Biomedical subjects

Y Nagamine

Publications and source records attributed to Y Nagamine.

At least 19 recordsLinked to original sources

PubMed1992

c-fos, c-jun and c-myc expressions are not growth rate limiting for the human MCF-7 breast cancer cells.

Insulin-like growth factor I (IGF-I) was 3 times more potent in pagating MCF-7 cell proliferation than epidermal growth factor (EGF). IGF-I stimulated c-fos mRNA expression about 5 times less than EGF. Both growth factors were equipotent in inducing c-jun and c-myc mRNA expressions. The protein level of c-Myc correlated with the mRNA level. IGF-I and EGF stimulated the transcriptional activity dependent on the phorbol 12-myristate 13-acetate-responsive element (TRE) to the same extent, when measured by the chloramphenicol acetyl transferase activity of a transiently transfected multiple TRE construct. These results strongly indicate that the expression levels of the measured proto-oncogenes do not correlate with the increase of growth stimulation by IGF-I and EGF and are not growth rate limiting for the human MCF-7 breast cancer cells.

Breast Neoplasms↗

PubMed1992

[Creatine kinase activity and its isozyme levels in cord blood].

We measured creatine kinase (CK) levels of cord blood and evaluated them in relation to the degree of maturity and distress. The reference value of CK of cord blood in normal neonates was 209 +/- 99 U/l (mean +/- SD) and the percent values of the isoenzymes, CK-MM, CK-MB and CK-BB, were 86.7 +/- 6.2%, 5.1 +/- 2.6%, and 8.1 +/- 5.4%, respectively. There was a positive correlation between the birth weight and CK, CK-MM, CK-MB and CK-BB levels of cord blood. The CK and CK-MM levels of cord blood in the neonates who were prematurely delivered and small-for-date were significantly lower than those in normal controls. The CK and CK-MM levels of cord blood in the neonates with low Apgar scores, 6-4 and 3-0, were 172 +/- 74, 145 +/- 73 and 104 +/- 63, 92 +/- 55 U/l, respectively, which were significantly lower than those in the neonates with the high scores (10-7), 208 +/- 104, 183 +/- 92 U/l. The neonates with distress showed the low CK and CK-MM values, 76 +/- 12 and 61 +/- 8 U/l. The CK and CK-MM levels of cord blood tended to decrease with prolongation of labor, but did not differ from each other among the neonates delivered by different modes. These results suggest that the CK and its isoenzyme levels are good indicators for the degree of maturity of neonates and the severity of neonatal distress.

Creatine Kinase↗

PubMed1991

Constitutive expression of the urokinase plasminogen activator gene in murine RAW264 macrophages involves distal and 5' non-coding sequences that are conserved between mouse and pig.

The 5' flanking regions of the mouse and pig urokinase plasminogen activator (uPA) genes were sequenced and sequence homology interrupted by repeat elements was found to extend to -4.6kb in pig and -6.6kb in mouse. A transient transfection procedure was devised for the murine macrophage cell line RAW264. Pig uPA promoter-CAT constructs were more active than mouse constructs in this assay. This contrast may involve sequence differences within 100 bp of the transcription start site. The selective deletion of distal regions of the promoter (greater than 2.6 kb upstream), and of a conserved element, 5'-AGGAGGAAATGAGG-TCA-3' around -2 kb greatly reduced the activity of reporter constructs in RAW264 cells. Electrophoretic mobility shift assays using the latter sequence identified a single nuclear protein complex. This element has been referred to as PEA3/AP1-like, but the complex did not comigrate with either AP1 or known proteins that bind polypurines (including the macrophage-specific factor PU-1) and was not competed by AP1 or polypurine oligonucleotides. uPA promoters contain multiple AP1 and AP2-like DNA sequences, which were recognised by nuclear proteins expressed constitutively in RAW264 cells. They also contain multiple binding sites for NF kappa B but activated NF kappa B was not expressed in RAW264 cells. The conserved, transcribed 5' non-coding sequences were also required for maximal gene expression. Hence, the uPA promoter contains multiple weak cis-acting elements distributed over 7.0 kb 5' to the translation start site.

Animals↗

PubMed1991

Protein kinase C down-regulation enhances cAMP-mediated induction of urokinase-type plasminogen activator mRNA in LLC-PK1 cells.

Expression of the urokinase-type plasminogen activator (uPA) gene in LLC-PK1 cells can be induced by signals mediated by both cAMP-dependent protein kinase (PKA) and Ca(2+)- and phospholipid-dependent protein kinase (PKC). We have utilized the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) to down-regulate PKC, in order to test for an effect on the PKA-mediated induction of the uPA gene expression. Incubation of cells for 24 h with 100 ng/ml TPA caused a marked decrease of PKC protein, both in cytosolic and particulate fractions, and an 85% reduction of total PKC activity. After down-regulation of PKC, uPA mRNA accumulation induced by 8-Br-cAMP was 5-10-fold higher than in control cells. Both uPA mRNA stability and uPA gene transcription rates induced by 8-Br-cAMP were increased by PKC down-regulation (6- and 1.8-fold, respectively). Although total PKA activity was reduced by 20% in extracts from PKC-depleted cells, activation of PKA by 8-Br-cAMP was 2.5-fold higher than in control cells. This enhanced activation of PKA in PKC-depleted cells also occurred in response to other cAMP derivatives and to cAMP induced endogenously by the activation of adenylate cyclase with forskolin, but was not due to down-regulation-associated changes in the rate of cAMP synthesis. Our results demonstrate that in LLC-PK1 cells, down-regulation of PKC results in an enhanced induction of uPA gene expression by cAMP-mediated signals without alterations in adenylate cyclase activity, suggesting a mechanism distal to adenylate cyclase.

1-Methyl-3-isobutylxanthine↗

PubMed1991

Protein synthesis inhibition stabilizes urokinase-type plasminogen activator mRNA. Studies in vivo and in cell-free decay reactions.

Inhibition of protein synthesis stabilizes a number of mRNAs, but little is known about the mechanism. To understand the relationship between protein synthesis and mRNA stability, we studied the degradation of calcitonin-induced urokinase-type plasminogen activator (uPA) mRNA in LLC-PK cells. uPA mRNA became highly stable by pretreatment with either cycloheximide or pactamycin, and the stabilizing effect of cycloheximide treatment was time dependent with the full effect exerted by 60 min. Stabilization was also observed with histone H4 mRNA but only partially with c-myc mRNA. To further analyze, we developed a cell-free decay reaction system based on post-mitochondrial supernatant (PMS). In this system, uPA mRNA was completely stable when fractions were obtained from cells pretreated with cycloheximide, but very unstable in control fractions, paralleling uPA mRNA stability in intact cells. However, in contrast to uPA mRNA and the in vivo observation, histone H4 mRNA was unstable whether or not the cells were pretreated with cycloheximide. These results suggest that inhibition of protein synthesis stabilizes mRNAs in at least two different ways in LLC-PK1 cells. When PMS from cycloheximide/calcitonin-treated cells was mixed with PMS from untreated cells, uPA mRNA was not destabilized. This suggests that a putative labile factor responsible for uPA mRNA degradation is not a soluble protein.

Animals↗

PubMed1991

Okadaic acid induction of the urokinase-type plasminogen activator gene occurs independently of cAMP-dependent protein kinase and protein kinase C and is sensitive to protein synthesis inhibition.

Urokinase-type plasminogen activator (uPA) gene expression in LLC-PK1 cells is induced by activation of cAMP-dependent protein kinase (cAMP-PK) or protein kinase C (PK-C). To determine whether protein phosphatases can also modulate uPA gene expression, we tested okadaic acid, a potent specific inhibitor of protein phosphatases 1 and 2A, in the presence and absence of cAMP-PK and PK-C activators. Okadaic acid by itself induced uPA mRNA accumulation. This induction was strongly attenuated by the inhibition of protein synthesis. In contrast, the inhibition of protein synthesis enhanced induction by 8-bromo-cAMP and only delayed induction by 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, down-regulation of PK-C by chronic treatment with TPA did not abrogate the okadaic acid-dependent induction. These results provide evidence for a novel signal transduction pathway leading to gene regulation that involves protein phosphorylation but is independent of both cAMP-PK and PK-C.

Animals↗

PubMed1991

Mechanisms of cAMP-mediated gene induction: examination of renal epithelial cell mutants affected in the catalytic subunit of the cAMP-dependent protein kinase.

The precise mechanistic role of the cAMP-dependent protein kinase (cAMP-PK) in cAMP-mediated gene induction remains unclear. Renal epithelial cell mutants were compared to the LLC-PK1 parental cell line for induction of the cAMP-responsive urokinase-type plasminogen activator (uPA) gene, as quantitated by the technique of mRNA solution hybridization. The FIB4 and FIB6 mutants, which possess less than 10% parental cAMP-PK catalytic (C) subunit activity, showed markedly diminished uPA mRNA induction in response to agents elevating intracellular cAMP such as the cAMP analogue 8-bromo-cAMP and the adenylate cyclase-stimulating hormones vasopressin and calcitonin. In contrast, the mutant cells responded to a similar or greater extent than the parental cells in terms of uPA mRNA induction following treatment with the Ca2+/phospholipid-dependent protein kinase activator phorbol 12-myristate 13-acetate (PMA). Elevation of intracellular cAMP was found to induce a translocation of the cAMP-PK C subunit from the perinuclear Golgi region to the nucleus in both parental and mutant cell lines, as shown by immunocytochemical techniques. Results argue for the role of the cAMP-PK C subunit activity and possibly nuclear translocation of the C subunit in cAMP-mediated uPA induction, which is mechanistically distinct from the PMA-stimulated response.

Animals↗

PubMed1991

Overexpression of the alpha-type protein kinase (PK) C in LLC-PK1 cells does not lead to a proportional increase in the induction of two 12-O-tetradecanoylphorbol-13-acetate-inducible genes.

Phorbol esters, by activating protein kinase C (PKC), induce the expression of the urokinase-type plasminogen activator (uPA) gene and the proto-oncogene c-fos in LLC-PK1 (PK1) porcine kidney epithelial cells. To investigate the role of PKC in the regulation of these two 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible genes, the alpha-type PKC, the predominant subtype present in the PK1 cells, was overexpressed in this cell line. Two clonal PK1 derivatives overexpressing the alpha PKC 15- and 20-fold, respectively, were established. Compared with the parental and control cells, only a modest but substantially sustained (2- to 3-fold) increase in the accumulation of uPA as well as c-fos mRNAs were observed by TPA in these cells. These results indicate that the extent of induction of these genes mediated by TPA was not proportional to the amounts of alpha-type PKC stably overexpressed in these cells, suggesting that factor(s) downstream of the activation of the alpha PKC appear to be rate limiting for the induction of both TPA-inducible genes in PK1 cells.

Animals↗

PubMed1991

Investigation of the active site of Bacillus macerans cyclodextrin glucanotransferase by use of modified maltooligosaccharides.

The active site of Bacillus macerans cyclodextrin glucanotransferase (CGTase) was examined by use of derivatives of phenyl alpha-maltopentaoside and phenyl alpha-glucoside as the substrates and acceptors, respectively. The active site of this enzyme is considered to be composed of tandem subsites (S4, S3, S2, S1, S1', S2', etc.) geometrically complementary to several glucose residues, and the alpha-1,4-glycosidic linkage of a substrate is split between S1 and S1'. The features of subsites S3 and S4 of the glycon binding site were estimated from the modes of the enzymatic action on phenyl alpha-maltopentaoside (G-G-G-G-G-phi; G, glucose residue; phi, phenyl residue; -, alpha-1,4-glycosidic bond) and its derivatives in which the CH2OH groups of the non-reducing-end glucose residues were converted to CH2I (IG-G-G-G-G-phi; IG, 6-deoxy-6-iodo-D-glucose residue), CH2NH2 (AG-G-G-G-G-phi; AG, 6-amino-6-deoxy-D-glucose residue), or COOH (CG-G-G-G-G-phi; CG, glucuronic acid residue). p-Nitrophenyl alpha-glucopyranoside (G-P; P, p-nitrophenyl residue) was used as an acceptor. HPLC analysis of the digests revealed that the CG residue of CG-G-G-G-G-phi was excluded from subsite S3, while it was accommodated in subsite S4. The Km and Vmax values for CG-G-G-G-G-phi were remarkably larger and smaller, respectively, than those for any other substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus↗

PubMed1990

Ca2+ potentiates cAMP-dependent expression of urokinase-type plasminogen activator gene through a calmodulin- and protein kinase C-independent mechanism.

In the porcine renal epithelial cell line, LLC-PK1, activation of the cAMP-dependent signal transduction pathway induces the urokinase-type plasminogen activator (uPA) gene. We show here that the cAMP response is enhanced when the intracellular calcium concentration is increased. When LLC-PK1 cells were treated with the calcium ionophore ionomycin alone, there was no uPA mRNA accumulation. However, in the presence of ionomycin the dose-response of 8-bromo-cAMP (Br-cAMP) with respect to uPA mRNA accumulation was shifted toward the lower concentrations of Br-cAMP. A Northern blot analysis after the inhibition of RNA synthesis and nuclear run-on assays showed that the synergistic effect of Ca2+ could be attributed to increases in uPA gene transcription and mRNA stability. In the presence of cycloheximide, a protein synthesis inhibitor, uPA mRNA was stabilized, but the effect of ionomycin on Br-cAMP-induced mRNA accumulation was still maintained. The result suggests that the Ca2+, at least on transcription, does not require new protein synthesis. Ionomycin treatment did not modify the activity of the cAMP-dependent protein kinase, suggesting that Ca2+ either affects a step in the pathway between the kinase and the uPA gene, or acts independently of the cAMP-dependent protein kinase pathway. The effect of ionomycin was not suppressed by protein kinase C down-regulation nor by inhibitors of calmodulin. Synergism was also observed when Br-cAMP was replaced with calcitonin, a peptide hormone which is coupled to adenylate cyclase, and when ionomycin was replaced with another ionophore A23187, suggesting that the synergism is due to an interaction between cAMP-dependent and Ca2(+)-dependent signal transduction pathways.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1990

Nordihydroguaiaretic acid inhibits urokinase synthesis by phorbol myristate acetate-stimulated LLC-PK1 cells.

Protein kinase C (PKC) activation is regulated by Ca2+, phospholipids, diacylglycerol (DAG) and fatty acids. Phorbol myristate acetate (PMA) which mimics the effect of DAG on PKC induces transcriptional activation of the urokinase-type plasminogen activator (u-PA) gene in LLC-PK1 cells. We examined in the present work the relationships between PKC activity, fatty acids, and u-PA synthesis in this cell line. We showed that H7, an inhibitor of PKC, inhibited the PMA-induced u-PA synthesis by LLC-PK1 cells. PMA-induced u-PA synthesis was enhanced by eicosatetraynoic acid (ETYA), a competitive inhibitor of both the lipoxygenase and cyclooxygenase pathways and inhibited by nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway. Three other unrelated lipoxygenase inhibitors (phenidone 100 microM, BW755 50 microM and diethylcarbamazine 50 microM) had no effect on u-PA biosynthesis. Two polyunsaturated fatty acids other than ETYA, arachidonic acid and linoleic acid, also potentiated the PMA effect and a lipoxygenase derivative, 12 hydroxyeicosatetraenoic acid (12 HETE), did not modify the basal and PMA-stimulated u-PA syntheses. PKC activity purified from cytosol of LLC-PK1 cells was stimulated by addition of 16 nM PMA in vitro and this effect was blunted by simultaneous addition of 5 microM NDGA. By Northern blot analysis using a pig u-PA cDNA probe we found that PMA increased the steady state level of u-PA mRNA after 2 h of incubation and that NDGA inhibited this effect. These data suggest that NDGA inhibits PMA-stimulated PKC activity in intact cells leading to a decrease of u-PA mRNA level and u-PA biosynthesis in PMA-stimulated LLC-PK1 cells. Polyunsaturated fatty acids have opposite effects.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1990

Disruption of cytoskeletal structures results in the induction of the urokinase-type plasminogen activator gene expression.

Urokinase-type plasminogen activator (uPA) is expressed at higher levels in many transformed cells as compared with their non-transformed counterparts. The transformed phenotype is associated with changes in the cytoskeleton. Therefore, we have investigated whether alterations in the cytoskeleton can trigger changes in the expression of the uPA gene. To this end we analyzed the expression of the uPA gene following exposure of porcine kidney cells, LLC-PK1, to agents that modify the organization of specific components of the cytoskeleton. These cells exhibited increased uPA mRNA and protein after disruption of microtubules by colchicine or nocodazole treatment or after disruption of microfilaments by cytochalasin B treatment. Colchicine, nocodazole, and cytochalasin B did not cause alterations in the level of cAMP-dependent protein kinase in LLC-PK1 cells. In contrast, down-regulation of protein kinase C by phorbol myristate acetate, reduced, but did not fully prevent the induction of uPA mRNA when LLC-PK1 cells were subsequently exposed to colchicine, nocodazole, or cytochalasin B. Apparently, a signal transduction pathway in part involving protein kinase C but not cAMP-protein kinase mediates the regulatory changes at the transcriptional level of the uPA gene. Inhibition of protein synthesis by cycloheximide prior to the exposure of LLC-PK1 cells to colchicine, nocodazole, or cytochalasin B, largely prevented the induction of uPA mRNA.

Actin Cytoskeleton↗

PubMed1990

Macromolecular interaction on a cAMP responsive region in the urokinase-type plasminogen activator gene: a role of protein phosphorylation.

We have studied the regulation of urokinase-type plasminogen activator gene expression by cAMP in LLC-PK1 cells. We found a cAMP responsive region 3.4 kb upstream of the transcription initiation site, which comprised three protein-binding domains designated A, B, and C. Domains A and B both contain a sequence, TGACG, homologous to a consensus cAMP response element (CRE; TGACGTCA). Effective cAMP-mediated induction was achieved when these two domains were linked with domain C, which by itself did not confer cAMP responsiveness to a heterologous promoter nor contained CRE-like sequence, suggesting a functional cooperation among these domains. Results of competition studies using gel retardation and DNase I footprinting assays suggest that there is a protein-protein interaction between a CRE binding protein and a domain C binding protein. In gel retardation assays, binding of a nuclear protein to domains A and B was strongly augmented by addition of the catalytic subunit of cAMP-dependent protein kinase, whereas the protein binding to domain C was slightly inhibited, suggesting that protein phosphorylation is involved in the regulation of protein-DNA interaction.

Base Sequence↗

PubMed1990

[Glossopharyngeal neurinoma causing symptomatic hemorrhage: case report].

A 86-year-old man was admitted to our hospital because of swallowing disturbance and deterioration of consciousness. He had been aware of hearing disturbance on the right side for twelve months. Computed tomography (CT) scan demonstrated an inhomogeneous hyperdense mass lesion, 3.2 X 2.3 cm in size, at the right cerebello-pontine angle, and ventricular enlargement with intraventricular hemorrhage. Skull tomogram revealed destructive enlargement of the right jugular foramen. The angiogram showed avascular mass with elevation of anterior inferior cerebellar artery, and downward shift of posterior inferior cerebellar artery. Operative and histological findings were compatible with glossopharyngeal neurinoma of Antoni type A dominance. This tumor had numerous abnormal vessels probably causing massive hemorrhage. Only fifteen cases of intracranial neurinoma with symptomatic hemorrhage have been reported in the world literature. This case is reported as the sixteenth one and the first glossopharyngeal neurinoma among them. Possible etiology of such hemorrhage is discussed.

Aged↗

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

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↗