Biomedical subjects

H Sawai

Publications and source records attributed to H Sawai.

At least 73 records · Page 4Linked to original sources

PubMed1997

The effect of extracellular Ca2+ on responses to purinoceptor agonists in cultured swine tracheal smooth muscle cells.

The effects of extracellular Ca2+ ions on purinergic responses were examined in swine tracheal smooth muscle cells (TSMCs) in primary culture. ATP (1 microM to 1 mM) and alpha, beta-methylene adenosine 5' triphosphate (alpha, beta-Me ATP) (100 microM and 1 mM) concentration-dependently increased [Ca2+]i in the presence and the absence of extracellular Ca2+. Responses to ATP (10 microM to 1 mM) in the presence of extracellular Ca2+ were significantly larger than those in its absence (n=8), whereas those to alpha, beta-Me ATP were not significantly different between the presence (n=7) and the absence (n=8) of extracellular Ca2+. Responses to ATP (1 mM) at extracellular Ca2+ concentration ([Ca2+]o) of 10 and 5 mM were significantly larger than that at extracellular EGTA concentration ([EGTA]o) of 1 mM (p< 0.01, n=5), whereas the responses to alpha, beta-Me ATP (1 mM) at 10 and 5 mM [Ca2+]o were significantly smaller than that at [EGTA]o of 1 mM (p<0.05, n=5). Increasing [Ca2+]o to 5 mM after the application of either 1 mM ATP (n=4) or 1 mM alpha, beta-Me ATP (n=4) in the absence of extracellular Ca2+ (1 mM [EGTA]o) further increased [Ca2+]i, though the increases in [Ca2+]i by agonists in 1 mM [EGTA]o had been already maximal. Incubating cells for 300 s in 5 mM [Ca2+]o before the application of ATP (1 mM) significantly increased the response to the drug than that obtained by incubating cells for 6 sec in 5 mM [Ca2+]o before the drug application (p< 0.01, n=4). However, alpha, beta-Me ATP (1 mM) induced similar responses by incubating cells for 30 or 300 s in 5 mM [Ca2+]o to that by incubating them for 6 s. These results suggest that the effect of alpha, beta-Me ATP in swine TSMCs in primary culture is mainly through Ca2+ release and that its effect on Ca2+ entry is smaller than other nucleotides.

Adenosine Triphosphate↗

PubMed1997

Survival of axotomized retinal ganglion cells in adult mammals.

As a nature of central neurons, retinal ganglion cells (RGCs) of adult mammals cannot regenerate spontaneously, but rather, degenerate retrogradely after optic nerve (ON) transection. Recent studies have clarified that some RGCs survive axotomy and regrow their axons through the graft when the peripheral nerve is transplanted to the cut stump. However, the numbers of regenerated fibers are still quite small and a great majority of RGCs die after axotomy. Studies on the rodent and cat retinas have revealed that vulnerability to axotomy differs among RGC types. Especially in the cat retina, X/beta cells are more vulnerable to axotomy than other cells. Several neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and neurotrophin-4 or 5 (NT-4/5) are effective to rescue RGCs from retrograde cell death, and promote growth of intraretinal axons. Localization of their receptors is also demonstrated in RGCs. On the other hand, evidence has been accumulated that regenerated ON fibers reform functional synapses with central visual neurons and thereby axotomized RGCs survive longer. These reformed retino-collicular synapses enabled the animals to recover the ability of light-dark discrimination.

Animals↗

PubMed1997

Synthesis of oligodeoxyribonucleotide containing novel C-5 reactive 2'-deoxyuridine derivative and its functional modification via post-synthetic technique.

2'-Deoxyuridine derivatives bearing an activated ester at C-5 position were synthesized and was examined their use for the preparation of modified oligodeoxyribonucleotides (ODNs) by a post-modification method. The ODNs containing cyanomethyl ester at C-5 position of the deoxyuridine residue reacted easily with a primary amine of several functional molecules under the mild condition to give the corresponding modified ODNs.

Base Sequence↗

PubMed1997

Synthesis and anti-HIV activity of phosphorothioate antisense DNA containing C-5 polyamine substituted 2'-deoxyuridine and/or acridine residues.

Novel phosphorotioate DNAs (S-ODNs) bearing polyamine moiety at the C-5 position of 2'-deoxyuridine in place of certain thymidine residues and/or an acridine moiety at the 5'-terminus were prepared. The sequence of the S-ODNs is complementary to the rev region of HIV-1 mRNA. The duplexes consisted of the modified S-ODNs and the complementary DNA exhibited the enhanced stability compared to that consisted of the corresponding unmodified S-ODN and the complement. The improved antisense activity against HIV-1 was also observed for all modified S-ODNs.

Acridines↗

PubMed1996

Brain-derived neurotrophic factor and neurotrophin-4/5 stimulate growth of axonal branches from regenerating retinal ganglion cells.

To investigate the influences of growth factors on axonal regeneration in the mammalian CNS, we used intracellular tracers to quantitate the effects of brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-4/5, or NT-3 on individual retinal ganglion cell (RGC) axons in the retinas of adult rats after optic nerve transection. A single injection of BDNF or the prolonged administration of NT-4/5 by mini-pump increased axon branch median lengths by eightfold but had no effect on the number of branches formed by the RGC axons. NT-3 did not significantly influence axonal regrowth. These specific in vivo effects of BDNF and NT-4/5 on axonal regeneration from injured RGCs may be used to promote growth and expand the abnormally small terminal arbors observed when RGCs regrow into their CNS targets.

Animals↗

PubMed1996

Conformational and stacking properties of 3'-5' and 2'-5' linked oligoribonucleotides studied by CD.

Comparative CD studies have been carried out to characterize the properties of 2'-5' and 3'-5' oligoriboadenylates and oligoribouridylates from dimer to decamer. The CD band of the 3'-5' oligoribonucleotides was larger than that of the 2'-5' oligoribonucleotides and increased with the increase in chain length, while the CD band of the 2'-5' oligoribonucleotides increased little beyond the dimer level. The CD analysis of the chain length dependency revealed that the 3'-5' oligoribonucleotides adopt mainly the base-base stacking interaction, while the base-sugar interaction is predominant in the 2'-5' oligoribonucleotides. The CD intensity of 3'-5' oligoribonucleotides decreased to a larger extent at elevated temperatures or in the presence of ethanol compared to that of the 2'-5' counterparts. Mg2+ or Mn2+ ion enhanced the magnitude of the CD of 3'-5' octariboadenylate, while a small decrease in the CD was observed by the presence of Mg2+ or Mn2+ ion to the 2'-5' octariboadenylate. The 3'-5' oligoribonucleotide is likely conformationally flexible and can form helical ordered structure with strong base-base stacking depending on changes in the environment such as temperature the presence of Mg2+ ion, or hydrophobicity of the solution.

Circular Dichroism↗

PubMed1996

Functional and morphological restoration of intracranial brachial lesion of the retinocollicular pathway by peripheral nerve autografts in adult hamsters.

Axons of adult mammals can regenerate through peripheral nerve grafts and restore the retinocollicular pathway if lesioned proximal to the retinal ganglion cell somata. Whether the grafting and subsequent reinnervation of the superior colliculus (SC) is possible in distal axotomy in the brain is a question of clinical relevance. We have deafferented the SC of adult hamsters at its brachium thus axotomizing the retinal ganglion cell axons rostral to its synaptic contact with the SC neurons. After unilateral brachium transection, a short segment of the autologous sciatic nerve was grafted to bridge the lesioned site to the SC (n = 28). As controls the brachium was transected and left ungrafted (n = 12). Functional restoration was examined 3 to 75 weeks later in grafted (n = 16) and control (n = 5) animals by recording visual evoked responses from the collicular cells. Prior to recording the grafts were visually evaluated and categorized into successfully (n = 8) and unsuccessfully (n = 8) grafted groups. To diffuse flash stimuli applied to the contralateral eye, visual evoked field potentials were recorded from all successfully grafted, but not in unsuccessfully grafted (with the exception of one animal) nor control animals. Unitary spike responses to diffuse flash stimuli were recorded exclusively from three successfully grafted animals. Morphological reinnervation was examined in the remaining grafted (n = 12) and control (n = 7) animals by anterogradely labeling the regenerating retinal axons with WGA-HRP. Axons in the grafts and their terminals in the superficial layers of the SC were clearly labeled in 8 of the grafted and none of the controls. From these results we conclude that the brachium of the SC is conducive to axonal regeneration and the peripheral nerve graft is indeed effective in restoring distally axotomized visual pathway in adult mammals.

Animals↗

PubMed1996

The effect of damage of the brachium of the superior colliculus in neonatal and adult hamsters and the use of peripheral nerve to restore retinocollicular projections.

Using horseradish peroxidase (HRP) tracing technique, we were able to confirm the critical age in hamsters as reported previously (SO et al., 1981). Thus, following transection of the retinal fibers at the brachium of the superior colliculus (BSC) on postnatal-day 4 (P4) or later, no retinocollicular projections were observed in the adult stage. However, the retinal fibers were observed to reinnervate the superior colliculus (SC) if the BSC was cut on P3 or earlier. Physiological recording showed a close to normal retinocollicular map following a BSC damage on P0. Although retinal fibers did not reinnervate the SC following a BSC cut on or after P4, they could be observed to grow along a membrane over the damaged site. Bridging the site of BSC damage in adult hamsters using a segment of peripheral nerve (PN), retinal fibers labelled with WGA-HRP were observed to reinnervate the SC along the PN graft and visual evoked responses could be recorded in the SC showing the PN graft is effective in restoring damaged central visual pathways in adult mammals.

Animals↗

PubMed1996

Differences in the spectrum of spontaneous mutations in the hprt gene between tumor cells of the microsatellite mutator phenotype.

We have determined the frequency and spectrum of spontaneous mutations at the hprt locus in LoVo, HCT116, LS180 and DLD-1 colon carcinoma cell lines exhibiting microsatellite genetic instability. Each cell line has a different mutator gene. LoVo and HCT116 cells have mutated hMSH2 and hMLH1 genes, respectively, which account for the majority of hereditary non-polyposis colorectal cancer (HNPCC). LS180 cells are wild type for these genes and also for hPMS1 and hPMS2 mismatch repair genes. DLD-1 cells harbor a mutated GTBP mismatch binding factor and a mutated DNA Polymerase delta. The mutation rate at the hprt locus was several hundred fold higher in these cell lines relative to control cell lines without microsatellite instability. The mutations were frameshifts (deletions and insertions of a single nucleotide in short repeats) and single base substitutions (transversions and transitions). Some mutations were shared by these four cell lines. However, every cell line also exhibited a distinctive spectrum of mutations suggesting that each mutator gene induces a particular mutator phenotype. These results also suggest that the frequency and spectrum of somatic mutations in tumor cells of the microsatellite mutator phenotype may have diagnostic applications to discriminate among the diverse underlying mutator genes.

Base Sequence↗

PubMed1996

Comparative studies of duplex and triplex formation of 2'-5' and 3'-5' linked oligoribonucleotides.

We have studied double and triple helix formation between 2'-5' or 3'-5' linked oligoriboadenylates and oligoribouridylates with chain length 7 or 10 by CD spectrometry. The complex formation depends on the type of linkage of oligoribonucleotides, chain length, concentration and molar ratio of the strands, temperature and the cationic concentration. Mixture of any linkage isomers of oligo(rA) and oligo(rU) in 1:1 molar ratio form duplex at 0.1 M NaCl. The duplex stability largely depends on the type of the linkages and is in the following order, [3'-5'] oligo(rA)-[3'-5'] oligo(rU) > [2'-5'] oligo(rA)-[3'-5'] oligo(rU) > [3'-5'] oligo(rA)-[2'-5'] oligo(rU) > [2'-5'] oligo(rA)-[2'-5'] oligo(rU). The higher cationic concentrations, 0.5 M MgCl2, stabilize the complex and either duplex or triplex is formed depending on the input strand ratio and the type of linkage. Thermodynamic parameters, DH and DS, for the complex formation between linkage isomers of oligo(rA) and oligo(rU) showed a linear relationship indicating an enthalpy-entropy compensation phenomena. The duplex and triplex composed of [2'-5'] oligo(rA) and [2'-5'] oligo(rU) exhibit different CD spectra compared to those of any others containing 3'-5' linkage, suggesting that the fully 2'-5' duplex and triplex may possess a unique conformation. We describe prebiological significance of the linkage isomers of RNA and selection of the 3'-5' linkage against 2'-5 linkage.

Circular Dichroism↗

PubMed1996

Effects of purinoceptor agonists on cytosolic Ca2+ concentration in swine tracheal smooth muscle cells in culture.

1. The effects of various purinoceptor agonists on intracellular Ca2+ concentration ([Ca2+]i) in swine tracheal smooth muscle cells in primary culture were examined to investigate the subtype of purinoceptors in these cells. 2. ATP (1 microM to 1 mM) concentration-dependently increased [Ca2+]i which was measured by monitoring the fluorescence signal of fura2. 3. alpha, beta-Me ATP at concentrations higher than 10 microM increased [ca2+]i in the presence of extracellular Ca2+. Responses to the drug were 12 +/- 5 and 61 +/- 4% of responses to ATP (100 microM) at 100 microM and 1 mM, respectively (n = 7). The response to 100 microM ATP was inhibited by 62% in the presence of 1 mM alpha, beta-Me ATP (n = 8), though the drug at concentrations lower than that did not affect the response to ATP. 4. ATP increased [Ca2+]i in the absence of extracellular Ca2+. The response to ATP in this condition was 40% of that in the presence of extracellular Ca2+ (n = 8). 5. Neither cibacron blue 3GA (10 microM) (n = 8) nor suramin (10 and 100 microM) (n = 10) affected the response to ATP (1 microM to 100 microM). 6. The rank order of potency in the absence of extracellular Ca2+ was UTP > ATP > adenosine 5'-o-(3-thiotriphosphate) > > ADP = alpha, beta-methylene adenosine 5'-triphosphate > 2-(methylthio)-adenosine 5'-(tetrahydrogen triphosphate). 7. UTP (1 microM to 100 microM) concentration-dependently increased inositol 1,4,5-triphosphate (IP3) production. 8. These results suggest that the increase in [Ca2+]i induced by purinoceptor agonists is mediated mainly via a nucleotide receptor in swine tracheal smooth muscle cells in primary culture.

Adenosine Triphosphate↗

PubMed1996

[DNA diagnosis for short limb dwarfism, possibility of prenatal diagnosis for achondroplasia].

Explore the source record for details and available documents.

Achondroplasia↗

PubMed1996

[Ceramide: a lipid mediator of apoptotic signal transduction].

Ceramide, the backbone of sphingolipids, is now recognized as an intracellular signal mediator of various cellular responses including cell differentiation and apoptosis. Tumor necrosis factor-alpha, anti-Fas antibody, anticancer drugs, radiation or heat shock induce apoptosis through generation of ceramide by activation of sphingomyelinase or ceramide synthase. The mechanism by which ceramide mediates apoptosis is unclear. We have found that ceramide induces the transcription of c-jun gene and increases the DNA binding activity of transcription factor AP-1 in human myelogenous leukemia HL-60 cells, and that activation of c-jun/AP-1 by ceramide(presumably through activation of Jun N-terminal kinase/stress-activated protein kinase) may be involved in the signaling pathway leading to apoptosis.

Apoptosis↗

PubMed1995

Requirement of AP-1 for ceramide-induced apoptosis in human leukemia HL-60 cells.

Ceramide has emerged as a novel lipid mediator in cell proliferation, differentiation, and apoptosis. In this work, we demonstrate that the levels of c-jun mRNA, c-Jun protein, and DNA binding activity of a nuclear transcription factor AP-1 to 12-o-tetradecanoylphorbol 13-acetate responsive elements all increased following treatment with the cell-permeable ceramide, N-acetylsphingosine in human leukemia HL-60 cells. N-Acetylsphingosine (1-10 microM) increased the levels of c-jun mRNA in a dose-dependent manner, and maximal expression was achieved 1 h after treatment. Increase of c-jun expression treated with 5 microM N-acetyldihydrosphingosine, which could not induce apoptosis, was one third of that with 5 microM N-acetylsphingosine. Ceramide-induced growth inhibition and DNA fragmentation were both prevented by treatment with curcumin, 1,7-bis[4-hydroxy-3-methoxy-phenyl]-1,6-heptadiene-3,5-dione (an inhibitor of AP-1 activation), or antisense oligonucleotides for c-jun. These results suggest that the transcription factor AP-1 is critical for apoptosis in HL-60 cells and that an intracellular sphingolipid mediator, ceramide, modulates a signal transduction inducing apoptosis through AP-1 activation.

Apoptosis↗

PubMed1995

High expression of c-kit in K562YO cells due to the prolonged half-life of its mRNA: the effects of modification with serine/threonine kinase signals.

We previously reported that the K562 cell line K562YO expressed a high level of the c-kit gene. In this study, we analyzed the mechanism of this expression and investigated the effects of the serine/threonine kinases such as protein kinase C (PKC) and cyclic adenosine 3',5'-monophosphate (cAMP)-dependent kinase (PKA) on it. The half-life of the c-kit mRNA in K562YO cells was greater than 10 hours, compared with 2 hours in the original K562 cells, which expressed a very low level of c-kit mRNA. This prolonged half-life can contribute to the high level of c-kit expression in K562YO cells. Cycloheximide (CHX), a protein synthesis inhibitor, caused increases in c-kit mRNA levels in K562YO cells. 12-O-tetradecanoylphorbol-13-acetate (TPA), by which PKC was activated at first and downregulated in a late phase, gradually decreased c-kit mRNA in K562YO cells until 9 hours and then returned to the control level 24 hours after treatment. TPA also rapidly decreased c-kit protein level on the membranes. In whole cells, c-kit protein was also decreased 6 hours after incubation with TPA. Calphostin C, a light-dependent PKC inhibitor, decreased c-kit mRNA levels within 30 minutes in a light-dependent manner. It also decreased c-kit protein in whole cells 2 hours after the addition. However, it increased the amount of c-kit protein on the cell surfaces. Dibutyryl cyclic AMP (dbc-AMP) increased c-kit mRNA as well as c-kit protein on membranes and in whole cells. Run-on transcriptional assay suggested that the agent (dbc-AMP) enhanced the transcription rate of the gene. These results suggest that c-kit protein on the membranes is downregulated by PKC activation and upregulated by PKC inhibition. In the whole cell lysate, c-kit proteins are decreased by PKC inhibition through downregulation of mRNA. On the other hand, the elevation of an intracellular cAMP level causes upregulation of both the mRNA and c-kit protein on membranes and in whole cells through enhanced transcription. Thus, c-kit gene expression is apparently modulated by PKC and PKA.

Bucladesine↗

PubMed1995

Number and dendritic morphology of retinal ganglion cells that survived after axotomy in adult cats.

Retinal ganglion cells (RGCs) of adult cats were labeled by injection of diI into the proximal stump of completely transected optic nerves. Approximately 2% to 5% of the RGC population appeared viable 2 months after these axotomies, based on diI retention. The morphological type and dendritic arbor of these surviving RGCs were examined after intracellular injections of Lucifer Yellow into diI-labeled RGCs. Postaxotomy survival rate was much higher for alpha-like cells than for beta-like cells. However, in one of four retinas examined, a large number of RGCs seemed to survive axotomy, and among these, beta cells survived at an unusually high rate. Dendritic arbors of surviving RGCs were also examined after intracellular injection of horseradish peroxidase. Some dendrites of these RGCs lacked branches and were thin in caliber. Other dendrites displayed many spiny processes and bulbous swellings. Essentially, these results confirm the previous suggestion that alpha cells survive axotomy longer than beta cells. The ability of alpha cells to regenerate axons may thus be attributable to their relatively high resistance to axotomy. The atypical dendritic profiles seen after optic nerve transection may reflect either degeneration or regrowth of dendrites.

Animals↗

PubMed1995

Circulating catecholamine and glucose concentrations in Japanese toads (Bufo japonicus) during the breeding season.

We investigated the relationship between catecholamine neurohormones and glucose during seasonal reproductive activity in Japanese toads (Bufo japonicus). Field studies found that plasma epinephrine concentration increased as toads migrated to their breeding ponds, where amplexus most frequently took place. Blood glucose concentration also increased as toads arrived at the ponds, even though these animals did not eat during the breeding season, and there was a positive correlation between epinephrine and glucose levels. Blood glucose concentration was higher in amplectic than in solitary males, whereas this relationship did not occur in females. For both males and females, plasma epinephrine concentration was elevated during amplexus. The plasma concentration of norepinephrine was lower than that of epinephrine and did not correlate with either the proximity of the animal to the breeding ponds or the blood glucose concentration. Laboratory experiments showed that systemic injection of [Trp7,Leu8]gonadotropin-releasing hormone (sGnRH) increased plasma epinephrine to levels characteristic of amplectic feral toads. These results suggest that a physiological role of GnRH-like peptides may be to stimulate epinephrine secretion and consequently to increase glucose production in toads under the starvation conditions associated with the breeding migration.

Animals↗

PubMed1995

Changes of compound action potentials in retrograde axonal degeneration of rat optic nerve.

Electrophysiological recordings of compound action potentials (CAPs) were made from the optic nerve (ON) following unilateral optic tract (OT) transection in adult rats. In response to optic chiasm stimulation CAPs recorded from the ON ipsilateral to the transected OT, i.e., the ON of control side, revealed three positive waves, termed n1, n2, and n3 components, corresponding to fast-, intermediate-, and slow-conducting fibers. Two weeks after OT transection, CAPs recorded from the ON contralateral to the transected OT, i.e., the ON of cut side, were generally smaller in amplitude than those recorded from the ON of control side. The degree of amplitude reduction was different among three components; n3 component was severely reduced and n2 component was moderately reduced, whereas n1 component relatively persisted. These tendencies were more marked in CAPs recorded at 4 weeks after OT transection; n3 component disappeared, whereas n1 and n2 components decreased in amplitude with elongation of latency. Cross sections of the ON after unilateral OT transection were examined electron microscopically, in which significant decrease in fiber density, demyelination, and distorted fibers were verified in the cut side. The present study suggests that slower-conducting ON fibers, that is, smaller-diameter ON fibers are initially and remarkably thrown into retrograde degeneration, while larger-diameter ON fibers are relatively resistant to axotomy.

Action Potentials↗