Biomedical subjects

Kazuhiro Nishimura

Publications and source records attributed to Kazuhiro Nishimura.

16 recordsLinked to original sources

PubMed2007

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↗

PubMed2006

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↗

PubMed2006

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↗

PubMed2006

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↗

PubMed2005

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↗

PubMed2005

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↗

PubMed2005

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↗

PubMed2005

Different intracellular polyamine concentrations underlie the difference in the inward rectifier K(+) currents in atria and ventricles of the guinea-pig heart.

The outward component of the strong inward rectifier potassium current, I(K1), is significantly larger in ventricles than in atria of the heart, resulting in faster repolarization at the final phase of the action potential in ventricles. However, the underlying mechanism of the difference in I(K1) remains poorly understood. I(K1) channels are composed of subunits from the Kir2 subfamily, and I(K1) amplitude is determined by the voltage-dependent blockade of the channel by the intracellular polyamines spermine and spermidine, and by Mg(2+). Using a perforated patch-clamp method, which minimizes changes in the intracellular polyamine and Mg(2+) concentrations, we detected repolarization-induced outward I(K1) transients, which are caused by competition between Mg(2+) and spermine to block the channel, in ventricular but not in atrial myocytes from guinea-pig heart. The contribution of the Kir2.3 subunit to the I(K1) channel was found to be minor in the guinea-pig heart, because the activation time course of the Kir2.3 currents was approximately 10-fold slower than those of I(K1), and the marked external pH sensitivity of the Kir2.3 currents was not found in I(K1). Both the Kir2.1 and Kir2.2 currents recorded from inside-out patches exhibited outward transients similar to those of ventricular I(K1) in the presence of 5-10 microM spermine and 0.6-1.1 mM Mg(2+), and their amplitudes were diminished by increasing the spermine or spermidine concentrations. The total and free polyamine concentrations in guinea-pig cardiac tissues were higher in atria than ventricles. These results strongly suggest that different intracellular polyamine concentrations are responsible for the difference in atrial and ventricular I(K1) of the guinea-pig heart.

Animals↗

PubMed2004

Defective function of GABA-containing synaptic vesicles in mice lacking the AP-3B clathrin adaptor.

AP-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking mu3B, a subunit of AP-3B. mu3B-/- mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of gamma-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in mu3B-/- mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.

Adaptor Protein Complex 3↗

PubMed2004

Upregulation of the viability of nucleus pulposus cells by bone marrow-derived stromal cells: significance of direct cell-to-cell contact in coculture system.

STUDY DESIGN: Upregulation of the viability of nucleus pulposus cells by coculture with bone marrow-derived stromal cells using a novel culture system. OBJECTIVES: The objective was to apply a novel coculture system having direct cell-to-cell contact between nucleus pulposus cells and bone marrow-derived stromal cells for stimulation of nucleus pulposus cells. SUMMARY OF BACKGROUND DATA: Reinsertion of nucleus pulposus cells was effective for treatment of intervertebral disc degeneration. However, obtaining highly viable nucleus pulposus cells was necessary to achieve successful results. Thus, an alternative method to upregulate the biologic and metabolic viabilities of nucleus pulposus cells was desired. METHODS: Nucleus pulposus cells and bone marrow-derived stromal cells were isolated from New Zealand white rabbits. A 6-well culture plate and insert with track-etched membrane having 0.4 microm pores at the bottom were used for coculture. Nucleus pulposus cells were monocultured, cocultured conventionally (having no direct cell-to-cell contact) with bone marrow-derived stromal cells, or cocultured having direct cell-to-cell contact with bone marrow-derived stromal cells. On day 4 of coculture, nucleus pulposus cells were evaluated for proliferation using WST-8 assay, deoxyribonucleic acid synthesis by measuring [H]-thymidine uptake, and proteoglycan synthesis by measuring [S]-sulfate uptake. We also quantified cytokines in supernatants from the culture system. RESULTS: Cell proliferation, deoxyribonucleic acid synthesis, and proteoglycan synthesis of nucleus pulposus cells were significantly upregulated in samples cocultured having direct cell-to-cell contact. Moreover, evaluations of supernatants revealed that growth factors associated with proliferation and cellular metabolism of nucleus pulposus cells were increased. CONCLUSIONS: Direct cell-to-cell contact in coculture system between nucleus pulposus cells and bone marrow-derived stromal cells accomplished significant upregulation in viability of nucleus pulposus cells.

Animals↗

PubMed2004

TATA-binding protein-associated factor 7 regulates polyamine transport activity and polyamine analog-induced apoptosis.

Identification of the polyamine transporter gene will be useful for modulating polyamine accumulation in cells and should be a good target for controlling cell proliferation. Polyamine transport activity in mammalian cells is critical for accumulation of the polyamine analog methylglyoxal bis(guanylhydrazone) (MGBG) that induces apoptosis, although a gene responsible for transport activity has not been identified. Using a retroviral gene trap screen, we generated MGBG-resistant Chinese hamster ovary (CHO) cells to identify genes involved in polyamine transport activity. One gene identified by the method encodes TATA-binding protein-associated factor 7 (TAF7), which functions not only as one of the TAFs, but also a coactivator for c-Jun. TAF7-deficient cells had decreased capacity for polyamine uptake (20% of CHO cells), decreased AP-1 activation, as well as resistance to MGBG-induced apoptosis. Stable expression of TAF7 in TAF7-deficient cells restored transport activity (55% of CHO cells), AP-1 gene transactivation (100% of CHO cells), and sensitivity to MGBG-induced apoptosis. Overexpression of TAF7 in CHO cells did not increase transport activity, suggesting that TAF7 may be involved in the maintenance of basal activity. c-Jun NH2-terminal kinase inhibitors blocked MGBG-induced apoptosis without alteration of polyamine transport. Decreased TAF7 expression, by RNA interference, in androgen-independent human prostate cancer LN-CaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to MGBG-induced growth arrest. Taken together, these results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis.

Animals↗

PubMed2004

Methotrexate differentially affects growth of suspension and adherent cells.

The effects of low concentrations of methotrexate (MTX) on the growth of suspension (FM3A, 2B4 and THP-1) and adherent (NIH3T3 and V79) cells were compared. The concentration of methotrexate to cause the inhibition of cell growth was lower in suspension cells than in adherent cells. The IC(50) for FM3A, 2B4, THP-1, NIH3T3 and V79 cells were 3.5, 5, 9, 30 and 50 nM, respectively. The inhibition of cell growth was reversed completely by tetrahydrofolate and was fully or significantly reversed by adenosine and thymidine, suggesting that the effects of low concentrations of methotrexate result from the inhibition of biosynthesis of purines and pyrimidines. In suspension cells but not in adherent cells there was a decrease in the levels of S-adenosylmethionine and polyamines after methotrexate treatment. Growth of suspension but not adherent cells was significantly recovered by treatment with S-adenosylmethionine. However, treatment with spermidine did not reverse the effects of methotrexate in any of the cell lines. The preferential inhibitory effect of methotrexate in suspension cells versus adherent cells was due mainly to a more rapid uptake of methotrexate. This may be relevant to the in vivo effects of low doses of methotrexate, which have immunosuppressive and anti-inflammatory effects, because lymphocytes are suspension cells.

Adenosylmethionine Decarboxylase↗

PubMed2004

Structural and functional relationship among diamines in terms of inhibition of cell growth.

Following the report that agmatine has an anti-proliferative effect on cell growth through induction of antizyme [Satriano et al. (1998) J. Biol. Chem. 273, 15313-15316], we examined the effects of 16 different diamines on cell growth. Many diamines had little or no effect on cell growth, but agmatine and 1,6-hexanediamine had anti-proliferative effects, with agmatine having the strongest effect. Inhibition of cell growth occurred after 2 days, and inhibitory effects paralleled the degree of antizyme induction. Decreased spermine levels indicated that induction of spermidine/spermine N(1)-acetyltransferase was also involved in the inhibition of cell growth by agmatine and 1,6-hexanediamine. The frameshift efficiency (ratio of antizyme synthesis with or without frameshift) measured in a rabbit reticulocyte cell-free system was also increased by 1,3-propanediamine and cis-1,4-cyclohexanediamine in addition to agmatine and 1,6-hexanediamine. However, the intracellular levels of 1,3-propanediamine and cis-1,4-cyclohexanediamine were low when these compounds were added to the cell-culture medium. Other diamines had no effect on cell growth or frameshift efficiency. The results suggest that the presence of two amino-groups separated by an appropriate distance is important for the enhancement of frameshifting by diamines.

Acetamides↗

PubMed2003

Transplantation of mesenchymal stem cells embedded in Atelocollagen gel to the intervertebral disc: a potential therapeutic model for disc degeneration.

Intervertebral disc degeneration is considered to be one of the major causes of low back pain. Despite this irreversible phenomenon, attempts to decelerate disc degeneration using various techniques have been reported. However, to date there has been no proven technique effective for broad clinical application. Based on previous studies, we hypothesize that maintenance of proteoglycan content in the disc is achieved by avoiding the depletion of nucleus pulposus and preserving the structure of the annulus is a primary factor in decelerating disc degeneration. One novel approach to solve the dilemma of intervertebral disc degeneration is found at the stem cell level. Mesenchymal stem cells (MSCs) are known to possess the ability to differentiate into various kinds of cells from mesenchymal origin. Although the majority of cells that contribute to disc formation are known to obtain chondrocyte-like phenotypes, no reported study has emphasized the correlation with mesenchymal stem cells. To evaluate the possible potential of MSCs in disc cell research and treatment of degenerative disc disease, autologous MSCs embedded in Atelocollagen gel were transplanted into the discs of rabbits which had undergone a procedure proven to induce degeneration. The results suggest that MSC transplantation is effective in decelerating disc degeneration in experimental models and provided new hopes for treatment of degenerative disc disease in humans. Atelocollagen gel served as an important carrier of MSCs in transplantation, permitting proliferation, matrix synthesis and differentiation of MSCs. This study strengthens the viable efficacy of practical application of MSCs in treatment of intervertebral disc disease.

Animals↗

PubMed2002

Inhibition of cell growth through inactivation of eukaryotic translation initiation factor 5A (eIF5A) by deoxyspergualin.

The mechanism of inhibition of cell growth by deoxyspergualin was studied using mouse mammary carcinoma FM3A cells. Results of studies using deoxyspergualin analogues showed that both the guanidinoheptanate amide and glyoxyspermidine moieties of deoxyspergualin were necessary to cause inhibition of cell growth. When deoxyspergualin was added to the medium, there was a strong inhibition of cell growth and formation of active eukaryotic translation initiation factor 5A (eIF5A) at the third day of culture. There was also a marked decrease in cellular putrescine content and a small decrease in spermidine content. Accumulation of decapped mRNA, which is typically associated with eIF5A deficiency in yeast, was also observed. The inhibition of cell growth and the formation of active eIF5A was not reversed by addition of spermidine. The activity of deoxyhypusine synthase, the first enzyme in the formation of active eIF5A, was inhibited by deoxyspergualin in a cell-free system. These results, taken together, indicate that inhibition of active eIF5A formation is strongly involved in the inhibition of cell growth by deoxyspergualin.

Animals↗

PubMed2002

Essential role of S-adenosylmethionine decarboxylase in mouse embryonic development.

BACKGROUND: S-Adenosylmethionine decarboxylase (AdoMetDC) is one of the key enzymes involved in the biosynthesis of spermidine and spermine, which are essential for normal cell growth. To examine the role of polyamines in embryogenesis, we carried out targeted disruption of the mouse Amd1 gene, encoding AdoMetDC, to generate mice that can not synthesize spermidine and spermine. RESULTS: Amd1 heterozygous mice were viable, normal and fertile. However, homozygous Amd1(-/-) embryos died early in embryonic development, between E3.5 and E6.5 days post-coitus. Homozygous (Amd1(-/-)) blastocysts at E3.5 arrested cell proliferation immediately after the onset of cell culture, and this arrest was rescued by the addition of spermidine. Chromosomal DNA breakage did not occur in Amd1(-/-) blastocysts at E3.5, as determined by TUNEL assay. CONCLUSIONS: These results indicate that AdoMetDC plays an essential role in embryonic development and that polyamines are required for cell proliferation in the embryo after E3.5.

Adenosylmethionine Decarboxylase↗