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Biomedical subjects

Keiko Kashiwagi

Publications and source records attributed to Keiko Kashiwagi.

At least 19 recordsLinked to original sources

Enhancement of the synthesis of RpoN, Cra, and H-NS by polyamines at the level of translation in Escherichia coli cultured with glucose and glutamate.

Proteins whose synthesis is enhanced by polyamines at the level of translation were identified in a polyamine-requiring mutant cultured in the presence of 0.1% glucose and 0.02% glutamate instead of 0.4% glucose as an energy source. Under these conditions, enhancement of cell growth by polyamines was almost the same as that in the presence of 0.4% glucose. It was found that synthesis of RpoN, Cra, and H-NS was enhanced by polyamines at the level of translation at the early logarithmic phase of growth (A(540) of 0.15). The effects of polyamines on synthesis of RpoN, H-NS, and Cra were due to the existence of unusual Shine-Dalgarno sequences (RpoN and H-NS) and an inefficient GUG initiation codon (Cra) in their mRNAs. Thus, rpoN, cra, and hns genes were identified as new members of the polyamine modulon. Because most of the polyamine modulon genes thus far identified encode transcription factors (RpoS [sigma(38)], Cya, FecI [sigma(18)], Fis, RpoN [sigma(54)], Cra, and H-NS), DNA microarray analysis of mRNA expressed in cells was performed. At the early logarithmic phase of growth, a total of 97 species of mRNAs that were up-regulated by polyamines more than twofold were under the control of seven polyamine modulon genes mentioned above.

Bacterial Proteins↗

Polyamine uptake by DUR3 and SAM3 in Saccharomyces cerevisiae.

It has been reported that GAP1 and AGP2 catalyze the uptake of polyamines together with amino acids in Saccharomyces cerevisiae. We have looked for polyamine-preferential uptake proteins in S. cerevisiae. DUR3 catalyzed the uptake of polyamines together with urea, and SAM3 was found to catalyze the uptake of polyamines together with S-adenosylmethionine, glutamic acid, and lysine. Polyamine uptake was greatly decreased in both DUR3- and SAM3-deficient cells. The K(m) values for putrescine and spermidine of DUR3 were 479 and 21.2 mum, respectively, and those of SAM3 were 433 and 20.7 mum, respectively. Polyamine stimulation of cell growth of a polyamine requiring mutant, which is deficient in ornithine decarboxylase, was not influenced by the disruption of GAP1 and AGP2, but it was diminished by the disruption of DUR3 and SAM3. Furthermore, the polyamine stimulation of cell growth of a polyamine-requiring mutant was completely inhibited by the disruption of both DUR3 and SAM3. The results indicate that DUR3 and SAM3 are major polyamine uptake proteins in yeast. We previously reported that polyamine transport protein kinase 2 regulates polyamine transport. It was found that DUR3 (but not SAM3) was activated by phosphorylation of Thr(250), Ser(251), and Thr(684) by polyamine transport protein kinase 2.

Amino Acid Sequence↗

Cyclophane and acyclic cyclophane: novel channel blockers of N-methyl-D-aspartate receptor.

The effects of cyclophanes (CPCn, CPPy and TGDMAP) and acyclic cyclophane (ATGDMAP) on various glutamate receptors were studied with these receptors expressed in Xenopus oocytes using voltage-clamp recording. CPCn, CPPy, TGDMAP and ATGDMAP were found to inhibit macroscopic currents at heteromeric NMDA receptors (NR1/NR2), but not Ca(2+)-permeable AMPA receptors (GluR1), Ca(2+)-nonpermeable AMPA receptors (GluR1/GluR2) and metabotropic glutamate receptors (mGluR1alpha). The inhibition of NR1/NR2A receptors by these compounds was more potent than those of the other NMDA receptor subtypes. At a resting potential (-70 mV), the IC(50) values of CPCn, CPPy, TGDMAP and ATGDMAP for NR1/NR2A receptors were 0.5+/-0.1, 1.0+/-0.2, 8.0+/-0.8 and 4.9+/-0.5 microM, respectively. The inhibition by these compounds was voltage-dependent, that is, the degree of inhibition was in the order of negative holding potentials, -100 mV>-70 mV>-20 mV. Results of experiments using mutant NR1 and NR2 subunits identified residues that influence block by CPCn. The inhibition by CPCn was not altered significantly in the mutants at the critical asparagines in the M2 loop, NR1 N616, NR2B N615 and NR2B N616, these residues are known to form the narrowest region of the channel and the binding site of Mg(2+). However, mutations at NR1 N650, located in the vestibule of channel pore, and NR1 D669, located in the extracellular region, reduced the inhibition by CPCn, suggesting that these amino acid residues interact with CPCn. These results suggest that CPCn interacts directly with the mouth or vestibule of the ion channel, like a lid.

Animals↗

Comparative studies of anthraquinone- and anthracene-tetraamines as blockers of N-methyl-D-aspartate receptors.

Anthraquinone spermine [N1-(anthraquinone-2-carbonyl)spermine; AQ343], anthraquinone homospermine [N1-(anthraquinone-2-carbonyl; AQ444], anthracene spermine [N1-(9-anthracenylmethyl)spermine; Ant343], and anthracene homospermine [N1-(9-anthracenylmethyl)homospermine; Ant444] were found to be potent antagonists of recombinant N-methyl-D-aspartate (NMDA) receptors (NRs). The effects of both anthraquinone (AQ)- and anthracene (Ant)-tetraamines were reversible and voltage-dependent. Results of experiments using mutant NR1 and NR2B subunits of NMDA receptor identified residues that influence block by AQ- and Ant-tetraamines. The results indicate that the polyamine tail is crucial for block by AQ- and Ant-tetraamines. Residues in the outer vestibule of the NR1 subunit were more strongly involved in block by AQ-and Ant-tetraamines than residues in the corresponding region of NR2B. Several amino acid residues in the inner vestibule, below the level of the selectivity filter of NR1 and NR2B, affected block by AQ444, Ant343, and Ant444, but they did not affect block by AQ343. AQ-tetraamines could permeate the channel at very negative membrane potentials when the narrowest constriction of the channel was expanded by replacing the Asn residue at Asn616 of NR1 and NR2B with Gly, whereas Ant-tetraamines did not easily pass through the channel, apparently because of differences in the relative position of the head groups on AQ- and Ant-polyamines.

Animals↗

Identification of the cadaverine recognition site on the cadaverine-lysine antiporter CadB.

Amino acid residues involved in cadaverine uptake and cadaverine-lysine antiporter activity were identified by site-directed mutagenesis of the CadB protein. It was found that Tyr(73), Tyr(89), Tyr(90), Glu(204), Tyr(235), Asp(303), and Tyr(423) were strongly involved in both uptake and excretion and that Tyr(55), Glu(76), Tyr(246), Tyr(310), Cys(370), and Glu(377) were moderately involved in both activities. Mutations of Trp(43), Tyr(57), Tyr(107), Tyr(366), and Tyr(368) mainly affected uptake activity, and Trp(41), Tyr(174), Asp(185), and Glu(408) had weak effects on uptake. The decrease in the activities of the mutants was reflected by an increase in the K(m) value. Mutation of Arg(299) mainly affected excretion, suggesting that Arg(299) is involved in the recognition of the carboxyl group of lysine. These results indicate that amino acid residues involved in both uptake and excretion, or solely in excretion, are located in the cytoplasmic loops and the cytoplasmic side of transmembrane segments, whereas residues involved in uptake were located in the periplasmic loops and the transmembrane segments. The SH group of Cys(370) seemed to be important for uptake and excretion, because both were inhibited by the existence of Cys(125), Cys(389), or Cys(394) together with Cys(370). The relative topology of 12 transmembrane segments was determined by inserting cysteine residues at various sites and measuring the degree of inhibition of transport through crosslinking with Cys(370). The results suggest that a hydrophilic cavity is formed by the transmembrane segments II, III, IV, VI, VII, X, XI, and XII.

Amino Acid Sequence↗

Docosahexaenoic acid inhibits cancer cell growth via p27Kip1, CDK2, ERK1/ERK2, and retinoblastoma phosphorylation.

Docosahexaenoic acid (DHA), a PUFA of the n-3 family, inhibited the growth of FM3A mouse mammary cancer cells by arresting their progression from the late-G(1) to the S phase of the cell cycle. DHA upregulated p27(Kip1) levels by inhibiting phosphorylation of mitogen-activated protein (MAP) kinases, i.e., ERK1/ERK2. Indeed, inhibition of ERK1/ERK2 phosphorylation by DHA, U0126 [chemical MAPK extracellularly signal-regulated kinase kinase (MEK) inhibitor], and MEK(SA) (cells expressing dominant negative constructs of MEK) resulted in the accumulation of p27(Kip1). MAP kinase (MAPK) inhibition by DHA did not increase p27(Kip1) mRNA levels. Rather, this fatty acid stabilized p27(Kip1) contents and inhibited MAPK-dependent proteasomal degradation of this protein. DHA also diminished cyclin E phosphorylation, cyclin-dependent kinase-2 (CDK2) activity, and phosphorylation of retinoblastoma protein in these cells. Our study shows that DHA arrests cell growth by modulating the phosphorylation of cell cycle-related proteins.

Animals↗

Effects of hypergravity environments on amphibian development, gene expression and apoptosis.

This study investigates how rearing under conditions of hypergravity affects amphibian development, Xotx2 and Xag1 gene expression and apoptosis. Uncleaved Xenopus laevis eggs 20 min after insemination, 2 cell stage embryos, and gastrula stage embryos were raised at 2G and 5G, while controls were raised in normal gravity. Apoptosis in brain and eye inner structures of hatching embryos was scored using the TUNEL staining method, and gene expression in tail-bud embryos was analyzed by whole-mount in situ hybridization. Results showed that: (1) 5G retarded the development of eggs and embryos and induced microcephaly and microphthalmia. (2) 5G suppressed the expression of the two genes, Xotx2 (involved in fore- and midbrain and eye development) and Xag1 (regulating cement gland formation). (3) Eggs and 2 cell stage embryos raised at 5G showed a greater extent of brain and eye apoptosis compared with controls, while those raised at 2G showed no significant difference. These findings suggest that high gravity suppresses certain gene functions and induces abnormal apoptosis in brain and eyes, resulting in developmental retardation and various morphological abnormalities.

Animals↗

Stable knock-down of vomeronasal receptor genes in transgenic Xenopus tadpoles.

Xenopus V2R (xV2R), a family of G-protein-coupled receptors with seven transmembrane domains, is expressed in the Xenopus vomeronasal organ (VNO). There are six subgroups of xV2R, one of which, xV2RE, is predominantly expressed in the VNO. To understand the function of xV2R during VNO development, we developed a new method to achieve stable siRNA-suppression of the V2RE genes by introducing siRNA expression transgenes into the genomes of unfertilized eggs. We found that some of the derived transgenic tadpoles lacked VNOs and that their olfactory epithelium was fused. With the exception of one tadpole, expression of xV2RE was not detected in morphologically abnormal mutant tadpoles, although the olfactory marker protein and the olfactory receptors were expressed. These results suggest that we successfully produced transgenic tadpoles in which xV2RE expression was stably suppressed by siRNA, and that xV2RE plays a role in the morphogenesis of olfactory organs.

Animals↗

Enhancement of +1 frameshift by polyamines during translation of polypeptide release factor 2 in Escherichia coli.

Polypeptide release factor 2 (RF2) in Escherichia coli is known to be synthesized by a +1 frameshift at the 26th UGA codon of RF2 mRNA. Polyamines were found to stimulate the +1 frameshift of RF2 synthesis, an effect that was reduced by excess RF2. Polyamine stimulation of +1 frameshift of RF2 synthesis was observed at the early logarithmic phase, which is the important phase in determination of the overall rate of cell growth. A Shine-Dalgarno-like sequence was necessary for an efficient +1 frameshift of RF2 synthesis, but not for polyamine stimulation. Spectinomycin, tetracycline, streptomycin, and neomycin reduced polyamine stimulation of the +1 frameshift of RF2 synthesis. The results suggest that a structural change of the A site on 30 S ribosomal subunits is important for polyamine stimulation of the +1 frameshift. The level of mRNAs of ribosomal proteins and elongation factors having UAA as termination codon was enhanced by polyamines, and OppA synthesis from OppA mRNA having UAA as termination codon was more enhanced by polyamines than that from OppA mRNA having a UGA termination codon. Furthermore, synthesis of ribosomal protein L20 and elongation factor G from the mRNAs having a UAA termination codon was enhanced by polyamines at the level of translation and transcription. The results suggest that some protein synthesis from mRNAs having a UAA termination codon is enhanced at the level of translation through polyamine stimulation of +1 frameshift of RF2 synthesis. It is concluded that prfB encoding RF2 is a new member of the polyamine modulon.

Base Sequence↗

Decrease in polyamines with aging and their ingestion from food and drink.

Changes in polyamine levels during aging were measured in 3-, 10- and 26-week-old female mice. The level of polyamines in pancreas, brain, and uterus was maintained during these periods. The level of spermidine slightly decreased in intestine, and decreased significantly in thymus, spleen, ovary, liver, stomach, lung, kidney, heart and muscle during these periods. In skin, the level of spermidine was maximal in 10-week-old mice and markedly reduced in 26-week-old mice. The results suggest that maintenance of polyamine levels may play important roles in the function of the pancreas, brain and uterus in 3- to 26-week-old mice. We next looked for polyamine-rich food materials as a dietary source of polyamines. Foods found to be rich in polyamines included wheat germ, rice bran, black rice, Philippine mango, green pepper, Japanese pumpkin, nuts, fermented pickles, pond smelt, turban shell viscera, whelk viscera, salted salmon roe, salted cod roe, beef intestine (boiled) and liver of eel, beef, pork and chicken; and, as previously reported, soybean, fermented soybean (natto), mushrooms, orange and green tea leaf. These results offer useful information when it becomes necessary to ingest polyamines from food.

Aging↗

Polyamine Modulon in Escherichia coli: genes involved in the stimulation of cell growth by polyamines.

We have recently proposed an idea to explain how polyamines enhance cell growth in Escherichia coli. Since most polyamines exist as polyamine-RNA complexes, our idea is that polyamines stimulate several kinds of protein synthesis which are important for cell growth at the level of translation. We found that synthesis of oligopeptide binding protein (OppA), which is important for nutrient supply, adenylate cyclase (Cya), RNA polymerase sigma(38) subunit (RpoS), transcription factor of iron transport operon (FecI), and transcription factor of growth-related genes including rRNA and some kinds of tRNA synthesis (Fis) was enhanced by polyamines at the level of translation. We proposed that a group of genes whose expression is enhanced by polyamines at the level of translation be referred to as a "polyamine modulon." By DNA microarray, we found that 309 of 2,742 mRNA species were up-regulated by polyamines. Among the 309 up-regulated genes, transcriptional enhancement of at least 58 genes might be attributable to increased levels of the transcription factors Cya, RpoS, FecI, and Fis. This unifying molecular mechanism is proposed to underlie the physiological role of polyamines in controlling the growth of Escherichia coli.

Adenylyl Cyclases↗

Polyamine oxidase and acrolein as novel biochemical markers for diagnosis of cerebral stroke.

BACKGROUND AND PURPOSE: We found previously that plasma levels of acrolein (CH2=CHCHO) and spermine oxidase (SMO) were well correlated with the degree of severity of chronic renal failure. The aim of this study was to test whether the levels of these 2 markers and of acetylpolyamine oxidase (AcPAO) were increased in the plasma of stroke patients. METHODS: The activity of AcPAO and SMO and the level of protein-conjugated acrolein in plasma of the stroke patients and normal subjects were measured by high-performance liquid chromatography and ELISA, respectively. Focal infarcts were estimated by MRI or computed tomography (CT). RESULTS: The levels of AcPAO, SMO, and acrolein were significantly increased in the plasma of stroke patients. The size of stroke was nearly parallel with the multiplied value of acrolein and total polyamine oxidase (AcPAO plus SMO). After the onset of stroke, an increase in AcPAO first occurred, followed by increased levels of SMO and finally acrolein. In 1 case, an increase in AcPAO and SMO preceded focal damage as detected by MRI or CT. Furthermore, stroke was confirmed by MRI in a number of mildly symptomatic patients (11 cases) who had increased levels of total polyamine oxidase and acrolein. Among apparently normal subjects (8 cases) who had high values of acroleinxtotal polyamine oxidase, stroke was found in 4 cases by MRI. CONCLUSIONS: The results indicate that increased levels of AcPAO, SMO, and acrolein are good markers of stroke.

Acrolein↗

Polyamine transport by mammalian cells and mitochondria: role of antizyme and glycosaminoglycans.

The role of antizyme (AZ) and glycosaminoglycans in polyamine uptake by mammalian cells and mitochondria was examined using NIH3T3 and FM3A cells and rat liver mitochondria. AZ is synthesized as two isoforms (29 and 24.5 kDa) due to the existence of two initiation codon AUGs in the AZ mRNA. Most AZ existed as the 24.5-kDa form translatable from the second AUG, but a portion of the 29-kDa AZ from the first AUG was associated with mitochondria because of the presence of a mitochondrial targeting signal between the first and the second methionine. The predominance of the 24.5-kDa isoform was mainly due to the presence of spermidine and a favorable sequence context (Kozak sequence) at the second initiation codon AUG. Spermine uptake by NIH3T3 cells was inhibited by both 29- and 24.5-kDa AZs, but uptake by rat liver mitochondria was not influenced by either form of AZ. Because spermine uptake by mitochondria caused a release of cytochrome c, an enhancer of apoptosis, we looked for inhibitors of mitochondrial spermine uptake other than AZ. Cations such as Na+, K+, and Mg2+ were inhibitors of the mitochondrial uptake. It has been reported that heparan sulfate on glypican-1 plays important roles in spermine uptake by human embryonic lung fibroblasts. Heparin, but not heparan sulfate, slightly inhibited spermine uptake by FM3A cells in the absence of Mg2+ and Ca2+ but had no effect under physiological conditions in the presence of Mg2+ and Ca2+.

Amino Acid Sequence↗

Development of metamorphosis assay using Silurana tropicalis for the detection of thyroid system-disrupting chemicals.

The West African clawed frog (Silurana tropicalis), which resembles the South African clawed frog (Xenopus laevis), but is somewhat smaller, has a diploid genome and a shorter generation time. Therefore, S. tropicalis has the potential for use as a new model in ecotoxicology. We demonstrated a S. tropicalis metamorphosis assay based on Xenopus Metamorphosis Assay (XEMA) using 1 microg/L thyroxine (T4) and 75 mg/L propylthiouracil (PTU). Tadpoles at developmental stages 48-50 were exposed to chemicals for 28 days and total body length, developmental stage, and hind limb length were recorded every 7 days. Significant differences in developmental stage and total body length were found for both T4 and PTU after 7-day exposure, which were similar to the results of the XEMA ring-test using the same chemicals. Moreover, in the present study, we measured hind limb length as a new endpoint of thyroid axis. Significant differences in the hind limb length were encountered in both T4 and PTU treatments after 7 days of exposure. These results suggest that S. tropicalis can be used in a XEMA-like protocol to detect agonist and antagonist effects of chemicals on the thyroid system. Hind limb length is also a suitable endpoint in such protocols. A new test protocol detecting both thyroid disruption and reproductive effects of chemicals using S. tropicalis should be established in the near future.

Animals↗

Uptake of putrescine and spermidine by Gap1p on the plasma membrane in Saccharomyces cerevisiae.

It has been reported that Gap1p on the plasma membrane of Saccharomyces cerevisiae can catalyze the uptake of many kinds of amino acids. In the present study, we found that Gap1p also catalyzed the uptake of putrescine and spermidine, but not spermine. The Km and Vmax values for putrescine and spermidine were 390 and 21 microM, and 4.6 and 0.59 nmol/min/mg protein, respectively. The uptake of putrescine was strongly inhibited by basic amino acids, lysine, arginine, and histidine, whose Ki values were 25-35 microM. Thus, it is deduced that spermidine and basic amino acids have almost the same affinity for Gap1p. When the concentrations of amino acids in the medium were reduced to one-third and 0.5 mM putrescine or 0.1 mM spermidine was added to the medium, accumulation of putrescine or spermidine by Gap1p was observed. Furthermore, when yeast was transformed with the GAP1 gene and cultured in the presence of 60 mM putrescine, cell growth was inhibited through overaccumulation of putrescine. GAP1 mRNA was found to be induced by polyamines. This is the first report of the identification, at a molecular level, of a polyamine uptake protein on the plasma membrane in eukaryotes.

Amino Acid Transport Systems↗

Independent roles of eIF5A and polyamines in cell proliferation.

To examine the roles of active hypusinated eIF5A (eukaryotic translation initiation factor 5A) and polyamines in cell proliferation, mouse mammary carcinoma FM3A cells were treated with an inhibitor of deoxyhypusine synthase, GC7 (N1-guanyl-1, 7-diaminoheptane), or with an inhibitor of ornithine decarboxylase, DFMO (a-difluoromethylornithine), or with DFMO plus an inhibitor of spermine synthase, APCHA [N1-(3-aminopropyl)-cyclohexylamine]. Treatment with GC7 decreased the level of active eIF5A on day 1 without affecting cellular polyamine content, and inhibition of cell growth occurred from day 2. This delay reflects the fact that eIF5A was present in excess and was very stable in these cells. Treatment with DFMO or with DFMO plus APCHA inhibited cell growth on day 1. DFMO considerably decreased the levels of putrescine and spermidine, and the formation of active eIF5A began to decrease when the level of spermidine fell below 8 nmol/mg of protein after 12 h of incubation with DFMO. The combination of DFMO and APCHA markedly decreased the levels of putrescine and spermine and significantly decreased the level of spermidine, but did not affect the level of active eIF5A until day 3 when spermidine level decreased to 7 nmol/mg of protein. The results show that a decrease in either active eIF5A or polyamines inhibits cell growth, indicating that eIF5A and polyamines are independently involved in cell growth

Animals↗

Excretion of putrescine and spermidine by the protein encoded by YKL174c (TPO5) in Saccharomyces cerevisiae.

The properties of the protein encoded by YKL174c (TPO5) were studied. It was found that TPO5 excretes putrescine effectively and spermidine less effectively. Gamma-aminobutyric acid slightly inhibited the excretion of putrescine, but basic amino acids did not affect excretion, suggesting that TPO5 preferentially recognizes polyamines. Accordingly, yeast cells transformed with the plasmid encoding YKL174c (TPO5) were resistant to toxicity caused by 120 mm putrescine or by 3 mm spermidine, and a mutant with a disrupted YKL174c (TPO5) gene was sensitive to toxicity by 90 mm putrescine. The growth of this mutant was faster than that of the wild-type strain. In parallel, there was an increase in putrescine and spermidine content of the YKL174c (TPO5) mutant compared with wild-type. It is noted that TPO5 functions as a suppressor of cell growth by excreting polyamines. The level of YKL174c (TPO5) mRNA was increased by the addition of polyamines to the medium. The degree of induction of the mRNA was spermine > spermidine > putrescine. The subcellular localization of TPO5 was determined by immunostaining of hemagglutinin-tagged TPO5, and it was found on Golgi or post-Golgi secretory vesicles. Excretion of putrescine and spermidine by TPO5 was reduced in cells that have mutations in the secretory or endocytic pathways, indicating that both processes are involved in the excretion of polyamines.

Blotting, Northern↗

Characteristics of the polyamine transporter TPO1 and regulation of its activity and cellular localization by phosphorylation.

The subcellular localization of the polyamine transporter TPO1 of Saccharomyces cerevisiae was determined by sucrose gradient centrifugation and indirect immunofluorescence microscopy. When expressed from a multi-copy vector, TPO1 was located mainly on the plasma membrane, but with some localization on the vacuolar membrane. Polyamine transport by TPO1 was dependent on pH. Uptake of spermidine and spermine occurred at alkaline pH (pH 8.0), whereas inhibition of spermidine uptake, but not spermine uptake, was observed at acidic pH (pH 5.0). This suggests that TPO1 catalyzes polyamine excretion at acidic pH, similar to the PotE transporter in Escherichia coli. Paraquat, a polyamine analogue, was excreted by TPO1 at a rate comparable with the excretion of spermidine (deduced from the inhibition of spermidine uptake) at pH 5.0. However, excretion of preloaded radiolabeled spermidine and spermine was not observed in intact cells, suggesting that preloaded spermidine (or spermine) exists mainly as spermidine (or spermine)-ribosome complex in cells. The transport activity of TPO1 was enhanced through phosphorylation at Ser19 by protein kinase C and at Thr52 by casein kinase 1. Sorting of TPO1 from the endoplasmic reticulum to the plasma membrane was enhanced through phosphorylation at Ser342 by cAMP-dependent protein kinases 1 and 2.

Amino Acid Sequence↗