A prototype of transition-metal-catalyzed carbothiolation of alkynes.
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Journal Article↗
Biomedical subjects
A Ohtaka
Publications and source records attributed to A Ohtaka.
Functional consensus for mammalian osmotic response elements.
The molecular mechanisms underlying adaptation to hyperosmotic stress through the accumulation of organic osmolytes are largely unknown. Yet, among organisms, this is an almost universal phenomenon. In mammals, the cells of the renal medulla are uniquely exposed to high and variable salt concentrations; in response, renal cells accumulate the osmolyte sorbitol through increased transcription of the aldose reductase (AR) gene. In cloning the rabbit AR gene, we found the first evidence of an osmotic response region in a eukaryotic gene. More recently, we functionally defined a minimal essential osmotic response element (ORE) having the sequence CGGAAAATCAC(C) (bp -1105 to -1094). In the present study, we systematically replaced each base with every other possible nucleotide and tested the resulting sequences individually in reporter gene constructs. Additionally, we categorized hyperosmotic response by electrophoretic mobility shift assays of a 17-bp sequence (-1108 to -1092) containing the native ORE as a probe against which the test constructs would compete for binding. In this manner, binding activity was assessed for the full range of osmotic responses obtained. Thus we have arrived at a functional consensus for the mammalian ORE, NGGAAAWDHMC(N). This finding should accelerate the discovery of genes previously unrecognized as being osmotically regulated.
Aldehyde Reductase↗
Effects of hyperosmolality on Na, K-ATPase gene expression in vascular smooth muscle cells.
Cultured vascular smooth muscle cells (VSMC) from rat thoracic aortas were exposed to hyperosmotic media to determine the effects on Na, K-ATPase alpha1- and beta1-mRNA expression. Hyperosmotic media (500 mOsm/kgH2O) supplemented with glucose or mannitol increased alpha1-mRNA levels threefold at 24 hr and beta1-mRNA levels sevenfold at 12 hr. In sharp contrast, hyperosmotic urea medium had no effect at any time. Both the protein synthesis inhibitor cycloheximide and the RNA transcription inhibitor actinomycin D reduced alpha1- and beta1-mRNA upregulation induced by hyperosmotic glucose or mannitol media. Protein kinase C (PKC) inhibitors (staurosporine A or calphostin C) or tyrosine kinase (TK) inhibitors (genistein or herbimycin A) had no effect on the alpha1-mRNA upregulation induced by hyperosmotic glucose or mannitol media. Hyperosmotic glucose or mannitol media (500 mOsm/kgH2O) significantly increased alpha1- and beta1-subunit protein levels and Na, K-ATPase activity, whereas hyperosmotic urea medium had no effect. Transfection experiments with the 5'-flanking sequences of the alpha1- or beta1-subunit genes linked to the luciferase reporter gene revealed that hyperosmolar glucose medium increased luciferase activity 2.9- and 3.7-fold, respectively. Similarly, hyperosmotic mannitol medium increased such activity 2.7- and 3.4-fold, respectively. These results demonstrate that: (i) hyperosmolality induced by the poorly permeating solutes (glucose and mannitol) stimulates alpha1- and beta1-mRNA accumulation, alpha1- and beta1-subunit protein accumulation, and Na, K-ATPase activity, whereas the rapidly permeating solute (urea) has no effect; (ii) the upregulation of alpha1- and beta1-mRNA in response to hyperosmotic glucose or mannitol media requires, at least in part, de novo synthesis of intermediate regulatory proteins; (iii) the hyperosmolality-induced alpha1-mRNA upregulation occurs through PKC- and TK-independent mechanisms, whereas the hyperosmolality-induced beta1-mRNA upregulation occurs through activation of PKC and TK; and (iv) hyperosmolality induced by glucose or mannitol increases promoter activities of the alpha1- and beta1-subunit genes.
Animals↗
Serum transcriptionally regulates Na(+)-K(+)-ATPase gene expression in vascular smooth muscle cells.
The present study was designed to examine the effects of serum on Na(+)-K(+)-ATPase alpha 1- and beta 1-subunit gene expression in cultured vascular smooth muscle cells (VSMC) from rat thoracic aortas. Addition of 10% serum to VSMC for 24 h increased Na(+)-K(+)-ATPase activity 1.5-fold and alpha 1- and beta 1-subunit protein levels 1.9-fold. Serum (10%) caused a 3.5-fold increase in alpha 1-mRNA levels and a 6.7-fold increase in beta 1-mRNA levels, with peak elevations at 12 h. The protein synthesis inhibitor cycloheximide abolished serum-mediated beta 1-mRNA induction but did not affect serum-mediated alpha 1-mRNA induction. Protein kinase C (PKC) inhibitors (staurosporine A or calphostin C) or tyrosine kinase (TK) inhibitors (genistein or herbimycin A) significantly reduced serum-mediated beta 1-mRNA induction but had no effect on serum-mediated alpha 1-mRNA induction. Transfection experiments with the 5'-flanking sequences of the alpha 1- or beta 1-subunit genes linked to the luciferase reporter gene revealed that 10% serum caused 2.8- and 6.5-fold increases in luciferase activity, respectively. Among growth factors, only basic fibroblast growth factor (FGF) enhanced luciferase activities for the alpha 1- and beta 1-subunit genes. We conclude that 1) serum stimulates alpha 1- and beta 1-mRNA expression, alpha 1- and beta 1-subunit protein accumulation, and Na(+)-K(+)-ATPase activity; 2) serum-mediated beta 1-mRNA induction partly requires de novo synthesis of intermediate regulatory proteins and activation of PKC and TK, whereas serum-mediated alpha 1-mRNA induction occurs through PKC- and TK-independent mechanisms; 3) the 5'-flanking regions of the alpha 1- and beta 1-subunit genes are serum responsive; and 4) FGF mimics stimulatory effects of serum on promoter activities for the alpha 1- and beta 1-subunit genes.
Animals↗
Differential regulation of Na+-K+-ATPase gene expression by corticosteriods in vascular smooth muscle cells.
To determine whether gluco- and mineralocorticoids have specific actions on Na+-K+-ATPase gene expression in vascular tissue, we used Northern blot analysis to compare the effects of dexamethasone (Dex) and aldosterone (Aldo) on Na+-K+-ATPase alpha1 and beta1-subunit mRNA expression in cultured vascular smooth muscle cells from rat aortae. Dex at 10(-6)M increased alpha1 -mRNA level 2.5-fold at 24 h and beta1-mRNA level 9.9-fold at 12 h. Aldo at 10(-6)M increased alpha1-mRNA 2.7-fold at 48 h and beta1-mRNA level 10.9-fold at 6 h. The half-maximal stimulation of both alpha1 and beta1-mRNA levels occurred at a concentration of 5-7 X 10(-9)M Dex, whereas it occurred at a concentration of 2-3 X 10(-9)M Aldo. The glucocorticoid receptor antagonist RU-38486 inhibited both Dex- and Aldo-mediated induction of beta1-mRNA. The mineralocorticoid receptor antagonist spironolactone inhibited Aldo-mediated induction of beta1-mRNA, whereas it had no effect on Dex-mediated induction of beta1-mRNA. Removal of Na+ from the extracellular medium (isosmotic replacement with choline) caused no effect on Dex-mediated induction beta1-mRNA, whereas it inhibited Aldo-mediated induction of beta1-mRNA. Addition of a specific inhibitor of the Na+/H+ exchange, ethylisopropylamiloride, had no effect on Dex-mediated induction of beta1-mRNA, whereas it resulted in a significant inhibition of Aldo-mediated induction of beta1-mRNA. We conclude that 1) both Dex and Aldo induce Na+-K+-ATPase alpha1- and beta1-mRNA expression in a time- and dose-dependent manner; 2) Dex-mediated induction of beta1-mRNA occurs only through glucocorticoid receptors, whereas Aldo-mediated induction of beta1-mRNA occurs through both gluco- and mineralocorticoid receptors; and 3) Dex-mediated induction of beta1-mRNA occurs through Na+-independent mechanisms, whereas Aldo-mediated induction of beta1-mRNA, at least in part, occurs through Na+-dependent mechanisms, including stimulation of the Na+/H+ exchange.
Adrenal Cortex Hormones↗
Hyperosmolality stimulates Na-K-ATPase gene expression in inner medullary collecting duct cells.
Primary cultures of inner medullary collecting duct (IMCD) cells of rats were incubated in hyperosmotic media to determine the effects on Na-K-ATPase alpha 1- and beta 1-subunit mRNA expression. Osmolality of the incubation media was raised from 300 up to 500 mosmol/kgH2O by adding NaCl, mannitol, raffinose, or urea. Hyperosmotic media supplemented with NaCl, mannitol, or raffinose caused two- to fourfold increases in the alpha 1-subunit mRNA accumulation and five- to eightfold increases in the beta 1-subunit mRNA accumulation, with peak elevations of both subunits at 12 h after addition. In sharp contrast, hyperosmolar urea medium had no effect at any time. When NaCl or mannitol was added to the media in amounts ranging from 300 to 600 mosmol/kgH2O, the maximal effects on both alpha 1- and beta 1-subunit mRNA accumulation occurred at 500 mosmol/kgH2O. In urea-supplemented medium, however, there was no significant change at any level of osmolality. The upregulation of alpha 1- and beta 1-subunit mRNA induced by hyperosmotic mannitol- or raffinose-supplemented media was markedly inhibited by removal of Na from the culture medium. Furthermore, pretreatment with a protein synthesis inhibitor cycloheximide partially inhibited the upregulation of alpha 1- and beta 1-subunit mRNA in IMCD cells exposed to hyperosmotic media treated with NaCl or mannitol. When IMCD cells were incubated with hyperosmotic media (500 mosmol/kgH2O) supplemented with NaCl or mannitol for 24 h, Na-K-ATPase activity increased by 78.6 and 82.8%, respectively. In contrast, hyperosmolar urea medium had no significant effect on Na-K-ATPase activity. These results demonstrate that 1) hyperosmolality induced by the poorly permeating solutes (NaCl, mannitol, and raffinose) but not the rapidly permeating solute (urea) stimulates both alpha 1- and beta 1-subunit mRNA accumulations in IMCD cells in a time- and an osmolality-dependent manner, 2) the hyperosmolality-induced upregulation of alpha 1- and beta 1-subunit mRNA leads to an increase in Na- K -ATPase activity; and 3) the above upregulation of alpha1- and beta 1-subunit mRNA in response to hyperosmotic media requires, at least in part, the presence of Na in the extracellular medium and the de novo synthesis of intermediate proteins.
Animals↗
Pulmonary hypertension in systemic lupus erythematosus: a report of an autopsied case.
An autopsied case of systemic lupus erythematosus with pulmonary hypertension is reported. A 29-year-old woman with a seven-year history of polyarthralgia, butterfly rash, nephrotic syndrome and Raynaud's phenomenon was admitted because of progressive dyspnea on exertion. Tests for antinuclear antibody, anti-cardiolipin antibody and lupus anticoagulant were positive. Echocardiographic examination revealed right ventricular hypertrophy and a moderate pericardial effusion. Estimated systolic pulmonary arterial pressure was 53 mmHg. Despite treatment with corticosteroids including pulse methylprednisolone therapy, lipo-PGE1 and warfarin, she died of progressive congestive heart failure. Postmortem examination of the pulmonary vasculature revealed findings consistent with plexogenic pulmonary arteriopathy, without evidence of vasculitis, fibrinoid necrosis, or thromboemboli.
Adult↗
Synthesis and characterization of the selenium analog of glutathione disulfide.
We synthesized the selenium analog of glutathione disulfide by a liquid phase method and named it glutaselenone (i.e., gamma-L-glutamyl-L-selenocysteinylglycine) diselenide. The selenol of selenocysteine was protected by the p-methoxybenzyl group, which was removed by acidolysis with trifluoroacetic acid in the presence of thioanisol. The overall yield of the final product, glutaselenone diselenide, was about 9% based on the starting compound, Se-(p-methoxybenzyl)-L-selenocysteine. Glutaselenone diselenide showed a broad absorption band between 270 and 400 nm and circular dichroism bands around 270 nm (positive) and 330 nm (negative), which were attributable to diselenide bond.
Chromatography, High Pressure Liquid↗
[Severe bleeding in a case of factor VIII inhibitor associated with intravascular lymphomatosis].
A 70-year-old man was admitted to Jichi Medical School Hospital on October 1, 1990, complaining of spontaneous severe hemorrhage in his extremities. He had prolonged APTT value and normal PT value. Factor VIII level was markedly diminished, and a high titer of factor VIII inhibitor was observed. He was diagnosed as having idiopathic factor VIII inhibitor because of no sign of underlying disorders. He received prednisolone and immunosuppressants, subsequently with plasma exchange and bolous factor VIII, resulting in disappearance of the inhibitor. He was admitted again in February 1991, because of the reappearance of factor VIII inhibitor. In June, furthermore, bone marrow smears showed infiltration of atypical B cells, although no evidence of organomegaly and adenopathy was observed. Therefore, he was suspected to have primary bone marrow lymphoma. He received CHOP therapy, but he died of multiorgan failure. Postmortem autopsy demonstrated multifocal intravascular lymphomatosis with marked intravascular involvement of the lung, liver, kidney, adrenal glands, spleen and bone marrow. Lymph nodes were not swollen, showing little evidence of intravascular lymphomatosis.
Aged↗
Interaction of human parathyroid hormone-related peptide with parathyroid hormone receptors in clonal rat osteosarcoma cells.
Synthetic peptides corresponding to the amino-terminal region of the human parathyroid hormone-related peptide (hPTHrp) were used to characterize the interaction of hPTHrp with parathyroid hormone (PTH) receptors in clonal rat osteosarcoma cells (ROS 17/2.8). Both hPTHrp-(1-34) and [Tyr40]hPTHrp-(1-40) showed full agonist activity in stimulating cyclic AMP accumulation in ROS cells; human PTHrp-(1-34) was approximately 2.5-fold as potent as hPTH-(1-34). Both [Tyr-40]hPTHrp-(3-40) and hPTH-(3-34) inhibited the cyclic AMP increase induced by either hPTHrp or PTH with parallel dose-inhibition curves. Binding to intact ROS cells of a 125I-labeled [Tyr40]hPTHrp-(1-40) (125I-[Tyr40]hPTHrp-(1-40)) which retains full biological activity was time- and temperature-dependent and reversible. Binding of 125I-[Tyr40]hPTHrp-(1-40) and 125I-labeled [Nle8, Nle18, Tyr34]bovine PTH-(1-34)NH2 to ROS cells was competed for, to the same extent and with the comparable potency, by either unlabeled hPTHrp or PTH peptides. The binding capacity and affinity of receptors in ROS cells were strikingly similar for hPTHrp and PTH. Affinity cross-linking with either radioligand resulted in high affinity, specific labeling of an apparently identical macromolecule centering at Mr = 80,000, which was detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in both reducing and nonreducing conditions. The data indicate that hPTHrp and PTH, their amino-terminal fragments at least, interact with the identical receptors with regard to affinity, capacity, specificity, and physicochemical characteristics in osteoblastic ROS 17/2.8 cells.
Animals↗