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N Koizumi

Publications and source records attributed to N Koizumi.

At least 37 records · Page 2Linked to original sources
PubMed2001

flaB-polymerase chain reaction (flaB-PCR) and its restriction fragment length polymorphism (RFLP) analysis are an efficient tool for detection and identification of Leptospira spp.

For establishment of a rapid-identification method of Leptospira species, a flaB gene of Leptospira was investigated and the following results were obtained. 1) HaeIII- or HindIII-restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products (793 bp) of flaB gene was effectual for the classification of species of Leptospira. 2) Twenty cells of Leptospira in 1 ml of coagulated blood and 100 cells of Leptospira in 1 ml of anti-coagulated blood could be detected by flaB-PCR. These results suggested that PCR-RFLP based on the flaB gene was an efficient tool for rapid detection and identification of species of infected Leptospira from clinical specimens.

DNA Primers↗
PubMed2001

[A case of leptospirosis infected in Borneo Island, Malaysia].

We report a case of leptospirosis infected in Sabah, Borneo island, Malaysia. The case is 25-year-old male who had participated in the EcoChallenge Sabah 2000 Expedition Race, a multisport event held during August 20 to September 3, 2000 at various sites in Sabah in Malaysian Borneo. He developed a high fever and headache on September 7, and he was admitted to our hospital on September 9. On admission he also had conjunctivitis and myalgias. Laboratory findings on admission revealed leukocytosis with left shift, slightly elevated transaminase levels, high CRP levels and proteinuria. Plasmodium spp. were negative on blood smears, and no bacteria were isolated from blood and feces cultures. We performed the laboratory tests for leptospirosis, based on the information about the probable leptospirosis outbreak among athletes who participated in the EcoChallenge Race, however both Leptospira antigens and antibodies were negative at that time. We diagnosed leptospirosis clinically because he manifested persistent symptoms, and minocycline 100 mg b.i.d. was administered intravenously resulting in excellent efficacy. Serum antibody tests by microscopic agglutination test (MAT) at convalescent stage revealed significant increased antibodies against Leptospira interrogans serovar hebdomadis, and the diagnosis of leptospirosis was confirmed. Infectious diseases have been global and it is important to have information concerning worldwide infectious disease situations as much as possible for accurate diagnosis.

Adult↗
PubMed2001

Pharmacokinetics of acetaminophen from rapidly disintegrating compressed tablet prepared using microcrystalline cellulose (PH-M-06) and spherical sugar granules.

The aim of the present study was to evaluate the bioavailability of a drug from rapidly disintegrating tablets prepared using fine spherical crystalline cellulose (PH-M-06) and spherical sugar granules (Nonpareil, NP). Rapidly disintegrating tablets containing acetaminophen as the model drug in combination with a mixture of NP-108 (purified n-mannitol) and PH-M-06 were prepared. Plasma concentration profiles and pharmacokinetic parameters of acetaminophen in rabbits were investigated after oral administration of the prepared tablets. No significant difference in Cmax and AUC(0-infinity) of acetaminophen between rapidly disintegrating tablets and conventional tablets was observed after direct administration of these tablets into the stomach of rabbits. However, tmax (15 min) of acetaminophen from rapidly disintegrating tablets was significantly (p<0.05) shorter than that from conventional tablets (130 min). The same tmax was observed for rapidly disintegrating tablets and solution. When suitable excipients such as fine spherical microcrystalline cellulose (PH-M series) and spherical sugar granules (NP series) were used, rapidly disintegrating tablets could be prepared by the conventional direct compression method. According to the results of moment analysis, the mean residence time (MRT) obtained between both rapidly disintegrating and conventional tablets indicates that the mean absorption time (MAT) from these tablets is approximately 60 and 90 min, respectively. This difference in MAT between the two tablets may be caused by the difference in the sum of the mean dissolution time (MDT) and the mean disintegration time (MDIT) of these tablets. Rapidly disintegrating tablets allow rapid absorption of the drug compared with conventional tablets.

Acetaminophen↗
PubMed2001

Molecular characterization of two Arabidopsis Ire1 homologs, endoplasmic reticulum-located transmembrane protein kinases.

A major response of eukaryotic cells to the presence of unfolded proteins in the lumen of the endoplasmic reticulum (ER) is to activate genes that encode ER-located molecular chaperones, such as the binding protein. This response, called the unfolded protein response, requires the transduction of a signal from the ER to the nucleus. In yeast (Saccharomyces cerevisiae) and mammalian cells, an ER-located transmembrane receptor protein kinase/ribonuclease called Ire1, with a sensor domain in the lumen of the ER, is the first component of this pathway. Here, we report the cloning and derived amino acid sequences of AtIre1-1 and AtIre1-2, two Arabidopsis homologs of Ire1. The two proteins are located in the perinuclear ER (based on heterologous expression of fusions with green fluorescent protein). The expression patterns of the two genes (using beta-glucuronidase fusions) are nearly nonoverlapping. We also demonstrate functional complementation of the sensor domains of the two proteins in yeast and show that the Ire1-2 protein is capable of autotransphosphorylation. These and other findings are discussed in relation to the involvement of these genes in unfolded protein response signaling in plants.

Amino Acid Sequence↗
PubMed2000

7-Methylxanthine methyltransferase of coffee plants. Gene isolation and enzymatic properties.

Caffeine is synthesized through sequential three-step methylation of xanthine derivatives at positions 7-N, 3-N, and 1-N. However, controversy exists as to the number and properties of the methyltransferases involved. Using primers designed on the basis of conserved amino acid regions of tea caffeine synthase and Arabidopsis hypothetical proteins, a particular DNA fragment was amplified from an mRNA population of coffee plants. Subsequently, this fragment was used as a probe, and four independent clones were isolated from a cDNA library derived from coffee young leaves. Upon expression in Escherichia coli, one of them was found to encode a protein possessing 7-methylxanthine methyltransferase activity and was designated as CaMXMT. It consists of 378 amino acids with a relative molecular mass of 42.7 kDa and shows similarity to tea caffeine synthase (35.8%) and salicylic acid methyltransferase (34.1%). The bacterially expressed protein exhibited an optimal pH for activity ranging between 7 and 9 and methylated almost exclusively 7-methylxanthine with low activity toward paraxanthine, indicating a strict substrate specificity regarding the 3-N position of the purine ring. K(m) values were estimated to be 50 and 12 microM for 7-methylxanthine and S-adenosyl-l-methionine, respectively. Transcripts of CaMXMT could be shown to accumulate in young leaves and stems containing buds, and green fluorescent protein fusion protein assays indicated localization in cytoplasmic fractions. The results suggest that, in coffee plants, caffeine is synthesized through three independent methylation steps from xanthosine, in which CaMXMT catalyzes the second step to produce theobromine.

Amino Acid Sequence↗
PubMed2000

Specific binding of a 14-3-3 protein to autophosphorylated WPK4, an SNF1-related wheat protein kinase, and to WPK4-phosphorylated nitrate reductase.

WPK4 is a wheat protein kinase related to the yeast protein kinase SNF1, which plays a role in catabolite repression. To identify proteins involved in signal transduction through WPK4, we performed yeast two-hybrid screens and isolated two cDNA clones designated as TaWIN1 and TaWIN2. Both encode 14-3-3 proteins that, upon autophosphorylation, bind the C-terminal regulatory domain of WPK4. Mutational analysis through amino acid substitution revealed that TaWIN1 and TaWIN2 primarily bind WPK4 through phosphoserines at the positions 388 and 418, both located in the C-terminal region. Mutations in the conserved residues of the TaWIN1 amphipathic groove impaired the ability of TaWIN1 to bind to WPK4. A screen for in vitro phosphorylation of proteins involved in nutrient metabolism revealed a putative WPK4 substrate, nitrate reductase; its hinge 1 region was efficiently phosphorylated by WPK4. Subsequent far Western blots showed that it specifically bound TaWIN1. Since nitrate reductase has been shown to be inactivated by phosphorylation upon 14-3-3 binding, the present findings strongly suggest that WPK4 is the protein kinase responsible for controlling the nitrogen metabolic pathway, assembling the nitrate reductase and 14-3-3 complex through its phosphorylation specificity.

14-3-3 Proteins↗
PubMed2000

Diverse response of rice and maize genes encoding homologs of WPK4, an SNF1-related protein kinase from wheat, to light, nutrients, low temperature and cytokinins.

The wheat gene WPK4 encodes a 56-kDa protein kinase that belongs to group 3 of the SNF1-related protein kinase family (SnRK3), and is up-regulated by light and cytokinins and down-regulated by sucrose. In order to determine whether or not this particular regulation pattern is general among plant species, we isolated and characterized homologous genes from rice and maize. Two rice genes, OsPK4 and OsPK7, encode proteins comprising 508 and 520 amino acids, and show, respectively, 75% and 76% sequence similarity to WPK4. OsPK4 and OsPK7 proteins produced in Escherichia coli were able to phosphorylate themselves and myelin basic proteins, the reaction requiring magnesium and/or manganese ions. Transcripts of OsPK4 were detected in all tissues tested, and amounts were increased upon illumination, nutrient deprivation and treatment with cytokinins. In contrast, transcripts of OsPK7 were not found in any tissues except in mature leaves at low levels, and did not accumulate under any of the stress conditions examined. A maize gene, ZmPK4, encodes a protein with 518 amino acids that shows 74% similarity to WPK4. Its transcripts were constitutively expressed in all tissues, regardless of light, nutrient and cytokinin status, but were increased upon exposure to low temperature. These results indicate that, despite the sequence similarity between their products, genes for SnRK3 proteins are differentially regulated in response to environmental stimuli.

Amino Acid Sequence↗
PubMed2000

Secreted proteins of tobacco cultured BY2 cells: identification of a new member of pathogenesis-related proteins.

Cultured cells of tobacco BY2 secrete more than 100 proteins into culture medium. Six major proteins were purified, and partial protein sequences were determined. Five of them were found to be similar to an ascorbic acid oxidase, three peroxidase isozymes and a beta-1,3-exoglucanase, respectively. A cDNA clone encoding the remaining polypeptide, whose amino acid sequence showed no similarity with earlier reported proteins, was isolated. It encoded a putative 27 kDa protein of 242 amino acids with resemblance to WCI-5, a wheat protein induced by benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) which activates genes involved in systemic acquired resistance. Transcripts of this clone accumulated upon tobacco mosaic virus infection, mechanical wounding and drought treatment, an induction profile that satisfies the definition of pathogenesis-related (PR) proteins by van Loon et al. (Plant Mol. Biol. Rep. 12 (1994) 245). No similar PR proteins have so far been reported, and therefore our newly designated NtPRp27 points to the existence of a novel PR protein family in tobacco plants.

Abscisic Acid↗
PubMed2000

Functional role of Ca(2+)-binding site IV of scallop troponin C.

Scallop troponin C (TnC) binds only one Ca(2+)/mol and the single Ca(2+)-binding site has been suggested to be site IV on the basis of the primary structure [K. Nishita, H. Tanaka, and T. Ojima (1994) J. Biol. Chem. 269, 3464-3468; T. Ojima, H. Tanaka, and K. Nishita (1994) Arch. Biochem. Biophys. 311, 272-276]. In the present study, the functional role of Ca(2+)-binding site IV of akazara scallop (Chlamys nipponensis akazara) TnC in Ca(2+)-regulation was investigated using a site-directed mutant with an inactivated site IV (TnC-ZEQ), N- and C-terminal half molecule mutants (TnC(N) and TnC(C)), and wild-type TnC (TnC(W)). Equilibrium dialysis using (45)Ca(2+) demonstrated that TnC(W) and TnC(C) bind 0.6-0.8 mol of Ca(2+)/mol, but that TnC-ZEQ and TnC(N) bind virtually no Ca(2+). The UV difference spectra of TnC(W) and TnC(C) showed bands at around 280-290 nm due to the perturbation of Tyr and Trp upon Ca(2+)-binding, while TnC-ZEQ and TnC(N) did not show these bands. In addition, TnC(W) and TnC(C) showed retardation of elution from Sephacryl S-200 upon the addition of 1 mM CaCl(2), unlike TnC-ZEQ and TnC(N). These results indicate that Ca(2+) binds only to site IV and that Ca(2+)-binding causes structural changes in both the whole TnC molecule and the C-terminal half molecule. In addition, TnC(W), TnC-ZEQ, and TnC(C), but not TnC(N), were shown to form soluble complexes with scallop TnI at physiological ionic strength. On the other hand, the Mg-ATPase activity of reconstituted rabbit actomyosin in the presence of scallop tropomyosin was inhibited by scallop TnI and recovered by the addition of an equimolar amount of TnC(W), TnC-ZEQ, or TnC(C), but not TnC(N). These results imply that the site responsible for the association with TnI is located in the C-terminal half domain of TnC. Ternary complex constructed from scallop TnT, TnI, and TnC(W) conferred Ca(2+)-sensitivity to the Mg-ATPase of rabbit actomyosin to the same extent as native troponin, but the TnC(N)-TnT-TnI and TnC-ZEQ-TnT-TnI complexes conferred no Ca(2+)-sensitivity, while the TnC(C)-TnT-TnI complex conferred weak Ca(2+)-sensitivity. Thus, the major functions of scallop TnC, such as Ca(2+)-binding and interaction with TnI, are located in the C-terminal domain, however, the full Ca(2+)-regulatory function requires the presence of the N-terminal domain.

Animals↗
PubMed2000

Screening of wound-responsive genes identifies an immediate-early expressed gene encoding a highly charged protein in mechanically wounded tobacco plants.

In order to identify genes that are temporally and spatially regulated during wound response, a cDNA population in mechanically wounded tobacco leaves was screened by the fluorescence differential display method. Of 28 clones initially identified to have altered levels of transcripts within 3 h of wounding, eight were characterized. Although each clone showed a unique pattern of transcript accumulation, one distinct clone was further characterized because of its immediate-early response. Its transcripts began to accumulate 10 min after wounding, reached a maximum level within 1 h and disappeared after 2 h. The response, which occurred repeatably and systemically, was observed by the treatment with propionic acid or erythrosin B, indicating that cytosolic acidification could be one of the signals for immediate-early response of this gene. The cDNA encodes a polypeptide of 513 amino acids with a relative molecular mass of 60,952. The putative polypeptide is rich in lysine (K), glutamic acid (E) and aspartic acid (D), which constitute up to 70% of total amino acids, and was therefore designated as KED. The KED polypeptide is composed of a highly hydrophilic N-terminal region and a relatively hydrophobic C-terminal region, suggesting that KED may function through electrostatic interactions with cellular components.

Amino Acid Sequence↗
PubMed2000

Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits.

PURPOSE: To examine the viability of using human amniotic membrane as substrate for culturing corneal epithelial cells and transplanting them onto severely injured rabbit eyes. METHODS: An ocular-surface injury was created in the right eye of eight rabbits by a lamellar keratectomy extending 5 mm outside the limbus. Next, from the limbal region of the uninjured left eyes of five of these animals, a small biopsy of corneal epithelial cells was taken and cultured on acellular human amniotic membrane. One month later, the invading conjunctiva that covered the corneal surface of all eight injured eyes was surgically removed. Five of the eyes then received grafts of amniotic membrane containing autologous cultured epithelial cells, whereas the other three received grafts of acellular amniotic membrane alone. RESULTS: A confluent primary culture of limbal corneal epithelial cells was established on acellular human amniotic membrane after 14 days. Cells were partially stratified and fairly well attached to the underlying amniotic membrane, although a fully formed basement membrane was not evident. The three rabbits that received amniotic membrane transplantation alone all had total epithelial defects on the graft in the early postoperative period. Eyes that were grafted with amniotic membrane that contained cultivated epithelial cells, however, were all successfully epithelialized up to 5 days after surgery. CONCLUSION: Autologous transplantation of cultivated corneal epithelium is feasible by using acellular amniotic membrane as a carrier.

Amnion↗
PubMed2000

Epithelial barrier function and ultrastructure of gelatinous drop-like corneal dystrophy.

PURPOSE: Recently, mutations in the M1S1 gene have been identified as responsible for gelatinous drop-like corneal dystrophy (GDLD). How the abnormal M1S1 gene product causes GDLD is not known, although evidence suggests that it may compromise corneal epithelial function. This investigation attempted to determine the effect of the abnormal M1S1 gene product by assessing epithelial barrier function and epithelial ultrastructure in GDLD corneas. METHODS: Epithelial barrier function was assessed on the basis of fluorescein uptake. The method used a modified slit-lamp fluorophotometer. High-resolution scanning electron and atomic force microscopy was used to investigate the amyloid deposits and epithelial cell structure. RESULTS: Epithelial permeability was orders of magnitude higher in GDLD corneas than normal. The structure of the amyloid deposits was characterized, and clear abnormalities in epithelial morphology and cell junctions were observed. CONCLUSIONS: The high epithelial permeability observed in GDLD corneas was directly correlated with abnormalities in epithelial structure, including irregular cell junctions. This suggests that the abnormal M1S1 gene product may affect epithelial cell junctions resulting in increased cell permeability in GDLD corneas.

Adult↗
PubMed2000

[Effects of low concentration of styrene monomer vapor on pregnancy].

To investigate the effects of exposure to styrene vapor on pregnancy, mice proved to be pregnant were exposed to 0, 2, 20 and 100 ppm styrene continuously for 24 hours during the period from day 0 to day 15 of gestation. A special small exposure chamber which can accurately maintain a constant low concentration of styrene vapor was made and used in the experiment. 1) In the 100 ppm group, hyperkinesis was presented in the chamber during the exposure period and inhibition of body weight gain was found (p < 0.01). No deaths occurred in any of the groups. 2) At necropsy of dams, no significant difference was found between the styrene exposed groups and the control group in any of the fertility indices, number of implantations, number of live fetuses, percentage of resorptions, or in the percentage of dead fetuses. The 100 ppm group showed lower fetal and placental weights (p < 0.01). 3) Gross necropsy of dams in the 100 ppm group showed significantly decreased adipose tissue. The liver, kidney and spleen weights were also low (p < 0.01). 4) When exposed to 100 ppm, which corresponds to 5 times the permissible concentration, i.e., 20 ppm, for 24 hours, non-pregnant dams showed no abnormality, while pregnant dams showed inhibition of body weight gain and significantly lower fetal and placental weights and maternal organ weights. Therefore, careful consideration should be given to exposure of women of childbearing age to styrene.

Animals↗
PubMed2000

[An experience with the Ross operation utilizing cryopreserved pulmonary homografts procured by and stored in our homograft valve bank].

Eight patients, 4 males and 4 females ranging in age from 10 to 54 years (mean 27 +/- 13 years) underwent the Ross operation using a cryopreserved pulmonary homograft harvested by and cryopreserved in our institutional "Tissue Bank". Seven patients had a congenital bicuspid aortic valve and 3 patients had had healed infective endocarditis of the aortic valve. Four young female patients wanted to have a baby after operation. The Ross procedure was carried out utilizing aortic root replacement techniques in all patients. All patients survived and are currently in NYHA class 1, but 2 cardiac events occurred in 2 patients during the mean follow-up term of 29 +/- 19 months. The one was the anastomic stenosis between the homograft and distal pulmonary artery treated by balloon dilatation and the other was ventricular tachycardia eventually managed by the insertion of an ICD. Pulmonary autograft valve regurgitation is present in 3 patients, but it is not progressive up to the present time. Pulmonary homograft valves function well in all patients. The Ross operation for adolescents and young adults should become more popular along with more easy availability of homograft valves based upon the establishment of the "Homograft Valve Bank" system in Japan.

Adolescent↗