Biomedical subjects

K Yamagami

Publications and source records attributed to K Yamagami.

At least 55 records · Page 3Linked to original sources

PubMed1995

Age-specific alterations in muscarinic stimulation of K(+)-evoked dopamine release from striatal slices by cholesterol and S-adenosyl-L-methionine.

The present experiments were carried out in order to test the hypothesis that age-related signal transduction (ST) deficits may occur as a result of structural changes in the membrane that are reflected partially as increased membrane microviscosity. Oxotremorine (oxo) enhancement of K(+)-evoked release of dopamine (K(+)-ERDA) was examined in superfused striatal slices from mature (6 months) and old (24 months) Wistar rats incubated (1 or 4 h, 37 degrees C) with graded concentrations of S-adenosyl-L-methionine (SAM) or cholesterol hemisuccinate (CHO) in a modified Krebs medium. Tissue was then assessed for one of the following: (a) the degree of oxo-enhanced K(+)-ERDA, (b) carbachol stimulated low Km GTPase activity, or (c) alterations in membrane microviscosity. In other experiments the tissue was incubated in CHO followed by SAM (or the reverse), and oxo-enhanced K(+)-ERDA examined. Results indicated that SAM treatment increased all the parameters in the striatal tissue from old animals, while CHO had selective, opposite effects in the striatal tissue obtained from young animals. CHO-SAM, or the reverse, produced the same pattern of results. These results suggest that ST deficits may involve age-related structural alterations in membranes that interfere with receptor-G protein coupling/uncoupling.

Aging↗

PubMed1995

Cloning of cDNAs for the precursor protein of a low-molecular-weight subunit of the inner layer of the egg envelope (chorion) of the fish Oryzias latipes.

cDNA clones for L-SF, the precursor of a low-molecular-weight subunit (ZI-3) of the inner layer of the Oryzias latipes egg envelope were isolated from Lambda ZAP cDNA libraries constructed from the poly(A)+ RNA of the liver of spawning female fish and estrogen-treated male fish. Among them, a clone, L-SF41, is 1473 bp long and contains an open reading frame encoding a signal peptide of 19 amino acids and L-SF protein of 420 amino acids. L-SF protein seems to be glycosylated, judging from the result of the glycanase digestion. L-SF protein contains a domain similar to ZP-domains in ZP3 of some mammalian species. Northern blot analysis employing XhoI-SmaI fragments of the cloned cDNA as probes revealed that expression of the L-SF gene occurred exclusively in the livers of spawning female fish and estrogen-treated male fish and that there was no mRNA encoding L-SF in the ovary of the spawning female fish.

Amino Acid Sequence↗

PubMed1995

Temporal and spatial patterns of gene expression for the hatching enzyme in the teleost embryo, Oryzias latipes.

The hatching enzyme of the medaka, Oryzias latipes, consists of two proteases, high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE). They are synthesized and accumulated in the same unicellular hatching glands and are secreted from them at the end of embryonic development to digest the egg envelope. Recently, these enzymes were purified, and their cDNA clones were isolated. In the present study, we examined temporal and spatial patterns of expression of the hatching enzyme genes during embryogenesis using cDNAs for HCE and LCE as probes. According to Northern blotting analysis, the expression of both genes started at the same time (stage 21-22 embryos: brain differentiation and lens formation) and the patterns of expression changed in parallel during development. In situ hybridization to whole embryo and the sections revealed that the expression of the HCE genes was detected first in the anterior end of the hypoblast layer in stage 16-17 (late gastrula) embryos. Distinct signals of the HCE gene expression were then detected in a group of cells located at the front of the head rudiment of embryos at stage 18-19 (1 somite). Treatment of the embryos with retinoic acid, which is known to affect the anterior differentiation of embryos, suppressed the hatching gland cell differentiation in accordance with the result of in situ hybridization. In stage 22 embryos, the HCE-positive cells dispersed in an ectodermal layer under the forebrain and optic vesicles. Thereafter, the hatching gland cells expressing the HCE mRNA were aligned along the branchial arches and finally rearranged to the inner wall of the pharyngeal cavity, following a marked elongation of the lower jaw. The results of in situ hybridization to whole embryos at consecutive developmental stages demonstrated that the hatching gland cells located at the most anterior portion of the hypoblast migrated posteriorward to endoderm (pharyngeal endoderm) by way of ectoderm, while they were expressing mRNA for the hatching enzyme. Retinoic acid treatment of embryos gave rise to aberrations in the final location of the hatching gland cells probably by disturbing their migration. Moreover, the number of hatching gland cells increased markedly during their migration. This fact strongly suggested a concurrence of gene expression and mitosis of a gland cell and/or a successive initiation of gene expression in maturing gland cells during migration.

Animals↗

PubMed1995

Interaction of the cell-binding domain of fibronectin with VLA-5 integrin induces monokine production in cultured human monocytes.

The effect of fibronectin on IL-1 alpha, IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), and IL-6 production was investigated with cultured monocytes isolated from human peripheral blood. Monokine concentrations were determined by both ELISA and bioassay. Fibronectin markedly stimulated the secretion of IL-1 alpha, IL-1 beta, TNF-alpha and IL-6 from cultured monocytes in a dose-dependent manner, with the maximal effect apparent within 24 h. Northern blot analysis revealed a marked increase in the abundance of mRNA specific for each monokine on exposure of monocytes to fibronectin. Monoclonal antibodies to the alpha chain of very late antigen (VLA)-5, the beta 1 integrin, the alpha chain of Mac-1, and the beta 2 integrin, as well as the synthetic peptide of GRGDSP (which corresponds to the cell-binding domain of fibronectin), inhibited (> 50%) fibronectin-induced monokine production. Monoclonal antibodies to the alpha chain of VLA-4, and the alpha chain of LFA-1, as well as the synthetic peptide CS-1 (which corresponds to the alternatively spliced connecting segment of fibronectin) and the control peptide GRADSP, had no inhibitory effect on monokine production. A MoAb, R60, that recognizes an epitope of the fibronectin molecule that includes the RGD sequence, inhibited monokine production, whereas the MoAb Y16, which recognizes another epitope of fibronectin not including RGD, did not. These results indicate that fibronectin-induced production of IL-1 alpha, IL-1 beta, TNF-alpha and IL-6 from cultured monocytes is mediated predominantly by interaction of the cell-binding domain of fibronectin with VLA-5, although Mac-1 also may contribute to this effect of fibronectin. Our results indicate that the interaction of fibronectin with integrins may contribute to the cytokine network in inflammatory response.

Binding Sites↗

PubMed1995

Xanthine oxidase mediates paraquat-induced toxicity on cultured endothelial cell.

The role of xanthine oxidase in paraquat toxicity was investigated using cultured bovine pulmonary artery endothelial cells. Exposure to paraquat 0.1 mM was done for 24 hr with or without tungsten pretreatment and in the presence or absence of xanthine oxidase inhibitors. Exposure to paraquat significantly increased O2- production and relative xanthine oxidase activity (xanthine oxidase activity divided by total xanthine dehydrogenase plus xanthine oxidase) while depressing cell growth. In contrast, tungsten and allopurinol inhibited the increase of xanthine oxidase activity and decreased O2- release. Cell injury was assessed by leakage of lactate dehydrogenase and by fluorescein diacetate staining; it was found that oxidase inhibitors (both allopurinol and tungsten) reduced paraquat cytotoxicity. Thus the toxicity of paraquat was at least partly due to intracellular O2- production mediated by xanthine oxidase and the subsequent formation of other free radicals.

Allopurinol↗

PubMed1995

Hypoxia alters the energy metabolism and aggregation of washed human platelets.

Washed human platelets were incubated under hypoxic and normoxic conditions to investigate the influence of oxygen deprivation on energy metabolism and aggregation. The maximum aggregation rate, oxygen consumption, lactate production, and adenine nucleotide content were measured. Hypoxic incubation decreased the oxygen burst and maximum aggregation induced by thrombin, and accelerated anaerobic glycolysis. ATP and ADP levels were preserved, but the hypoxanthine level increased in the incubation medium and the platelet AMP level and adenylate energy charge decreased compared with normoxic incubation. Thus, anaerobic glycolysis failed to compensate for impaired oxidative phosphorylation during hypoxic incubation, suggesting that oxidative energy is essential for full platelet function.

Adenine Nucleotides↗

PubMed1995

Association of epidermal growth factor-related peptides and type I receptor tyrosine kinase receptors with prognosis of human colorectal carcinomas.

The frequency of expression and localization of cripto-1 (CR-1), amphiregulin (AR), transforming growth factor alpha (TGF alpha), epidermal growth factor receptor (EGFR) and erbB-2 were examined by immunohistochemistry in 45 carcinomas and adjacent non-involved normal colon mucosa. Thirty (66.7%), 24 (53.3%), 23 (51.1%), 23 (51.1%) and 13 (28.9%) of the 45 carcinomas showed positive staining for CR-1, AR, TGF alpha, EGFR and erbB-2, respectively, whereas 7 (15.5%), 17 (37.7%), 15 (33.3%), 20 (44.4%) and 0 (0%) of the corresponding non-involved normal mucosa specimens were reactive. Among 13 carcinomas with lymph node involvement, 10 (76.9%), 8 (61.5%), 10 (76.9%), 8 (61.5%) and 7 (53.8%) exhibited positive staining for CR-1, AR, TGF-alpha, EGFR and erbB-2, respectively. There was a statistically significant association between the frequency of either TGF alpha (P < 0.05) or erbB-2 (P < 0.05) expression and lymph node metastasis. In addition, a significantly higher frequency of positive staining for TGF alpha was observed in Dukes' grade C carcinomas (P < 0.05). Finally, significant trends for coexpression of EGFR and either TGF alpha (P < 0.01) or AR (P < 0.05) were detected in carcinomas. These data suggest that AR and TGF alpha may play an important role in the development of colorectal carcinomas through an autocrine mechanism involving EGFR, and demonstrate that TGF alpha and erbB-2 may be more reliable indicators of metastasis or prognosis than CR-1, AR or EGFR in human colon cancers.

Adenocarcinoma↗

PubMed1994

Area specific alterations in muscarinic stimulated low Km GTPase activity in aging and Alzheimer's disease: implications for altered signal transduction.

Carbachol-stimulated low Km GTPase activity (an index of muscarinic receptor-G protein coupling) was examined in hippocampus, basal ganglia, orbital frontal gyrus and superior frontal gyrus obtained from mature, aged and Alzheimer's Disease (AD) groups. Results indicated that carbachol-stimulated low Km GTPase activities in basal ganglia were as follows: mature controls > aged > AD, and there was a trend toward a similar pattern of decline in the hippocampus. No differences were seen in the two cortical areas examined; however, carbachol-stimulated low Km GTPase activity was small in the mature controls. Importantly, there were significant negative correlations between disease duration and carbachol-stimulated low Km GTPase activity in all areas examined except the orbital frontal gyrus. The longer the duration of the disease the lower the carbachol-stimulated low Km GTPase activity. Results suggest that age and disease-related changes in mAChR-G protein interactions in the basal ganglia may contribute to reduced signal transduction (ST). In addition, since decreased carbachol-stimulated low Km GTPase activity has also been observed in the aged rat; thus, investigations of the factors involved in decrements in signal transduction in the aged rat may be useful for understanding these alterations in aged humans or victims of AD.

Aged↗

PubMed1994

Cd2+ and Co2+ at micromolar concentrations stimulate catecholamine secretion by increasing the cytosolic free Ca2+ concentration in cat adrenal chromaffin cells.

Cd2+ and Co2+ at micromolar concentrations increased the cytosolic free Ca2+ concentration, which was measured by fura-2 microfluorometry, in cat adrenal chromaffin cells. Simultaneously, these divalent cations stimulated catecholamine secretion from the perfused adrenal. The present findings suggest that these cations increase the Ca2+ influx by depolarizing the cell membrane and consequently stimulate catecholamine secretion.

Adrenal Medulla↗

PubMed1994

Synchronous production of the low- and high-molecular-weight precursors of the egg envelope subunits, in response to estrogen administration in the teleost fish Oryzias latipes.

Estrogen stimulation of production and accumulation of L-SF and H-SF, the low- and high-molecular-weight precursors of the egg envelope subunits in Oryzias latipes, was analyzed by immunohistochemical and immunoblotting methods. Both SF substances were detected in the liver of the male fish as quickly as 16-24 hr after estrogen treatment, and their production and accumulation in the liver and blood plasma increased as estrogen treatment continued. Immunohistochemical analysis revealed that the number of cells producing both SF substances was increased in the liver as estrogen treatment continued. These results strongly suggest the occurrence of synchronous production of L-SF and H-SF in response to estrogen. In liver extracts, but not in blood plasma, an additional L-SF-like immunoreactive protein was detected on immunoblots. Preliminary data indicate parallel appearance of vitellogenin and SF substances in the liver of estrogenized fish.

Animals↗

PubMed1994

Inability of Ca2+ influx through nicotinic ACh receptor channels to stimulate catecholamine secretion in bovine adrenal chromaffin cells: studies with fura-2 and SBFI microfluorometry.

The concentration of cytosolic Ca2+ ([Ca]in) and catecholamine (CA) secretion were examined in bovine adrenal chromaffin cells to determine whether Ca2+ influx through nicotinic ACh receptor (nAChR) channels contributes to CA secretion induced by nAChR stimulation. Nicotine added under Na(+)-free conditions caused a marked increase in [Ca]in and quenching of fura-2 fluorescence in the presence of Mn2+, suggesting the stimulated entry of divalent cations through nAChR channels. However, nicotine-induced increase in CA secretion occurred only at a non-physiologically high external Ca2+ concentration under Na(+)-free conditions. Both the nicotine-induced increase in [Ca]in and CA secretion under Na(+)-free conditions were reduced in the presence of hexamethonium, methoxyverapamil (D600), nifedipine, Bay-K-8644, clonidine, and guanethidine. All of these agents inhibited the nicotine-induced increase in cytosolic Na+ concentration in a dose-dependent manner, as measured by SBFI microfluorometry. The present results suggest that Ca2+ influx through nAChR channels under physiological conditions may not contribute to CA secretion.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1994

[An analysis of autopsy cases with cancer of unknown primary site].

A retrospective review of autopsy cases with cancer of clinically unknown primary site seen at Shikoku Cancer Center Hospital from 1959 to 1992 was performed. Fifty three cases (3.1%) were registered as having cancer of unknown primary site among 1690 consecutive autopsy cases with malignancies except for leukemias and brain tumors. Major discrepancies on the primary site between the clinical diagnoses and the autopsy reports were present in 30 cases (1.8%). Twelve cases (0.7%) with malignancy of peritoneum or retroperitoneum primary were found that were clinically regarded as peritonitis carcinomatosa. Latent cancer, in which a new primary cancer was subsequently identified at autopsy, was recognized in 190 cases (11.2%). The mean age in latent cancer group was significantly higher than in control group, but patients with cancer of unknown primary site tended to be somewhat younger. A high male: female ratio was found in latent cancer group, whereas it was reverse in cases with clinically undetermined primary cancer. Survival overall was poor, with a median survival of 5 months. As diagnostic techniques (such as sonography, computerized tomography scanning and nuclear scans) have been improved, cases with cancer of unknown primary site have tended to decrease, but the percentage of cases presenting latent cancer has remained relatively stable over the years. Precise autopsy examination revealed that the primary sites of clinically undetermined cancers were ovary, pancreas, stomach, gallbladder and peritoneum in order of decreasing frequency. The metastatic pattern and histologic types were not different from that in the control group.

Aged↗

PubMed1993

Isolation of H-SF substances, the high-molecular-weight precursors of egg envelope proteins, from the ascites accumulated in the oestrogen-treated fish, Oryzias latipes.

High- and low-molecular weight spawning-female-specific (H-SF and L-SF) substances are liver-derived putative precursors of the inner layer of egg envelope of the medaka Oryzias latipes, and are also found in the ascites which are accumulated in the male fish on prolonged treatment with oestrogen. In the present study, the H-SF substances purified from the ascites of the oestrogen-treated male medaka were found to consist of three proteins. Their molecular weights ranged from 74,000 to 76,000, which corresponds to those of ZI-1 and ZI-2, the major components of medaka egg envelope. The amino acid composition of the H-SF substances was characterised by high contents of proline (17.58 mol%) and glutamic acid/glutamine (13.34 mol%). The characteristics were almost identical to those of ZI-1 and ZI-2 but differed from those of L-SF substance, a precursor of ZI-3, another major component of the egg envelope. These results confirm our view that H-SF substances are the precursors of ZI-1 and ZI-2. The average amino acid compositions of H-SF substances and L-SF substance coincided with the amino acid composition of whole egg envelope. This result strongly suggests that an equal amount of H-SF substances and L-SF substance would construct the inner layer of egg envelope after a little modification.

Amino Acids↗

PubMed1993

Effects of point mutation in a flexible loop on the stability and enzymatic function of Escherichia coli dihydrofolate reductase.

To elucidate the role of a flexible loop in the stability and function of Escherichia coli dihydrofolate reductase, glycine-121 in the flexible loop (117-131) was substituted to valine and leucine by site-directed mutagenesis. Despite the increased hydrophobicity of the side chains, the free energy changes of unfolding of the two mutants (G121V and G121L) determined by urea denaturation at 15 degrees C were decreased by 1.22 and 0.38 kcal/mol, respectively, compared with that of the wild-type. Thermal denaturation temperature, as monitored by differential scanning calorimetry, was decreased by 2.4 and 5.2 degrees C for G121V and G121L, respectively, accompanying the decrease in enthalpy change of denaturation. These findings indicate that the structure of DHFR is destabilized by the mutations, predominantly due to the large decrease in enthalpy change of denaturation relative to entropy change of denaturation. The steady-state kinetic parameter in the enzyme reaction, Km, was not influenced but kcat was greatly decreased by these mutations, resulting in 240- and 52-fold decreases in kcat/Km for G121V and G121L, respectively. The main effect of the mutations appeared to be modification of the flexibility of the loop due to overcrowding of the bulky side chains, overcoming the enhancement of hydrophobic interaction.

Base Sequence↗

PubMed1993

Tungsten modulates the toxicity of paraquat for epithelial cells.

The modulation of paraquat toxicity by tungsten was studied in vitro using cultured MDCK epithelial cells. MDCK cells were cultured in minimal essential medium with or without 1 ppm tungsten. Proliferation of cells cultured with tungsten was not inhibited after exposure to 0.25 mM or 0.5 mM paraquat. In addition, lactate dehydrogenase release into the culture medium was lower for tungsten-treated cells than for cells cultured without tungsten. Cells cultured in medium alone showed reduced viability compared with controls after exposure to 0.5 mM paraquat, but 0.25 mM paraquat did not decrease cell viability. Tungsten-treated cells showed no decrease viability in after exposure to either concentration of paraquat. Cells exposed to paraquat developed a honeycomb morphology with scanty cytoplasm and abnormal nucleoli. However, these major structural changes were not observed in cells cultured with tungsten. Our study showed that cell damage after paraquat exposure was modulated by addition of tungsten to the culture medium. It is suggested that cytosolic xanthine oxidase activity was reduced by tungsten, leading to less production of superoxide and other radicals and thus conferring resistance to paraquat toxicity.

Animals↗

PubMed1992

The effects of aging on muscarinic receptor/G-protein coupling in the rat hippocampus and striatum.

In the striatum and hippocampus, there is a loss of sensitivity to muscarinic agonists with age which has been traced to events early in the signal transduction pathway. Our laboratory has therefore focussed on investigations at this level. The current experiments investigate the effects of age on G-protein/receptor interactions by using competitive binding assays to measure the ability of GppNHp to decrease the proportion of receptors bound to G-proteins in the absence and the presence of added Mg2+. L-[3H]Quinuclidinyl benzilate was used as a nonselective ligand and [3H]pirenzepine as an M1 selective ligand. We find that: (1) muscarinic receptors and G-proteins in the striatum appear to become loosely coupled with age, with no change in Mg2+ sensitivity. (2) M1-receptor/G-protein complexes in the hippocampus display increased sensitivity to the presence of Mg2+ with age, with those from old but not young tissue requiring added Mg2+ in order to uncouple. This effect, however, may not be M1 specific.

Aging↗

PubMed1992

Decrement of muscarinic receptor-stimulated low-KM GTPase in striatum and hippocampus from the aged rat.

Previous studies have shown that there is an age-related loss of responsiveness in several different receptor systems (e.g. beta-adrenergic, dopaminergic and muscarinic). Our research, using perifused striatal slices and examining muscarinic agonist enhancement of K(+)-evoked dopamine release, has determined that at least part of the loss of sensitivity in muscarinic receptors (mAChR) may occur early in the post-receptor signal transduction process. The present study was carried out to further characterize and localize this deficit by examining carbachol- and oxotremorine-stimulated low-KM guanosine triphosphatase (GTPase) activity in striatal as well as hippocampal tissue obtained from adult (6 months) and old (24 months) Wistar rats. Receptor stimulated low-KM GTPase catalyzes the conversion of GTP to GDP to end the signal transduction cycle and is an indicator of receptor-G-protein coupling/uncoupling. The results showed that stimulated GTPase activity was significantly reduced in hippocampal and striatal tissue from the old animals. These findings suggest that there may be an age-related coupling/uncoupling deficit between muscarinic receptor and G-proteins, and that this deficit may contribute to the reduced mAChR responsiveness in senescence.

Aging↗

PubMed1992

Possible role of surface potential in the gating mechanism of Ca2+ channels in cat adrenal chromaffin cells: studies with fura-2 microfluorometry.

The cytosolic free Ca2+ concentration ([Ca]in) in isolated cat chromaffin cells was measured by fura-2 microfluorometry. During 30 mM KCl depolarization or sucrose substitution for NaCl, a reduction in external Ca2+ concentration under optimal conditions paradoxically caused a rise in [Ca]in and, in separate experiments, in catecholamine secretion. The results support a previously suggested role of surface potentials in the gating mechanism of Ca2+ channels.

Adrenal Medulla↗