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Biomedical subjects

Y Nagamine

Publications and source records attributed to Y Nagamine.

At least 91 records · Page 5Linked to original sources
PubMed1989

Staurosporine stimulates expression of the urokinase-type (u-PA) plasminogen activator in LLC-PK1 cells.

In LLC-PK1 porcine epithelial cells, the urokinase-type plasminogen activator (u-PA) mRNA and protein can be induced either by stimulation of the protein kinase C (PKC) pathway using a tumor promoter (PMA) or by stimulation of the protein kinase A (PKA) pathway with calcitonin (SCT). By contrast, addition of 10(-7) M staurosporine, an inhibitor of PKC, to LLC-PK1 cells also stimulated urokinase production. In contrast to the in vitro situation (where staurosporine inhibited PKC activity), in the cell-culture system the microbial agent caused an early translocation of PKC and inhibited PKA. Addition of staurosporine together with PMA or with SCT further increased urokinase mRNA and protein synthesis. Maximal stimulation was obtained when all 3 agents were added together. We thus assume that in LLC-PK1 cells the PKA and PKC signal-transferring pathways can function independently.

Alkaloids↗
PubMed1989

[Changes in normal reference ranges for serum chemical analyses with ageing].

To get normal reference ranges for blood chemistry in elderly subjects, laboratory data on serum chemical analytes obtained from 1822 male and 1870 female outpatients were statistically analyzed with a non-parametric method with iterative truncation. Regarding the difference in the test data between male and female, the values for most analyses except for inorganic phosphorus and total protein were significantly higher in male than in female when an adult group including subjects of age ranging from 21 to 50 years was compared. The values for lactic dehydrogenase, albumin, sodium and calcium were higher in female of over 50 years of age than in their counterpart. When male and female were combined, the normal reference ranges for lactic dehydrogenase, alkaline phosphatase, uric acid, blood urea nitrogen, creatinine and potassium tended to be elevated while those for total protein, albumin and calcium to be declined with ageing. The values for total cholesterol and triglyceride reached a peak at a certain age. Total and direct bilirubin, sodium and chloride values were constant throughout whole age categories.

Adolescent↗
PubMed1988

Multiple nuclear factors interact with promoter sequences of the urokinase-type plasminogen activator gene.

To characterize proteins that bind to the cyclic AMP inducible promoter of the urokinase-type plasminogen activator gene, we performed a DNAase I footprinting analysis. Within 500 nucleotides upstream of the transcription start site we found eight protected regions due to at least four different binding proteins. Among these is a single binding site for the transcription factor CTF/NF1, which is flanked on each side by two conserved binding sites for the transcription factor Sp1. A region at -380, which shares a similarity with sequences observed in the corresponding regions of other cyclic AMP regulated genes, was protected. This binding site contains a sequence of ten nucleotides which is repeated further upstream at -480 and also protected against DNAase I digestion. Comparisons of extracts from four different cell lines revealed that all DNA binding factors are present in nuclei of uPA expressing and nonexpressing cells. Mechanism underlying hormonal regulation of the gene is discussed.

Animals↗
PubMed1988

hsp70 mRNA accumulates in LLC-PK1 pig kidney cells treated with calcitonin but not with 8-bromo-cyclic AMP.

In LLC-PK1 pig kidney cells, treatment with a cAMP-elevating peptide hormone, calcitonin, induces the accumulation of urokinase-type plasminogen activator (uPA) mRNA. When we used the method of differential hybridization to isolate uPA cDNA clones, we also obtained several calcitonin-inducible clones that were unrelated to uPA. Sequence analysis revealed 60% sequence homology between one of these clones and that of a Drosophila hsp70 gene. The uPA and the hsp70 cDNA clones were used as probes to compare the effects of various treatments on the accumulation of uPA mRNA and hsp70 mRNA in LLC-PK1 cells. Calcitonin or 8-bromo-cAMP treatment induced uPA mRNA accumulation, which was negligible in untreated cells. Heat treatment (42 degrees C) was ineffective. Calcitonin or heat treatment increased hsp70 mRNA accumulation, which was already high in untreated cells, but 8-bromo-cAMP was ineffective. Nuclear transcription of the hsp70 gene was increased by calcitonin but not by 8-bromo-cAMP treatment. These results suggest that calcitonin induces hsp70 mRNA accumulation in LLC-PK1 cells by a pathway apart from the activation of adenylate cyclase and through, at least partly, the activation of the gene transcription. Furthermore, induction of uPA mRNA accumulation by calcitonin or 8-bromo-cAMP treatment did not require protein synthesis. In contrast, induction of hsp70 mRNA accumulation by calcitonin or heat treatment did require protein synthesis. Other reports showed that protein synthesis is not required for heat induction of hsp70 mRNA in different cells, suggesting that the mechanism of induction of hsp70 mRNA accumulation in LLC-PK1 cells is not the same as in other cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Transcriptional regulation of a plasminogen activator gene by cyclic AMP in a homologous cell-free system. Involvement of cyclic AMP-dependent protein kinase in transcriptional control.

We have developed a homologous cell-free transcription system using extracts from the porcine kidney cell line LLC-PK1 to study the molecular mechanisms by which cAMP regulates urokinase-type plasminogen activator (uPA) gene transcription. We demonstrated accurate initiation of transcription using a cloned fragment of the uPA gene as template. The in vitro transcription rate was stimulated by up to 10-fold by the addition of cAMP (greater than 10 microM). This effect of cAMP on the transcription was greater for closed circular than for linear templates. Furthermore, addition of the purified catalytic subunit of cAMP-dependent protein kinase stimulated the in vitro transcription in the absence of cAMP to levels 2-fold higher than those observed with cAMP. Addition of cAMP had no stimulatory effect on the transcription of the rat heme oxygenase gene promoter tested under identical conditions. HeLa whole cell extract by itself showed no stimulation of transcription of the uPA gene by cAMP. Results of reconstitution experiments using HeLa whole cell extracts and nuclear lysates from LLC-PK1 cells suggest the presence of putative cAMP regulatory factor(s) as well as general transcription factor(s) in the nucleus of LLC-PK1 cells. These results provide experimental evidence directly implicating cAMP-dependent protein kinase in the regulation of gene transcription.

Animals↗
PubMed1988

Investigation of the active site of human salivary alpha-amylase from the modes of action on modified maltooligosaccharides.

The modes of action of two isozymes of human salivary alpha-amylase on phenyl alpha-maltopentaoside, phenyl alpha-maltotetraoside, and their derivatives which have an iodo or an amino or a carboxyl group at their first or penultimate glucopyranosyl residues from the non-reducing-end were examined. It is conceivable that the active site of this enzyme is composed of tandem subsites (S4,S3,S2,S1,S1',S2', and S3') geometrically complementary to several glucose residues, and that the glucosidic bonds of the substrates are split between S1 and S1'. Product analysis of each digest strongly suggested the presence of a hydrophobic amino acid residue at subsite S3 in the active site of the enzyme. No difference in the modes of action on the substrates was found between the two isozymes, indicating that the three-dimensional structures of their active site areas are, at the least, similar.

Binding Sites↗
PubMed1988

A comparison of the modes of actions of human salivary and pancreatic alpha-amylases on modified maltooligosaccharides.

The actions of three isozymes of human pancreatic alpha-amylase (HPA) on phenyl alpha-maltopentaoside, phenyl alpha-maltotetraoside, and their derivatives which have an iodo, an amino, or a carboxyl group at their first or penultimate glucopyranosyl residue from the non-reducing-end were examined. The results revealed that there was no difference in the actions of the three isozymes on the modified substrates and suggested the presence of five subsites (S3, S2, S1, S1', and S2') and a hydrophobic amino acid residue at subsite S3 in the active site of HPA. As compared with the action of human salivary alpha-amylase (HSA) on the same substrates, HPA had a tendency to release more phenyl alpha-glucoside from every substrate; however, an iodo, an amino, and a carboxyl group of the substrates had the same effects on the binding modes of the substrates to the active site of HPA as seen in the case of the salivary enzyme. This result indicates that the three-dimensional structures of the active sites of both alpha-amylases are quite similar except for some minor changes at subsites S3 and S2'.

Amylases↗
PubMed1988

An autopsy case of histiocytic medullary reticulosis presenting with marked hepatosplenomegaly for 13 years before the onset.

A 32-year-old male was hospitalized with high fever, pancytopenia and hepatosplenomegaly. No atypical cells were found in the peripheral blood. Bone marrow aspiration resulted in dry taps. Superficial lymph node swelling was not observed. He had been treated twice for high fever, hepatosplenomegaly and leukopenia that were very similar to the present illness, 13 and 3 years before the onset and hepatosplenomegaly had been noted by the patient for 13 years. The patient died after a rapid course of 20 days. Histiocytic medullary reticulosis (HMR) was diagnosed at the autopsy, which revealed atypical histiocytic infiltration showing erythrophagocytosis in the liver, spleen, left adrenal, and mesenterial and pulmonary hilar lymph nodes. This patient had shown the same clinical signs and hepatosplenomegaly 13 years before the onset of HMR, which suggest a possible latent stage and acute exacerbation of HMR.

Adult↗
PubMed1988

Asymptomatic hyperbromidaemia detected as pseudohyperchloridaemia measured with an ion selective electrode meter.

Six patients were found to have increased serum chloride concentrations when these concentrations were determined with an ion-selective electrode, but not when determined by continuous flow mercuric thiocyanate colorimetry or amperometric-coulometric titration. Their serum bromide levels of 1.8-8.0 mmol/l were much higher than those of 0.07-0.13 mmol/l in normal controls. The urinary bromide excretion, measured in two of these patients, was higher than that in normal subjects. No common symptoms or abnormalities in laboratory findings except hyperbromidaemia were found in these patients, who claimed not to have taken any drugs containing bromide. For determination of the incidence of subclinical hyperbromidaemia, the serum bromide concentrations were measured in sera of 1,323 outpatients sent to Tokushima University Hospital for routine measurements of blood chemistry over a one-month period. Five samples showed abnormally high bromide levels. It is concluded that subclinical hyperbromidaemia is not as rare as generally thought, though the aetiology of this state is unknown. Chloride determination with an ion-selective electrode can be used to screen for hyperbromidaemia, since increased levels of bromide ion result in apparently high chloride values.

Adult↗
PubMed1987

Inhibition of protein synthesis in LLC-PK1 cells increases calcitonin-induced plasminogen-activator gene transcription and mRNA stability.

The peptide hormone calcitonin induces the accumulation of urokinase-type plasminogen activator (uPA) mRNA in pig kidney LLC-PK1 cells. By itself, inhibition of protein synthesis had a negligible effect on uPA mRNA accumulation. Inhibition of protein synthesis led to two superinductive effects: an increase in calcitonin-induced uPA mRNA accumulation over time, and a shift in the dose-response curve so that lower calcitonin doses became more potent. To explain these two superinductive effects of protein-synthesis inhibition on calcitonin treatment, we demonstrated that the inhibition of protein synthesis increased both calcitonin-induced uPA-gene transcription and uPA-mRNA stability. Different protein-synthesis inhibitors had similar actions, arguing against the possibility that the results were attributable to an anomalous action of a particular inhibitor. The superinductive effects of protein-synthesis inhibition could not be mimicked when a tumour promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), was used instead of calcitonin as an inducer. Calcitonin and TPA exert their effects through different pathways, suggesting a clue to the mechanism of superinduction. Although inhibition of protein synthesis has been reported to increase transcription and mRNA stability in a number of other systems, the one described here appeared unique in combining both effects in the context of hormonal regulation.

Animals↗
PubMed1987

Dexamethasone coordinately inhibits plasminogen activator gene expression and enzyme activity in porcine kidney cells.

The peptide hormone, calcitonin, induces urokinase-type plasminogen activator (uPA) enzyme activity in cultured LLC-PK1 pig kidney cells. This induction occurs as a consequence of transcriptional activation of the uPA gene. Treatment with the synthetic glucocorticoid hormone, dexamethasone, was found to inhibit calcitonin induction of uPA enzyme activity by as much as 80%. The inhibitory effect of dexamethasone was attributed to at least two mechanisms: induction of an inhibitor of uPA enzyme activity, and reduction in uPA mRNA levels. Study on reduction of uPA mRNA levels showed that dexamethasone significantly reduced the transcription rate of the calcitonin-induced uPA gene, without affecting the half-life of uPA mRNA. Although dexamethasone has been reported to induce inhibitors of plasminogen activator enzyme activity and to inhibit transcription of various genes, the system described here appears novel in that both actions are coordinated.

Animals↗
PubMed1987

An experimental study of vascular damage in estrogen-induced embolization.

Temporal sequences of estrogen-induced embolization were studied in both in vivo and in vitro models. Infusion of the estrogen compound into rat mesenteric artery caused local spherocytosis and severe rapid degeneration of endothelial cells, followed by injury to the underlying muscle cells and fibroblasts. These changes were regarded as mainly the results of the embolization effect of conjugated estrogen. A study of cultured vascular endothelial cells suggested narrow margins of effective drug concentrations for cell damage. Also, nuclear disintegration with relative sparing of cytoplasmic constituents seemed to be characteristic of estrogen-induced cell damage in both in vivo and in vitro models.

Animals↗
PubMed1987

Studies on the active site of Taka-amylase A: its action on phenyl maltooligosides with a charge at their non-reducing-ends.

Five modified moltooligosaccharides, phenyl O-6-amino-6-deoxy-alpha-D- glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)- alpha-D-glucopyransoide (AG4P), phenyl O-(alpha-D-glucopyranosyluronic acid)-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-d-glucopyran osy l- (1----4)-alpha-D-glucopyranoside (CG4P), phenyl O-6-amino-6-deoxy-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyra nos yl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)- alpha-D-glucopyranoside (AG5P), phenyl O-(alpha-D-glucopyranosyluronic acid)-(1----4)-O-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)- alpha-D-glucopyranoside (CG5P), and phenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)- O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-a lph a-D- glucopyranoside (FG4P), were prepared to examine the active site of Taka-amylase A (TAA) [EC 3.2.1.1, Aspergillus oryzae]. Phenyl alpha-maltotetraoside (G4P) was predominantly hydrolyzed by TAA to maltose and phenyl alpha-maltoside (G2P). While G2P, phenyl alpha-glucoside (GP), and phenol were liberated from AG4P in the ratio of 7:63:30. G4P, phenyl alpha-maltotrioside (G3P), G2P, and GP were liberated from G5P in the ratio of 1:20:73:6, but AG5P was almost completely hydrolyzed to modified maltotriose and G2P. On the hydrolysis of CG4P and CG5P, no remarkable change was observed except for a decrease in the relative reaction rates compared with G4P and G5P, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1987

A new genetic approach for studying hormonal regulation of urokinase-type plasminogen activator gene expression in LLC-PK1 cells.

In LLC-PK1 cells, a cyclic AMP (cAMP)-elevating peptide hormone, calcitonin, induces urokinase-type plasminogen activator (uPA) gene transcription without concomitant protein synthesis. To understand the molecular mechanism of the uPA gene regulation by cAMP, we developed a system which allows us to obtain mutant cells with modified regulatory proteins. A uPA-gpt hybrid gene was constructed, in which the regulatory region of the uPA gene was linked to a bacterial xanthine-guanine phosphoribosyltransferase gene (gpt), and it was transfected into LLC-PK1 cells. A stably transformed cell line, which expressed gpt only in the presence of calcitonin, was obtained, and then these cells were treated with a chemical mutagen, ethyl methanesulfonate. Cells were screened for constitutive gpt expression and, as mutations in regulatory proteins should affect the two genes at the same time, cells were further screened for an increased basal uPA mRNA level. Several such clones were obtained and none of them had modified cAMP-dependent protein kinase activity, suggesting that mutations were in the post-protein kinase step in the pathway of hormone action. Five clones were fused with the parent LLC-PK1 cells, and all of the fusion cells showed reduced basal uPA mRNA levels, indicating that they were recessive mutants. One clone was analyzed further for sensitivity to calcitonin in the induction of uPA mRNA, and it showed a significantly different dose-response pattern compared with parent cells. These results suggest that the uPA gene is regulated, at least partly, by a negatively regulating factor and that the action of cAMP is linked to this factor.

Animals↗
PubMed1987

[Chemical embolization using conjugated estrogen--a clinical study of 40 cases].

For the treatment of dural arteriovenous malformation (AVM), meningioma and other external carotid lesions, a method of chemical embolization using a conjugated estrogen is reported. A total of 40 cases were treated, including 11 cases of dural AVM, 20 cases of meningioma and 9 other cases such as of skull tumor, aneurysmal bone cyst, nasopharyngeal tumor, traumatic AV fistula, scalp AVM and venous angioma of the scalp. The conjugated estrogen was administered continuously through a cannula inserted into the external carotid artery. For venous angioma of the scalp, it was injected directly into the lesion. Disappearance or reduction of the abnormal vasculature of the lesion and/or the feeding arteries was confirmed on angiograms in 9 of 11 cases (81.9%) of dural AVM, in 17 of 20 cases (85%) of meningioma and in all cases of other external carotid lesions. Diminution of enhancement on CT scan was obtained in 8 of 20 cases (40%) of meningioma and in 6 of 7 cases (85.7%) of other external carotid lesions. There has been no recurrence of the dural AVM as long as 10 years. Eighteen cases of meningioma were operated on, and in 16 cases the lesion was totally removed without massive hemorrhage. In 2 cases of skull tumor and a case of nasopharyngeal tumor as well, the lesions were totally removed. Complications in 40 cases were as follows: parotitis in 17 cases, liver dysfunction in 3 cases, and each of gynecomastia, facial palsy, trismus and convulsion in one case. All of them cleared up after the interruption or reduction of administration of the conjugated estrogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1987

[An experimental study of chemical embolization using conjugated estrogen--ultrastructural observations on rat mesenteric vessels].

We previously reported a new embolization method using conjugated estrogen and also a basic study which demonstrated that conjugated estrogen produced microcirculatory disturbance. To clarify the mechanism of this microcirculatory disturbance following the administration of conjugated estrogen, the changes of vascular endothelial cells and red blood cells were studied. After intra-arterial infusion of conjugated estrogen into rat mesenteric arteries, microcirculatory changes were observed with a biomicroscope, and also ultrastructural changes of vascular endothelial cells and red blood cells were examined. As a result, biomicroscopically, mesenteric microcirculatory disturbance in proportion to the doses of conjugated estrogens was seen and stasis of the microcirculation was finally observed in all rats. Ultrastructural observations revealed an appearance of spherocytes soon after the infusion and injury of vascular endothelial cells perfused with conjugated estrogen. Vascular endothelial cells were injured remarkably, including their plasma membrane, cytoplasm, and nuclei when mesenteric microcirculation showed sludging under the biomicroscope. At the stage of stasis, endothelial cells were severely injured and most of the vessels were filled with spherocytes. From these results, it was presumed that vascular endothelial injury and alterations of red blood cells into spherocytes induced by conjugated estrogen were the major factors of embolization effect of the conjugated estrogen. Furthermore, conjugated estrogen was considered as an effective embolic material because not only main arteries but microcirculations could be occluded by its intraarterial infusion.

Animals↗