protein kinase C of a human megakaryoblastic leukemic cell line (MEG-01). Analysis of subspecies and activation by diacylglycerol and free fatty acids.

Protein kinase C (PKC) from a human megakaryoblastic leukemic cell line (MEG-01) was resolved into two fractions by hydroxyapatite column chromatography, which are indistinguishable from the brain type II (beta I/beta II) and type III (alpha) subspecies, by biochemical and immunoblot analysis. In the presence of both phosphatidylserine and diacylglycerol, several free unsaturated fatty acids (FFA's), such as arachidonic, oleic, linoleic and linolenic acids, further enhanced the enzyme activation, and allowed the enzyme to exhibit almost full activity at nearly basal levels of Ca2+ concentration. The concentration of unsaturated FFA's giving rise to the maximum enzyme activation was around 2 x 10(-5) M. Palmitic and stearic acids were inactive. The result implies that, in addition to diacylglycerol, the receptor-mediated release of unsaturated FFA's from membrane phospholipids may also take part in the activation of PKC.

Arachidonic Acid

Identification of the promoter region of the rat protein phosphatase 2A alpha gene.

We have cloned a genomic fragment containing the promoter region of the rat protein phosphatase 2A alpha gene (PP-2A alpha). A 1.6 kb fragment of the 5' flanking region was sequenced. Three major transcriptional initiation sites were identified by the primer extension method using rat liver mRNA and found to be located 225, 222 and 220 bases upstream of the translational initiation site, respectively. Bacterial chloramphenicol acetyltransferase (CAT) assay revealed that a 503 bp SmaI fragment containing the transcriptional initiation sites had promoter activity, which was stronger than that of the SV40 early promoter on the pSV2CAT plasmid when introduced into NIH3T3 cells. Deletion of a 119 bp SacII fragment decreased its promoter activity considerably. The promoter region has an extremely high GC content and does not contain either a 'TATA box' or a 'CAAT box' suggesting that this promoter can be classified as that of a 'house keeping' gene, although there is only one typical GC-box (GGGCGG) immediately preceding the transcriptional initiation sites. There is a 10 base pair palindrome, 5'-GTGACGTCAC-3', 26 base pairs upstream of the +1 transcriptional initiation site, which is highly conserved in many other genes, whose expression is regulated by cAMP. The promoter activity was shown to be increased by forskolin treatment (10 microM) in NIH3T3 cells.

Amino Acid Sequence

Hypoxic depression of mitochondrial mRNA levels in HeLa cell.

Steady-state levels of 12 S and 16 S mitochondrial (mt) rRNAs and four mRNAs (NADH dehydrogenase subunits 3 and 4/4L, cytochrome c oxidase subunit III, H(+)-ATPase subunits 6/6L) were estimated by hybridization of cellular RNA with cloned human mt DNA fragments during hypoxia of HeLa cells. When the partial pressure of oxygen (pO2) was shifted from 135 to 15 Torr, the level of all mRNAs coordinately decreased more than 95% in 48 h, while that of rRNAs remained virtually unchanged. mRNA levels recovered within 4-6 h of reexposure to normoxia. The depression was observed at pO2 less than 40 Torr, the physiological pO2 in peripheral tissues. During these transitions, the growth rate of HeLa cells and the copy number of mt DNA per cell remained unchanged. The degradation rate of mt mRNAs in the presence of cordycepin was not affected by pO2. In contrast to the in vivo results, the potential activity of transcription in isolated mitochondria assayed under optimum conditions was not affected by previous hypoxic exposure of the cells. These observations provide evidence for the existence of a new mechanism controlling mitochondrial gene transcription.

Adenosine Triphosphate

Islet cells but not thyrocytes are susceptible to lysis by NK cells.

BB rats develop both pancreatic insulitis and lymphocytic thyroiditis, but whereas spontaneous autoimmune diabetes is common, hypothyroidism is rare. Splenic natural killer (NK) cells from acutely diabetic (AD) BB rats and from athymic nude rats are known to be cytotoxic to rat islet cells in vitro. To investigate possible differential tissue susceptibility to lysis by NK cells or their cytokines such as cytolysin (perforin) or NK cytotoxic factor (NKCF), we used an in vitro 51Cr-release assay to measure the cytotoxicity of splenocytes, cytolysin or NKCF against Wistar Furth (WF) and Fischer 344 (F-344) rat islet cells, and FRTL-5 F-344-derived and WRT Wistar-derived rat thyrocytes. The results demonstrated that spleen cells from AD-BB (RT1u) rats and athymic F-344 nude (RT11) rats are cytotoxic to WF (RT1u) islets and F-344 (RT11) islets, but not to FRTL-5 (RT11) or WRT (class I RT11) thyrocytes. WF and F-344 rat spleen cells were not cytotoxic to any of these cells. Thyrocytes are known to express class II molecules on their surface in chronic thyroiditis. We found that treatment of thyrocytes with interferon-gamma (IFN-gamma) induced class II expression but did not increase the cytotoxicity of splenocytes against these cells. Cytolysin and NKCF were both cytotoxic to islets in a dose dependent manner, but FRTL-5 thyrocytes were resistant to killing by these cytokines. These findings suggest that islet cells and thyrocytes in vitro are differentially susceptible to lysis by NK cells.

Animals

Three-dimensional structure of Cu,Zn-superoxide dismutase from spinach at 2.0 A resolution.

The three-dimensional structure of Cu,Zn-superoxide dismutase from spinach leaves has been determined by X-ray crystal structure analysis. The atomic coordinates were refined at 2.0 A resolution using the Hendrickson and Konnert program for stereochemically restrained refinement against structure factors, which allowed the use of non-crystallographic symmetry. The crystallographic residual error for the refined model was 24.9%, with a root mean square deviation of 0.03 A from the ideal bond length and an average atomic temperature factor of 9.6 A. A dimeric molecule of the enzyme is comprised of two identical subunits related by a non-crystallographic 2-fold axis. Each subunit of 154 amino acid residues is composed primarily of eight anti-parallel beta-strands that form a flattened cylinder, plus three external loops. The main-chain hydrogen bonds primarily link the beta-strands. The overall structure of this enzyme is quite similar to that of the bovine dismutase except for some parts. The single disulfide bridge (Cys57-Cys146) and the salt bridge (Arg79-Asp101) may stabilize the loop regions of the structure. The Cu2+ and Zn2+ ions in the active site lie 6.1 A apart at the bottom of the long channel. The Cu2+ ligands (ND1 of His-46, and NE2 of His-48, -63, and -120) show an uneven tetrahedral distortion from a square plane. The Zn2+ ligands (ND1 of His-63, -71, and -80 and OD1 of Asp-83) show an almost tetrahedral geometry. The imidazole ring of His-63 forms a bridge between the Cu2+ and Zn2+ ions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence

Crystal structure of basic fibroblast growth factor at 1.6 A resolution.

We have determined the crystal structures of two types of human basic fibroblast growth factor, the serine analogue and the wild-type, at 1.6 and 2.5 A resolution, respectively. Two good heavy atom derivatives were found and used for multiple isomorphous replacement phasing. The atomic coordinates were refined using the Hendrickson & Konnert program for stereochemically restrained refinement against structure factors. The crystallographic R factors were reduced to 15.3% for the serine analogue structure and 16.0% for the wild-type structure. The serine analogue and wild-type structures have been found to be almost identical, the root-mean-square deviation between the corresponding C alpha atoms being 0.11 A. Their structures are composed of twelve beta-strands forming a barrel and three loops. Their molecules have an approximate threefold internal symmetry and are similar in architecture to that of interleukin-1 beta. A possible heparin-binding site, which comprises five basic residues, Lys119, Arg120, Lys125, Lys129, and Lys135, has been revealed by calculating the electrostatic potential energy.

Amino Acid Sequence

Phosphorylation of type II (beta) protein kinase C by casein kinase II.

Rat brain type II (beta) protein kinase C (PKC) was phosphorylated by rat lung casein kinase II (CK-II). Neither type I (gamma) nor type III (alpha) PKC was significantly phosphorylated by CK-II. CK-II incorporated 0.2-0.3 mol of phosphate into 1 mol of type II PKC. This phosphate was located at the single seryl residue (Ser-11) in the V1-variable region of the regulatory domain of the PKC molecule. A glutamic acid cluster was located at the carboxyl-terminal side of Ser-11, showing the consensus sequence for phosphorylation by CK-II. The velocity of this phosphorylation was enhanced by the addition of Ca2+, diolein, and phosphatidylserine, which are all required for the activation of PKC. Phosphorylation of casein or synthetic oligopeptides by CK-II was not affected by Ca2+, diolein, or phosphatidylserine. Available evidence suggests that CK-II phosphorylates preferentially the activated form of type II PKC. It remains unknown, however, whether this reaction has a physiological significance.

Amino Acid Sequence