Biomedical subjects

Y Machida

Publications and source records attributed to Y Machida.

At least 19 recordsLinked to original sources

PubMed1995

Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana.

Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic beta-glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F1 and F2 progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F1 progeny and most of the F2 progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F2 individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F1 generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.

Arabidopsis↗

PubMed1995

A tobacco protein kinase, NPK2, has a domain homologous to a domain found in activators of mitogen-activated protein kinases (MAPKKs).

A cDNA (cNPK2) that encodes a protein of 518 amino acids was isolated from a library prepared from poly(A)+ RNAs of tobacco cells in suspension culture. The N-terminal half of the predicted NPK2 protein is similar in amino acid sequence to the catalytic domains of kinases that activate mitogen-activated protein kinases (designated here MAPKKs) from various animals and to those of yeast homologs of MAPKKs. The N-terminal domain of NPK2 was produced as a fusion protein in Escherichia coli, and the purified fusion protein was found to be capable of autophosphorylation of threonine and serine residues. These results indicate that the N-terminal domain of NPK2 has activity of a serine/threonine protein kinase. Southern blot analysis showed that genomic DNAs from various plant species, including Arabidopsis thaliana and sweet potato, hybridized strongly with cNPK2, indicating that these plants also have genes that are closely related to the gene for NPK2. The structural similarity between the catalytic domain of NPK2 and those of MAPKKs and their homologs suggests that tobacco NPK2 corresponds to MAPKKs of other organisms. Given the existence of plant homologs of an MAP kinase and tobacco NPK1, which is structurally and functionally homologous to one of the activator kinases of yeast homologs of MAPKK (MAPKKKs), it seems likely that a signal transduction pathway mediated by a protein kinase cascade that is analogous to the MAP kinase cascades proposed in yeasts and animals, is also conserved in plants.

Amino Acid Sequence↗

PubMed1995

DNA rearrangement associated with the integration of T-DNA in tobacco: an example for multiple duplications of DNA around the integration target.

Transferred DNA (T-DNA) of the tumor-inducing (Ti) plasmid is transferred from Agrobacterium tumefaciens to plant cells and is stably integrated into the plant nuclear genome. By the inverse polymerase chain reaction DNA fragments were amplified that contained the T-DNA/plant DNA junctions from the total DNA of a transgenic tobacco plant that had a single copy of the T-DNA in a repetitive region of its genome. A DNA fragment containing the target site was amplified from the total DNA of non-transformed tobacco by the polymerase chain reaction using high-stringency conditions. Comparison of the nucleotide sequence of the target site with those of the T-DNA/plant DNA junctions revealed that various duplications of short stretches of nucleotide sequences around the target and in the incoming T-DNA had accompanied the integration of the T-DNA. A deletion of 16 bp at the target site was also found and the target site was similar, in terms of nucleotide sequence, to regions around the breakpoints of the T-DNA. This finding provides a clear example of the occurrence of complex rearrangements during the integration of T-DNA.

Agrobacterium tumefaciens↗

PubMed1995

Plant homologues of components of MAPK (mitogen-activated protein kinase) signal pathways in yeast and animal cells.

As they respond to numerous extracellular and intracellular stimuli, plants develop various morphological features and the capacity for a large variety of physiological processes during their growth. If we are to understand the molecular basis of such developments, we must elucidate the way in which signals generated by such stimuli can be transduced into plant cells and transmitted by cellular components to induce the appropriate terminal events. In yeast and animal systems, signal pathways that are known collectively as MAPK (mitogen-activated protein kinase) cascades have been shown to play a central role in the transmission of various signals. The components of these pathways include the MAPK family, the activator kinases of the MAPK family (the MAPKK family) and the activator kinases of the MAPKK family (the MAPKKK family). The members of each respective family are structurally conserved and signals are transmitted by similar phosphotransfer reactions at corresponding steps that are mediated by a specific member of each family in turn. Both cDNAs and genes that encode putative homologues of these components have recently been isolated from plant sources. Some of them have been shown to be related not only structurally but also functionally to members of the MAPK cascades of other organisms. These findings suggest that plants have signal pathways that are analogues to the MAPK cascades in yeast and animal cells but it remains to be proven that plant homologues do in fact constitute kinase cascades.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Effects of dose, pH and osmolarity on intranasal absorption of recombinant human erythropoietin in rats.

The effects of dose, pH and osmolarity on the intranasal absorption of a recombinant human erythropoietin (rEPO) solution were studied in male Wistar rats. The intranasal administration of rEPO was evaluated by measuring percentage circulating reticulocytes of red blood cells on a stained blood smear (smear method), and also by measuring residual circulating reticulocyte counts using a microcell counter (sysmex method). Both results suggest that rEPO solution was absorbed through the nasal mucosa of rats without enhancers after a single intranasal administration. The pharmacological availabilities of rEPO after intranasal administration compared with intravenous administration were about 7% and 4%, when estimated by smear method and sysmex method, respectively. The pharmacological activity was enhanced in low pH and hypotonic mannitol solution.

Absorption↗

PubMed1994

NPK15, a tobacco protein-serine/threonine kinase with a single hydrophobic region near the amino-terminus.

A cDNA clone (cNPK15) was isolated from tobacco cells in suspension culture, which encodes a predicted protein kinase of 422 amino acids. The predicted NPK15 protein consists of a hydrophobic region near the amino-terminus, a linker domain and the catalytic domain of a protein-serine/threonine kinase in the carboxyl-half. NPK15 was not found to be closely related to any reported protein, but its putative catalytic domain shares some structural similarity with those of receptor-like protein kinases of plants, such as ZmPK1 from Zea mays and TMK1 from Arabidopsis, even though no receptor-like domain is found in NPK15. Recombinant NPK15 expressed in Escherichia coli as a fusion protein was found capable of autophosphorylation and of phosphorylation of the histone H1 protein on both serine and threonine residues. Upon overexpression of cNPK15 under control of the promoter of cauliflower mosaic virus 35S RNA in tobacco cells, into which it had been introduced by Agrobacterium-mediated transformation, the NPK15 gene acted as a "suicide" gene and blocked proliferation of the host cells. By contrast, such a suicide effect was not observed with the gene for a kinase-negative mutant protein in which the nucleotide sequence for the ATP-binding site had been mutated or with a mutant derivative encoding a protein in which the hydrophobic region had been deleted. Thus, the protein kinase activity of NPK15 and the hydrophobic region of the protein are responsible for the suicide effect. The NPK15 protein kinase seems to be associated with specific cellular functions. Southern blot analysis with cNPK15 as the probe detected several fragments in restriction digests of genomic DNAs from both tobacco and other members of the Solanaceae. This results suggests that NPK15-related genes constitute a small gene family in the genomes of Solanaceae.

Amino Acid Sequence↗

PubMed1994

Determination of D,L-propargylglycine and N-acetylpropargylglycine in urine and several tissues of D,L-propargylglycine-treated rats using liquid chromatography-mass spectrometry.

An experimental animal model with cystathioninuria was obtained by the injection of D,L-propargylglycine into rats. The concentrations of D,L-propargylglycine in urine, several tissues and serum at different times after the injection were measured by liquid chromatography-mass spectrometry. The propargylglycine accumulated rapidly in several tissues and serum of the rats, and reached its maximum level at about 2 h after the injection. Approximately 21.2% of the administered propargylglycine was excreted in urine. N-Acetylpropargylglycine was identified as a new metabolite of propargylglycine in urine. The concentration of propargylglycine was 100 times that of N-acetylpropargylglycine in urine.

Alkynes↗

PubMed1994

Organ-specific and auxin-inducible expression of two tobacco par A-related genes in transgenic plants.

We have isolated four genomic DNA clones that contain the transcription initiation site of the parA gene(s) from a tobacco genomic library by hybridization with the 5' segment of the parA cDNA previously isolated. They were classified into two types on the basis of their nucleotide sequences. Southern blot analysis indicated that two types of clones were respectively derived from the two parental species of tobacco, Nicotiana tomentosiformis and Nicotiana sylvestris. The genes corresponding to these clones were designated as parAt and parAs, respectively, and the parA cDNA clone was shown to code for mRNA from parAt on the basis of its nucleotide sequence. The 5' regions about 400 nucleotides upstream from the transcription initiation sites of the parAt and parAs genomic clones were highly homologous to one another, but regions further upstream showed no significant similarity. The coding sequence of the GUS (beta-glucuronidase) reporter gene was linked to the 5'-upstream regions of parAt and parAs, and the sites of expression of these fusion genes were examined in transgenic tobacco plants. In the absence of auxins, both fusion genes were expressed in capsules at a late stage of seed development, mature seeds, a root apex and a root-hair zone whereas no significant expression was seen in other organs. Their expression was enhanced by 2,4-dichlorophenoxyacetic acid in most of the organs of tobacco. The results show that expression of these genes is regulated by both organ-specific and auxin-inducible mechanisms.

Amino Acid Sequence↗

PubMed1994

A longitudinal study from prepuberty to puberty of gingivitis. Correlation between the occurrence of Prevotella intermedia and sex hormones.

A longitudinal study of 24 subjects progressing normally from prepuberty to puberty was undertaken to evaluate the effects of sex hormone levels on clinical and microbiologic parameters and on serum antibodies. During elementary school, at the beginning of the longitudinal monitoring, 2 groups, 12 subjects with gingivitis and 12 gingivitis-free subjects, were selected and observed through puberty. Bone ages and self-assessment of secondary sex characteristics were used to confirm puberty. A statistically significant increase in the proportions of Prevotella intermedia including Prevotella nigrescens and serum antibody levels against P. intermedia was seen in gingivitis group throughout the longitudinal study. Serum levels of testosterone in boys and estradiol and progesterone in girls was positively correlated with levels of P. intermedia and P. nigrescens. In puberty, a slight but significant increase in GI scores over prepuberty has been shown; however, there was no significant change in PlI from prepuberty to puberty. Our study confirmed that there was a statistically significant increase in gingival inflammation and in the proportion of P. intermedia and P. nigrescens in puberty relative to the baseline value, except in the gingivitis-free male group. Our findings suggest that these increases are correlated with elevation in systemic levels of the sex hormones.

Adolescent↗

PubMed1994

Infection by Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, and antibody responses at different ages in humans.

This study examined the serum IgG and IgM responses against Porphyromonas gingivalis and 3 serotypes of Actinobacillus actinomycetemcomitans, and the correlations of these responses with age and homologous infection. A total of 90 individuals were included in this study: 40 subjects with gingivitis, 40 periodontally healthy subjects, and 10 adult periodontitis subjects. The subjects in the gingivitis and periodontally healthy groups were divided into 4 stages based on their physiological age: early childhood, school age, puberty, and adult. In the gingivitis group, there was a positive correlation between increase in age and increase in serum IgG antibody levels against P. gingivalis until puberty. However, no statistically significant difference was found between the puberty stage and the adult stage. The average level of IgG antibodies against A. actinomycetemcomitans in the school age gingivitis group was significantly higher than that in the early childhood gingivitis group for all serotypes (p < 0.01). In serotype c, IgG antibody levels in the school age gingivitis group were significantly higher than in the early childhood gingivitis group or the adult gingivitis group (p < 0.01). With both P. gingivalis and A. actinomycetemcomitans, positive correlations between elevated IgG level and infections by these microorganisms were found in the puberty gingivitis and adult periodontitis groups.

Adolescent↗

PubMed1994

Characterization of tobacco protein kinase NPK5, a homolog of Saccharomyces cerevisiae SNF1 that constitutively activates expression of the glucose-repressible SUC2 gene for a secreted invertase of S. cerevisiae.

We have isolated a cDNA (cNPK5) that encodes a protein kinase of 511 amino acids from suspension cultures of tobacco cells. The predicted kinase domain of NPK5 is 65% identical in terms of amino acid sequence to that of the SNF1 serine/threonine protein kinase of Saccharomyces cerevisiae, which plays a central role in catabolite repression in yeast cells. SNF1 positively regulates transcription of various glucose-repressible genes of the yeast, such as the SUC2 gene for a secreted invertase, in response to glucose deprivation: snf1 mutants cannot utilize sucrose as a carbon source. Expression of cNPK5 in yeast cells allowed the snf1 mutant cells to utilize sucrose for growth and caused constitutive expression of the SUC2 gene in wild-type cells even in the presence of glucose, an indication that the NPK5 protein is present in a constitutively active form in S. cerevisiae. On the other hand, expression of cNPK5 failed to suppress the growth defect of the snf4 mutant cells in the presence of sucrose and to induce expression of the SUC2 gene. These results indicate that SNF4 is required for the induction of SUC2 expression by NPK5, as by SNF1, even if NPK5 is constitutively active in S. cerevisiae. The recombinant NPK5 protein is capable of autophosphorylation in vitro in a reaction that requires Mn2+ rather than Mg2+ ions but is inhibited by Ca2+ ions. Both dicotyledonous and monocotyledonous plants have several copies of the NPK5-related gene, which probably constitute a small gene family. NPK5-related genes were found to be expressed in the roots, leaves, and stems of tobacco plants. The high degree of structural conservation and the functional similarity of NPK5 to SNF1 lead us to speculate that NPK5 (or a related kinase) also plays a role in sugar metabolism in higher plants.

Amino Acid Sequence↗

PubMed1993

Genetic evidence for an interaction between the VirA sensor protein and the ChvE sugar-binding protein of Agrobacterium.

Most vir genes of Agrobacterium, which are required for tumorigenicity of the bacterium, are expressed in response to plant phenolics. The induction of vir is markedly enhanced by specific monosaccharides. Signals generated by both types of compound are transduced into Agrobacterium cells via the functions of the VirA membrane-bound sensor protein. A putative sugar-binding protein, known as ChvE, also functions at a step of the enhancement of vir induction by monosaccharides. To investigate the signal pathway of the enhancement by the sugars, we first isolated a virA mutant of Agrobacterium with a base substitution mutation that caused a single amino acid change in the periplasmic domain. The mutant exhibited no enhancement of vir induction by sugar and had severely attenuated tumorigenicity on Kalanchoe leaves. We then isolated two chvE mutants that restored sugar enhancement on the background of this virA mutation. One chvE mutant, which exhibited a higher level of sugar enhancement, restored the tumorigenicity of the virA mutant. Wild-type and suppressor ChvE proteins were localized in the periplasmic space. These results provide genetic evidence for the physical interaction between VirA and ChvE proteins in the periplasmic space of Agrobacterium, which enhances the cytoplasmic signal generated by phenolics. We also discuss the molecular architecture of the operon to which the chvE gene belongs.

Amino Acid Sequence↗

PubMed1993

Molecular characterization of a short interspersed repetitive element from tobacco that exhibits sequence homology to specific tRNAs.

We have characterized a family of tRNA-derived short interspersed repetitive elements (SINEs) in the tobacco genome. Members of this family of SINEs, designated TS, have a composite structure and include a region structurally similar to a rabbit tRNA(Lys), a tRNA-unrelated region, and a TTG repeat of variable length at the 3' end. Southern blot hybridization, together with a search of the GenBank data base, showed that various plants belonging to the families Solanaceae and Convolvulaceae contain sequences homologous to the TS family in the introns and flanking regions of many genes, whereas Arabidopsis in the family Cruciferae and several species of monocoytledonous plants do not. The TS family is widely involved in structural and genetic variations in the genomes of many plants that belong to the order Tubiflorae. All of nine sequences identified in a data base search are truncated at their 5' regions and lack the tRNA-related region of the TS family. We characterized the entire sequence of the members of the TS family and found that this family can be categorized as a member of a group of SINEs with a tRNA(Lys)-like structure, as can several animal SINEs. The TS family can be divided into two major subfamilies by analysis of diagnostic positions, and one of the subfamilies is clearly younger than the other. Amplification of many copies of the full sequence of the younger subfamily occurred during the recent evolution of the tobacco lineage. We also discuss mechanisms that could be involved in the generation of SINEs in animals and also in plants.

Base Sequence↗

PubMed1993

NPK1, a tobacco gene that encodes a protein with a domain homologous to yeast BCK1, STE11, and Byr2 protein kinases.

We have isolated a cDNA (cNPK1) that encodes a predicted protein kinase of 690 amino acids from suspension cultures of tobacco cells. The deduced sequence is closely related to those of the protein kinases encoded by the STE11 and BCK1 genes of Saccharomyces cerevisiae and the byr2 gene of Schizosaccharomyces pombe. STE11 and Byr2 function in the yeast mating pheromone response pathways, and BCK1 acts downstream of the yeast protein kinase C homolog encoded by the PKC1 gene, which is essential for normal growth and division of yeast cells. Overexpression in yeast cells of a truncated form of cNPK1, which encodes only the putative catalytic domain, replaced the growth control functions of BCK1 and PKC1 but not the mating pheromone response function of STE11. Thus, the catalytic domain of NPK1 specifically activates the signal transduction pathway mediated by BCK1 in yeast. In tobacco cells in suspension culture, the NPK1 gene is transcribed during logarithmic phase and early stationary phase but not during late stationary phase. In a tobacco plant, it is also transcribed in stems and roots but not in mature leaves, which rarely contain growing cells. The present results suggest that a signal transduction pathway mediated by this BCK1- and STE11-related protein kinase is also conserved in plants and that a function of NPK1 is controlled at least in part at a transcriptional level.

Amino Acid Sequence↗

PubMed1993

Effect of pretreatment of skin with cyclic monoterpenes on permeation of diclofenac in hairless rat.

The promoting effect of d-limonene and l-menthol on the percutaneous absorption of nonionized and ionized diclofenac (DF) was investigated employing a pretreatment method. After the pretreatment of hairless rat skin with an ethanol buffer solution containing terpenes, the permeation study was performed in vitro. The permeability coefficients of nonionized (Pn) and ionized diclofenac (Pi) were calculated under the assumption that the total flux was composed of individual fluxes of nonionized and ionized DF. In the case of pretreatment with d-limonene, both Pn and Pi values were increased dramatically. However, the promoting magnitude was not affected by an extension of the pretreatment period. In contrast, when the skin was pretreated with l-menthol, the Pn and Pi values increased gradually as the pretreatment period increased. Based on the measurement of the solubility of terpenes in the pretreatment solution, the difference in promoting efficiency could arise from the difference in thermodynamic activity of the terpenes.

Animals↗

PubMed1993

Evaluation of bioavailability upon oral administration of phytonadione preparations in beagle dogs.

Bioavailability of phytonadione was investigated after oral administration to beagle dogs. The administrations of phytonadione in a surfactant solution (preparation a) and in an oleic acid solution (preparations b) greatly increased the bioavailability of phytonadione. The AUCs of preparations a and b were about 2.5 times larger than those of commercially available tablets A and C. This result well corresponded to the results of the dissolution test previously reported. The absorption of phytonadione from the gastro-intestinal tract was affected by food and the bioavailability was largely increased under non-fasted conditions. However, a large scatter was observed in the data, and it was found that the evaluation of the bioavailability of phytonadione preparations could not be done in non-fasted animals.

Administration, Oral↗

PubMed1993

Site-dependent effect of aprotinin, sodium caprate, Na2EDTA and sodium glycocholate on intestinal absorption of insulin.

In order to determine an advantageous site for intestinal insulin absorption, the hypoglycemic effects of insulin after administration to the duodenum, the jejunum, the ileum and the colon were investigated using an in situ loop method. Insulin solution was administered to the various loops of fasted rats with or without aprotinin (AP) as a protease inhibitor, or absorption enhancers such as sodium caprate, Na2EDTA or sodium glycocholate. An obvious hypoglycemic effect of insulin alone was seen only in the ileum loop washed with phosphate buffered saline. When coadministered with AP, the most remarkably amplified effect was again observed in the ileum. In the ileum, the area under the serum insulin levels vs. time curve from 0 to 4 h was linearly related to the logarithm of the AP dose. Both sodium caprate and Na2EDTA significantly promoted the hypoglycemic effect of insulin at all sites, and their intensity increased towards the distal regions of the intestine. On the other hand, sodium glycocholate improved only colonic insulin efficacy. These results suggest that the ileum seems to be the most useful region in the small intestine for insulin absorption; however, insulin must be protected from proteolysis to enhance its absorption. In addition, the insulin efficacy could be increased by absorption promoters more effectively in the colon than in the small intestine.

Animals↗

PubMed1993

[Development of topical drug delivery systems utilizing polymeric materials].

Topical drug delivery is important from the view points of improvement of therapeutic effect and reduction of systemic side effects. Utilization of polymeric materials seemed to be as a key for the development of new topical dosage forms including targeting drug delivery systems. Adriamycin ointment for local chemotherapy to breast cancer prepared using polyethylene glycol, ammonium polyacrylate and hydroxypropyl cellulose (HPC) according to an optimum formulation showed an excellent clinical effect in spite of a decreased drug content. Double-layered mucoadhesive sticks for the treatment of uterine cervix cancer were prepared by direct compression of powder mixture of bleomycin, HPC and carboxyvinyl polymer (CP). Drug release property of the sticks could be controlled by the weight of outer layer, drug combining ratio to each layer and coating of core layer. The results suggested a possibility of a "once-a-week" treatment that is preferable for the patients. Magnetic granules for the treatment of esophageal cancer were prepared using ferrite, HPC and CP. Magnetic guidance and retainment of the granules on esophageal mucosa were confirmed using rabbits in vivo. Buoyant sustained release preparations were prepared using chitosan, soybean protein, HPC and other polymers. Usefulness of the buoyant preparations was suggested from the results in vitro and in vivo. Insulin microspheres (IMS) for targeting delivery to the small intestine were prepared by the newly developed method. Employment of enteric coating material (Eudragit) and combination of protease inhibitor protected insulin from enzymatic attack and gave decreased levels of blood glucose by oral administration.

Administration, Topical↗