Biomedical subjects

Y Seyama

Publications and source records attributed to Y Seyama.

At least 73 records · Page 4Linked to original sources

PubMed1996

Two distinct long-chain-acyl-CoA synthetases in guinea pig Harderian gland.

Two distinct long-chain-acyl-CoA synthetases which have different kinetic properties were identified in the guinea pig Harderian gland. One was localized in the microsomes and the other in the mitochondria. The relative V(max) values of the microsomal enzyme were 8.1, 1.7 and 1 and the apparent Km values were 66.7, 12.0 and 30.0 microM for palmitic, linoleic and arachidonic acids, respectively. The relative V(max) values of the mitochondrial enzyme were 2.7, 3.5 and 1 and the apparent Km values were 33.3, 29.9 and 30.0 microM for palmitic, linoleic and arachidonic acids, respectively. The relative V(max) values for the liver microsomal enzyme were 2.0, 2.5 and 1, while those of the liver mitochondrial enzyme were 4.1, 3.9 and 1 with palmitic, linoleic and arachidonic acids, respectively. There were no difference between the microsomal and the mitochondrial enzymes in the liver, regarding apparent Km values; these were 38.4, 29.9 and 22.0 microM for palmitic, linoleic and arachidonic acids, respectively. Thus, the substrate specificity and catalytic rate of the mitochondrial enzyme in Harderian gland for palmitic, linoleic and arachidonic acids were similar to the liver enzyme, but not to the microsomal enzyme in Harderian gland. On the other hand, the antiserum raised against the rat liver enzyme immune-titrated and immuno-blotted the enzymes from Harderian gland microsomes and liver, but not so the enzyme from Harderian gland mitochondria. Thus, the microsomal enzyme in Harderian gland had a common immunogenic epitope(s) with the liver enzyme, but the mitochondrial enzyme did not. The Harderian gland mitochondrial enzyme was a distinct protein from liver enzymes. The catalytic and immunogenic characteristics suggest that the enzyme proteins in the Harderian gland are unique, that is, different from that in the liver. The large V(max) value of the Harderian gland microsomal enzyme for palmitic acid suggests that it contributes to the synthesis of a large amount of the secretory lipid and the high Km value to maintenance of cellular lipid in this organ. The evidence that long-chain-acyl-CoA synthetase in the mitochondria is distinct from that in the microsomes was first found in guinea pig Harderian gland.

Animals↗

PubMed1996

Sexual diversity of the lipid metabolism in the Harderian gland of the golden hamster.

Fatty acid and alkyl compositions of 1-alkyl-2,3-diacylglycerols (ADG) in the Harderian gland of the golden hamster were determined. Fatty acids of male ADG consisted of straight chain saturated acids ranging from C12 to C22. Both even- and odd-numbered acids were observed, indicating that acetyl- and propionyl-CoAs were equally used as primers in the fatty acid synthesis. In female ADG a large amount of iso- and anteiso-branched fatty acids were detected. Odd-numbered acids (C15, 17, 19, and 21) contained iso- together with anteiso-branchings, and even-numbered acids (C16 and 18) contained iso-branchings. These findings suggested that isobutyryl-, isovaleryl-, and 2-methylbutyryl-CoAs were used as primers in addition to acetyl- and propionyl-CoAs in fatty acid synthesis in the female gland. Such unusual primers are catabolic intermediates of valine, leucine, and isoleucine, respectively. Male ADG contained straight chain saturated alkyl residues ranging from C14 to 21. In the case of female ADG, though the main component was C18:O, most alkyl components had branchings at the iso- and anteiso-positions. NMR data suggested the presence of propionic acid at the C3 position of glycerol in the male ADG and of isovaleric and 2-methylbutyric acids at the same position in the female ADG. These results suggest that the sexual differences of the lipids in the Harderian gland of the golden hamster are determined at the step of fatty acid synthesis depending on the available precursors in the male and female glands. We speculate that sex hormones affect the metabolism of branched chain amino acids and bring about the dramatic diversities of the lipid compositions in the male and female glands.

Amino Acids↗

PubMed1996

Tumor necrosis factor alpha and lymphotoxin stimulate human myeloblastic leukemia cell (ML-1) invasion through a reconstituted basement membrane (Matrigel) with concomitant induction of 92 kDa gelatinase secretion.

We have examined the effects of tumor necrosis factor alpha (TNF) and lymphotoxin (LT) on gelatinase (72 kDa and 92 kDa) and tissue inhibitor of metalloprotease 1 (TIMP1) secretion by human myeloblastic leukemia cells (ML-1) in vitro. TNF (0.1-30 ng/ml) significantly stimulated 92 kDa gelatinase secretion in a dose-dependent manner, but did not significantly stimulate 72 kDa gelatinase secretion. LT also significantly stimulated 92 kDa gelatinase secretion, but the stimulation was less effective compared to TNF. TNF, but not LT, concentrations at 30 ng/ml slightly stimulated TIMP1 secretion. Because 92 kDa gelatinase is thought to play a pivotal role in tumor invasion, we examined the effect of TNF or LT on ML-1 cell invasion through a reconstituted basement membrane (Matrigel). Exposure of ML-1 cells to TNF (3, 10, and 30 ng/ml) or LT (3, 10, and 30 ng/ml) stimulated ML-1 cell invasion through Matrigel in a dose-dependent manner in vitro. The data suggest that TNF- and LT-stimulated 92 kDa gelatinase secretion could play an important role in TNF- or LT-stimulated ML-1 cell invasion.

Antibodies, Monoclonal↗

PubMed1996

Endothelin-1 and vasopressin activate Ca(2+)-permeable non-selective cation channels in aortic smooth muscle cells: mechanism of receptor-mediated Ca2+ influx.

The effects of vasopressin and endothelin-1 on cultured aortic smooth muscle cell lines (A7r5) were investigated by measurements of intracellular calcium [Ca2+]i and the patch-clamp techniques. Vasopressin and endothelin-1 (100 nM) evoked an initial peak followed by a smaller sustained rise of [Ca2+]i in the presence of extracellular calcium [Ca2+]o. In the absence of [Ca2+]o, only the initial peak of [Ca2+]i was observed. Therefore, the initial peak of [Ca2+]i was mainly due to calcium release from the storage sites, whereas the later sustained rise of [Ca2+]i was due to the calcium entry from outside. The sustained rise of [Ca2+]i was unaffected by nifedipine (10 microM) significantly, but was completely abolished by La3+ (1 mM). Under current clamp conditions with K(+)-internal solution, vasopressin and endothelin-1 (100 nM) produced hyperpolarization, then followed by depolarization. Under voltage clamp conditions at a holding potential of -40 mV, both vasopressin and endothelin-1 first activated the outward current, then followed by a long-lasting inward current with a high noise level. The first outward current was abolished by charybdotoxin (100 nM), Cs+ in the patch pipette and high EGTA (10 mM) in the pipette, suggesting that it was a Ca(2+)-sensitive K+ current (IK.Ca). The inward current was still elicited with the patch pipette containing Cs(+)-internal solution, and reversed at about 0 mV. The reversal potential was not significantly altered by the replacement of [Cl-]i or [Cl-]o, proposing that the inward current is a cation selective channel (IN.S.). The inward current was also observed even when extracellular cations are Ca2+. La3+ (1 mM), Cd2+ (1 mM) completely abolished the vasopressin-induced (IN.S.), however, nifedipine (10 microM) failed to inhibit it significantly. Single channel activities were recorded in the cell-attached configurations when vasopressin or endothelin-1 was applied to the bathing solution. The unitary conductance of the channels was approximately 20 pS with 140 mM Na+, Cs+, or K+ in the pipette, but was 15 pS with 110 mM Ca2+ in the pipette. Permeabilities sequence calculated from the reversal potentials was Na+ not equal to Cs+ not equal to K+ > Ca+. These results provide evidence that calcium entry and membrane depolarization elicited by vasopressin or endothelin-1 are mediated by a receptor-mediated Ca(2+)-permeable non-selective cation channel in aortic smooth muscle cells.

Aorta, Thoracic↗

PubMed1996

Isolation of three novel cholinergic neuron-specific gangliosides from bovine brain and their in vitro syntheses.

In the present study, three extremely minor but novel Chol-1 antigens, termed X1, X2, and X3 have been isolated from bovine brain gangliosides. Based on the results of sialidase degradation, TLC-immunostaining with anti-Chol-1 antibody and fast atom bombardment mass spectrometry, their chemical structures were identified as: III6NeuAc-GgOse4Cer (X1: GM1 alpha) III6NeuAc,II3NeuAc-GgOse4Cer (X2: GD1a alpha) III6NeuAc,II3NeuAc-NeuGc-GgOse4Cer (X3: GT1b alpha) The yields of GM1 alpha, GD1a alpha, and GT1b alpha, were approximately 150, 20, and 10 micrograms, respectively, from 10 g of the bovine brain ganglioside mixture. In conjunction with our previous observations, all gangliosides with anti-Chol-1 reactivity were found to contain a common sialyl alpha 2-6 N-acetylgalactosamine residue, indicating that this unique sialyl linkage is the specific antigenic determinant. We subsequently examined the biosynthesis of the three novel Chol-1 gangliosides using rat liver Golgi fraction as an enzyme source. The results showed that GM1 alpha, GD1a alpha, and GT1b alpha were synthesized from asialo-GM1, GM1a, and GD1b, respectively, by the action of a GalNAc alpha 2-6sialyltransferase.

Animals↗

PubMed1996

PET analysis of a case of cerebrotendinous xanthomatosis presenting hemiparkinsonism.

We describe a 34-year-old Japanese woman presenting gait difficulty and Achilles tendon swelling. The patient was diagnosed as having cerebrotendinous xanthomatosis (CTX) based on the high serum cholestanol level and diminished enzymatic activity of 27-hydroxylase of fibroblasts from her skin. Her clinical presentation was atypical regarding the presence of hemiparkinsonism and absence of apparent cataract, dementia, and cerebellar ataxia. Although MRI studies could not detect any abnormality in the basal ganglia or midbrain, PET analysis using [18F]-6-fluoro-L-dopa revealed reduced uptake of dopamine into the putamen, suggesting the impairment of presynaptic dopaminergic neurons.

Adult↗

PubMed1996

Analysis of connective tissue macromolecular components in Ishibashi rat skin: role of collagen and elastin in cutaneous aging.

The Ishibashi (IS) rat, established from cross-breeding between Wistar and wild rats, has a unique skin appearance, with wrinklings and furrows indicative of skin aging appearing at the age of 12 weeks. To understand the underlying mechanism of the formation of wrinkles, macromolecular components of connective tissue, collagen and elastin, in the young (5-6-week-old) and the aged (23-30-week-old) IS rat skins were biochemically analyzed. Hydroxyproline and isodesmosine contents in the aged IS rats were reduced 22% (P < 0.05) and 37% (P < 0.05) compared to the young rats, whereas no significant differences in the contents of both macromolecules in control Sprague-Dawley (SD) rats were seen. The relative content of type III collagen was unaltered between the young and aged skins of both IS and SD rats. A relative decrease in the intact elastin molecule (65 kDa) and a relative increase in the elastin fragments with lower molecular weights were observed in the aged IS rat skin by immunoblotting method. These results indicate that the reduction in collagen and elastin contents and increased degradation of elastin molecules in the aged IS rat skin could be related to the formation of wrinkles. Thus, the IS rat may provide a useful model for the study of skin aging.

Aging↗

PubMed1996

Analytical studies of spontaneous and vasopressin-induced calcium oscillations in cultured vascular smooth muscle cells.

Spontaneous and vasopressin-induced Ca2+ oscillations in cultured vascular smooth muscle (A7r5) cells were further examined and characterized. Intracellular Ca2+ concentrations ([Ca2+]i) were measured by use of a high-performance laser cytometer. When the oscillatory patterns in [Ca2+]i were analyzed with a power spectrum method, about 80% of cells exhibited spontaneous Ca2+ oscillations with the frequency of 0.02-0.5 Hz. Nifedipine abolished these repetitive spikes, whereas pinacidil partially attenuated their amplitude and frequency. When vasopressin (100 nM) was applied to A7r5 cells, there was an initial rise in [Ca2+]i, followed by a delayed sustained increase in [Ca2+]i. The one-pool, nonoscillatory model was employed to fit this biphasic change, and the difference between the observed response and the simulated response was then analyzed with a power spectral method. About 50% of cells were noted to display oscillatory patterns in [Ca2+]i after sustained increase in [Ca2+]i. The present study indicates that spontaneous Ca2+ oscillations in A7r5 cells are modulated by the activity of ATP-sensitive K+ channels and are not related to pertussis toxin-sensitive GTP-binding protein(s). On the basis of the one-pool, nonoscillatory model, it is suggested that the buffering capacity of internal stores appears to be stronger in the cells with spontaneous Ca2+ oscillations than in those in a quiescent state, and the vasopressin-mediated inhibition of accumulation by internal stores was attenuated when the cells exhibited spontaneous Ca2+ oscillations. The implementation of this minimum kinetic model integrated with a power spectrum method would be an alternative to understand the oscillating behavior in [Ca2+]i.

Adenosine Triphosphate↗

PubMed1996

Androgenic control of 1-alkyl-2,3-diacylglycerol in the harderian gland of the golden hamster, Mesocricetus auratus.

Harderian glands of golden hamsters produce a copious lipid secretion, most of which is in the form of 1-alkyl-2,3-diacylglycerol (ADG). Sexual differences are seen in the composition of golden hamster ADG and in the morphology of secretory lipid droplet. ADGs from females contained abundant iso- and anteiso-branched chain alkyl groups and fatty acids [Seyama, Y., Otsuka, H., Ohashi, K., Vivien-Roels, B., and Pevet, P. (1995) J. Biochem. 117, 661-670]. Female hamsters were either untreated or given subcutaneous testosterone pellets. Treatment of females with testosterone led to the disappearance of such branched chain alkyl groups and fatty acids. Intact males had ADGs with entirely saturated straight chain alkyl groups and fatty acids. Castration led to the appearance of iso- and anteiso-branched chain alkyl groups and fatty acids. These observations suggested that the production of branched chain fatty acids in the Harderian gland of golden hamster is inhibited by testosterone at the step of isovaleryl-CoA dehydrogenase and 2-methyl branched-chain acyl-CoA dehydrogenase

Alkenes↗

PubMed1996

Localization of porphyrin in mouse Harderian glands.

Lipid secreting Harderian glands in the ocular region of rodents contain a large amount of metal-free porphyrins. The localization of these porphyrins and the areas of formation of the lipidporphyrin complexes in the glandular lumina have not been clearly defined. We therefore examined the intracellular localization of porphyrin fluorescence both in tissue sections and in cultured cells of Harderian glands of 5 weeks old female ICR mice, using confocal UV-laser scanning microscopes. The fluorescence appeared in glandular cells containing lipid droplets, but not in non-glandular cells (e.g. myoepithelial or interstitial) without these droplets. Fluorescence in the cells was recognized in the cytoplasm, but there was no evidence indicating that porphyrins were restrictively stored in mitochondria. Nuclei and lipid droplets lacked fluorescence. It is thus reasonable to conclude that porphyrins accumulate in the cytoplasm, and that porphyrins and lipids are secreted separately from glandular cells. The lipid-porphyrin complex is therefore probably formed in the glandular lumina and not within the glandular cells.

Animals↗

PubMed1996

Effect of vitamin K2 on experimental calcinosis induced by vitamin D2 in rat soft tissue.

The effect of vitamin K2 on calcium (Ca) and inorganic phosphorus (P) levels in the aorta and kidney obtained from experimental calcinosis induced by vitamin D2(2.5 x 10(5) I.U./ kg b.w.) of male rats was investigated. A high dose of vitamin K2 (100 mg/kg b.w.) inhibited the increase in the aortic Ca and P or in the renal Ca and P induced by vitamin D2, and a low dose of vitamin K2 (10 mg/kg b.w.) showed the same tendency, but the degree of the efficacy was small. It may be suggested that a high dose of vitamin K2 suppressed experimental calcification of soft tissues induced by vitamin D2. Therefore, a pharmacological dose of vitamin K2 might have a usefulness for the prevention and treatment of arteriosclerosis with calcification.

Animals↗

PubMed1995

Dolichyl phosphate, a potent inducer of apoptosis in rat glioma C6 cells.

Exposure of rat glioma C6 cells to dolichyl phosphate resulted in cell shrinkage followed by nuclear fragmentation and internucleosomal cleavage of genomic DNA, yielding ladder patterns of oligonucleosomal fragments, all characteristics of apoptosis. This phenomenon occurred in a dose and time dependent manner. Dolichol and prenol failed to induce apoptosis. The inhibitors of N-glycosylation, tunicamycin and swainsonine had no apparent effect on dolichyl phosphate-induced apoptosis. Apoptotic changes were also observed in HL-60 cells, SIRC cells and HeLa cells. Thus, dolichyl phosphate functions as a potential apoptosis inducer as well as an essential carrier lipid in the biosynthesis of N-linked glycoprotein.

Animals↗

PubMed1995

Stimulation of cell proliferation and autoregulation of elastin expression by elastin peptide VPGVG in cultured chick vascular smooth muscle cells.

Synthetic elastin peptides, VPGVG or its polymer (VPGVG)n, enhanced the proliferation of smooth muscle cells 1.5-fold during 48 h treatment at the concentrations over 10(-6) M or 1.0 microgram/ml, respectively. Monomeric and polymeric VPGVG sequences reduced elastin synthesis and its mRNA level to one-third and one-half of control respectively under the conditions in which the proliferation of cells were enhanced, but did not change collagen synthesis as measured by bacterial collagenase digestion. The elastin-specific autoregulation by elastin fragments may reflect the feedback regulation of elastin expression which may play an essential role in elastin metabolism under the normal and diseased conditions.

Amino Acid Sequence↗

PubMed1995

Cell cycle-dependent regulation of elastin gene in cultured chick vascular smooth-muscle cells.

A study was made of the relationship between elastin expression and the proliferative state of chick vascular smooth-muscle cells. Confluent cells of primary culture brought to a quiescent state by the deprivation of serum for 72 h exhibited a 5-, 3.5- and 2-fold increase in elastin synthesis, elastin mRNA level and transcriptional activity of elastin gene respectively over those in the proliferative state. On re-addition of serum in serum-deprived culture, cells started to proliferate, and elastin synthesis, its mRNA level and transcription of the gene decreased to the level of a proliferative state within 24 h, indicating that elastin expression in smooth-muscle cells was controlled by their growth states at least in part at a transcriptional level. A comparable increase in elastin mRNA level was observed when the cell growth was arrested by suspension culture for 72 h. When the cells were synchronized at the G1/S phase with thymidine/hydroxyurea treatment, elastin expression at the G1/S phase was greater than that at the G2/M phase during cell cycling. Elastin mRNA level at the G0 phase brought about by serum-deprivation or suspension culture predominated over that at the G1/S phase during cell cycling. These results indicate that gene expression of elastin and cell cycle are tightly coupled, which is independent of the presence of serum or adhesive state, and that elastin expression could be a biochemical marker for the growth states of smooth-muscle cells.

Animals↗

PubMed1995

Enhanced tropoelastin-degrading activity during cell passages in cultured smooth muscle cells.

Tropoelastin expression in vascular smooth muscle cells during serial cell passages from the primary to the tertiary culture was studied. The level of tropoelastin was found to be greatly reduced as the number of cell passages increased. The translational activity and level of elastin mRNA were essentially unchanged throughout the cell passages. The reduction in tropoelastin expression was not due to the repetitive trypsin treatment nor to the prolyl hydroxylation level of the newly-synthesized elastin. A comparable decline in tropoelastin expression was also found with increasing cell division in the primary cultures plated at different cell densities. A pulse-chase experiment revealed that the newly-synthesized elastin in the tertiary culture degraded more rapidly than that in the primary culture. The culture medium harvested from the tertiary culture exhibited a higher tropoelastin-degrading activity than that from the primary culture in the test-tube. The degrading activity of the tertiary culture was inhibited by the addition of 1 mM ethylenediaminetetraacetic acid or 1 mM phenylmethylsulfonyl fluoride, but not by 1 mM N-ethylmaleimide. These results suggest that the reduction in tropoelastin expression during the cell passages from the primary to the tertiary culture is due to the enhanced tropoelastin-degrading activity of the tertiary culture. The transition to tropoelastin-degrading phenotype during cell passages may explain the biological mechanisms of smooth muscle cell migration from the media to the intima observed in the pathological conditions.

Animals↗

PubMed1995

Ornithine decarboxylase is directly involved in mouse mammary carcinoma cell invasion in vitro.

To elucidate the role of ornithine decarboxylase (ODC) in cancer cell invasion, we have compared the invasiveness of mouse mammary carcinoma, FM3A and its variant cell line, EXOD, which overproduces ODC. We have found that EXOD cells showed about 5.6-fold more invasiveness through a reconstituted basement membrane (Matrigel) compared to FM3A cells. To elucidate the underlying mechanism of increased invasiveness of EXOD cells, we analyzed gelatinase activity in conditioned media derived from FM3A and EXOD cells. EXOD cells secreted approximately 3-fold 72kDa gelatinase compared to FM3A cells. Cell attachment ability to Matrigel was also studied. Although FM3a and EXOD cells showed increased attachment to Matrigel in a dose-dependent manner, EXOD cells showed higher cell attachment ability compared to FM3A cells. Anti-72kDa gelatinase neutralizing antibody suppressed EXOD cell invasion through Matrigel. Further, antisense oligonucleotides of ODC suppressed EXOD cell invasion through Matrigel. Although the causal relationship among ODC expression, gelatinase secretion and cell attachment ability remains to be elucidated, the results suggest that both overproduction of ODC and 72kDa gelatinase secretion are directly involved in increased invasiveness of EXOD cells.

Animals↗

PubMed1995

Comparison of ELISA and HPLC for the determination of desmosine or isodesmosine in aortic tissue elastin.

We developed a rapid and simple method for estimating tissue elastin content by measuring desmosine (D) in tissue hydrolysates by competitive ELISA. We compared the ELISA previously reported HPLC methods. When D or isodesmosine (ID) in hydrolysate of the same elastin preparation were measured by the two different methods, a good linear relationship was obtained (r = 0.854 for human aorta or r = 0.938 for rabbit aorta, respectively). The ELISA method can detect as little as 6 pmol/ml and it may be useful in monitoring elastin metabolism in patients with various connective tissue diseases.

Animals↗

PubMed1995

Induction of Ca2+ oscillations by vasopressin in the presence of tetraethylammonium chloride in cultured vascular smooth muscle cells.

The change of cytosolic Ca2+ concentration ([Ca2+]i) caused by vasopressin was examined in indo-1-loaded A7r5 smooth muscle cells by use of the high-performance laser cytometer and ratiometric fluorescence method. Vasopressin (100 nM) caused an initial rapid rise and a delayed increase in [Ca2+]i (n = 6). However, in the presence of tetraethylammonium chloride (10 mM), vasopressin consistently triggered sustained Ca2+ oscillations which were preceded by a large peak of [Ca2+]i. The latency for the development of this huge increase in [Ca2+]i prior to the occurrence of sustained Ca2+ oscillations was always the same. The frequency and amplitude of this type of Ca2+ oscillation varied depending upon the extracellular Ca2+ concentration. Ca(2+)-free solution did not completely suppress the sustained Ca2+ oscillations, but caffeine (20 mM) effectively abolished them. The present findings indicate that in A7r5 smooth muscle cells, the sustained Ca2+ oscillations triggered by vasopressin in the presence of tetraethylammonium chloride were mainly due to Ca2+ release from IP3-sensitive Ca2+ stores and Ca2+ influx from extracellular space, and did not require the pacemaker activity derived from the surface membrane. Moreover, the vasopressin-induced change in [Ca2+]i appeared to be linked to pertussis toxin-insensitive GTP-binding protein(s).

Animals↗