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1.
Diabetologia ; 48(2): 336-44, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15654601

ABSTRACT

AIMS/HYPOTHESIS: SHIP2 is a physiologically important negative regulator of insulin signalling hydrolysing the PI3-kinase product, PI(3,4,5)P3, which also has an impact on insulin resistance. In the present study, we examined the effect of inhibiting the endogenous SHIP2 function on the insulin resistance caused by chronic insulin treatment. METHODS: The endogenous function of SHIP2 was inhibited by expressing a catalytically inactive SHIP2 (DeltaIP-SHIP), and compared with the effect of treatments designed to restore the levels of IRS-1 in insulin signalling systems of 3T3-L1 adipocytes. RESULTS: Chronic insulin treatment induced the large (86%) down-regulation of IRS-1 and the modest (36%) up-regulation of SHIP2. Subsequent stimulation by insulin of Akt phosphorylation, PKClambda activity, and 2-deoxyglucose (2-DOG) uptake was markedly decreased by the chronic insulin treatment. Coincubation with the mTOR inhibitor, rapamycin, effectively inhibited the proteosomal degradation of IRS-1 caused by the chronic insulin treatment. Although the coincubation with rapamycin and advanced overexpression of IRS-1 effectively ameliorated subsequent insulin-induced phosphorylation of Akt, insulin stimulation of PKClambda activity and 2-DOG uptake was partly restored by these treatments. Similarly, expression of DeltaIP-SHIP2 effectively ameliorated the insulin-induced phosphorylation of Akt without affecting the amount of IRS-1. Furthermore, the decreased insulin-induced PKClambda activity and 2-DOG uptake following chronic insulin treatment were ameliorated by the expression of DeltaIP-SHIP2 more effectively than by the treatment with rapamycin. CONCLUSIONS/INTERPRETATION: Our results indicate that the inhibition of endogenous SHIP2 is effective in improving the state of insulin resistance caused by chronic insulin treatment.


Subject(s)
Adipocytes/physiology , Insulin Resistance/physiology , Insulin/pharmacology , Phosphoric Monoester Hydrolases/antagonists & inhibitors , 3T3 Cells , Adipocytes/drug effects , Animals , Biological Transport/drug effects , Deoxyglucose/metabolism , Insulin Receptor Substrate Proteins , Isoenzymes/metabolism , Mice , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphoproteins/metabolism , Protein Kinase C/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Sirolimus/pharmacology , src Homology Domains
2.
Acta Neurochir Suppl ; 87: 75-8, 2003.
Article in English | MEDLINE | ID: mdl-14518528

ABSTRACT

We used fMRI to study brain activation with facilitative rehabilitation techniques (passive hand movements and visual feedback) in two patients with subcortical lesions. Two tasks were given in a sequence. The first task (trial 1) was repetitive hand grasping by the paretic hand at a rate of 0.5 Hz with the eyes closed. The second task (trial 2), the facilitative rehabilitation technique, included task 1 plus support by a trainer to move the paretic hand with the eyes open to get visual feedback of the movement. The data were analyzed by a subtractive method. When task 1 was subtracted from task 2, it was found that the bilateral visual cortex, contralateral premotor cortex and posterior parietal cortex were involved with the passive hand movement and visual feedback. These facilitative rehabilitation techniques may integrate networks between sensory information and motor commands, and lead to functional reorganization.


Subject(s)
Brain Mapping/methods , Cerebral Cortex/physiopathology , Magnetic Resonance Imaging/methods , Paresis/diagnosis , Paresis/rehabilitation , Subtraction Technique , Adult , Association , Female , Hand Strength , Humans , Male , Middle Aged , Paresis/physiopathology , Photic Stimulation/methods , Treatment Outcome
3.
Nihon Rinsho ; 59(11): 2179-85, 2001 Nov.
Article in Japanese | MEDLINE | ID: mdl-11712404

ABSTRACT

The advancement of molecular biology in the field of insulin signal transduction is remarkable and the knowledge acquired through the recent research can be applied to the development of new antidiabetic drugs. There are several serine-threonine kinases and tyrosine phosphatases which can decrease insulin action at the state of diabetes and adipocytokines, which are produced from enlarged adipocytes, may affect insulin signal transduction. To prevent these proteins and cytokines from suppressing insulin action, specific molecular targets could be identified and the new agents can be developed for the normal insulin signaling and the development of new antidiabetic drugs is ongoing at present time.


Subject(s)
Drug Design , Hypoglycemic Agents , Insulin/physiology , Muscle Proteins , Signal Transduction , Thiazolidinediones , Adipocytes/metabolism , Animals , Cytokines/metabolism , Diabetes Mellitus/etiology , Glucose Transporter Type 4 , Humans , Hypoglycemic Agents/pharmacology , Monosaccharide Transport Proteins/metabolism , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphoric Monoester Hydrolases/physiology , Protein Serine-Threonine Kinases/physiology , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/physiology , Signal Transduction/physiology , Thiazoles/pharmacology
4.
J Exp Psychol Gen ; 130(3): 479-92, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11561922

ABSTRACT

Preattentive models of early vision have not been supported by the evidence. Instead, an input filtering system, which is dynamically reconfigured so as to optimize performance on the task at hand, is proposed. As a case in point, the authors examined Sagi and Julesz's (1985a) claim that detection tasks are processed preattentively and efficiently (shallow search slopes), whereas discrimination tasks require focal attention and yield inefficient steep slopes. In 5 visual search experiments, efficiency was found to depend not on the nature of the task but on whether the task is single or dual. The second component of a dual task, whether detection or discrimination, is performed inefficiently if it does not fit the configuration of the input system, which had been set optimally for the first component. But, even the second component is processed efficiently if there is enough time to reconfigure the system after processing the first component.


Subject(s)
Attention , Discrimination Learning , Pattern Recognition, Visual , Field Dependence-Independence , Humans , Orientation , Psychophysics , Reaction Time
5.
Horm Metab Res ; 33(7): 402-6, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11507676

ABSTRACT

Osmotic shock induces GLUT4 translocation and glucose uptake through a mechanism independent of PI 3-kinase, but dependent on tyrosine phosphorylation of cellular proteins. To identify the tyrosine phosphorylated proteins required for osmotic shock-stimulated glucose uptake, we examined tyrosine phosphorylation of candidate proteins, and found that the 60-80kDa species including paxillin and the 120-130kDa species including p130Cas, PYK2, FAK and Gab1 were tyrosine-phosphorylated in response to osmotic shock. Inhibition of actin polymerization by cytochalasin D significantly decreased the tyrosine phosphorylation of paxillin, p130Cas, PYK2 and FAK but not Gab1, but had no effect on 2-deoxyglucose (DOG) uptake, suggesting a role for Gab1 in osmotic shock-induced glucose transport. Also, we found that osmotic shock increases the association of phospholipase C-gamma (PLC-gamma) with Gab1 and stimulates tyrosine phosphorylation of PLC-gamma itself. The PLC inhibitor, U73122, inhibited osmotic shock-induced 2-DOG uptake. These results suggest that tyrosine phosphorylation of Gab1 and subsequent recruitment and activation of PLC-gamma may play a role in osmotic shock-induced glucose transport.


Subject(s)
Adipocytes/metabolism , Glucose/metabolism , Isoenzymes/physiology , Phosphoproteins/physiology , Type C Phospholipases/physiology , 3T3 Cells , Adaptor Proteins, Signal Transducing , Adipocytes/drug effects , Animals , Cytochalasin D/pharmacology , Deoxyglucose/metabolism , Immunoblotting , Mice , Nucleic Acid Synthesis Inhibitors/pharmacology , Osmotic Pressure , Phosphatidylinositol 3-Kinases/metabolism , Phospholipase C gamma , Phosphorylation , Precipitin Tests , Tyrosine/metabolism
6.
J Exp Psychol Hum Percept Perform ; 27(4): 969-84, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11518157

ABSTRACT

Perception of the 2nd of 2 targets (T1 and T2) is impaired if the lag between them is short (0-500 ms). The authors used this attentional blink (AB) to index attentional requirements in detection and identification tasks, with or without backward masking of T2, in 2 stimulus domains (line orientation, coherent motion). With masking, the AB occurred because T2 was masked during the attentional dwell time created by T1 processing (Experiments 1, 2, and 3). Without masking, an AB occurred only in identification because during the attentional dwell time, T2 decayed to a level that could support simple detection but not complex identification. However, an AB occurred also in detection if T2 was sufficiently degraded (Experiment 4). The authors drew 2 major conclusions: (a) Attention is required in both identification and detection, and (b) 2 factors contribute to the AB, masking of T2 while attention is focused on T1 and decay of the T2 trace while unattended.


Subject(s)
Attention , Blinking , Signal Detection, Psychological , Visual Perception/physiology , Humans , Random Allocation , Reaction Time
7.
Diabetes ; 50(8): 1891-900, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11473053

ABSTRACT

Growth hormone (GH) is well known to induce in vivo insulin resistance. However, the molecular mechanism of GH-induced cellular insulin resistance is largely unknown. In this study, we demonstrated that chronic GH treatment of differentiated 3T3-L1 adipocytes reduces insulin-stimulated 2-deoxyglucose (DOG) uptake and activation of Akt (also known as protein kinase B), both of which are downstream effects of phosphatidylinositol (PI) 3-kinase, despite enhanced tyrosine phosphorylation of insulin receptor substrate (IRS)-1, association of IRS-1 with the p85 subunit of PI 3-kinase, and IRS-1-associated PI 3-kinase activity. In contrast, chronic GH treatment did not affect 2-DOG uptake and Akt activation induced by overexpression of a membrane-targeted form of the p110 subunit of PI 3-kinase (p110(CAAX)) or Akt activation stimulated by platelet-derived growth factor. Fractionation studies indicated that chronic GH treatment reduces insulin-stimulated translocation of Akt from the cytosol to the plasma membrane. Interestingly, chronic GH treatment increased insulin-stimulated association of IRS-1 with p85 and IRS-1-associated PI 3-kinase activity preferentially in the cytosol. These results indicate that cellular insulin resistance induced by chronic GH treatment in 3T3-L1 adipocytes is caused by uncoupling between activation of PI 3-kinase and its downstream signals, which is specific to the insulin-stimulated PI 3-kinase pathway. This effect of GH might result from the altered subcellular distribution of IRS-1-associated PI 3-kinase.


Subject(s)
Adipocytes/physiology , Human Growth Hormone/pharmacology , Insulin Resistance/physiology , Insulin/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/physiology , 3T3 Cells , Adipocytes/cytology , Adipocytes/drug effects , Animals , Biological Transport/drug effects , Cell Line , Cytosol/metabolism , Deoxyglucose/metabolism , Humans , Insulin Receptor Substrate Proteins , Insulin-Like Growth Factor I/pharmacology , Janus Kinase 2 , Kinetics , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Mice , Microsomes/metabolism , Phosphoproteins/metabolism , Phosphotyrosine/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Recombinant Proteins/metabolism , Signal Transduction/drug effects , Swine , Transfection
8.
Mol Cell Biol ; 21(15): 5050-62, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11438661

ABSTRACT

A pathway sensitive to rapamycin, a selective inhibitor of mammalian target of rapamycin (mTOR), down-regulates effects of insulin such as activation of Akt (protein kinase B) via proteasomal degradation of insulin receptor substrate 1 (IRS-1). We report here that the pathway also plays an important role in insulin-induced subcellular redistribution of IRS-1 from the low-density microsomes (LDM) to the cytosol. After prolonged insulin stimulation, inhibition of the redistribution of IRS-1 by rapamycin resulted in increased levels of IRS-1 and the associated phosphatidylinositol (PI) 3-kinase in both the LDM and cytosol, whereas the proteasome inhibitor lactacystin increased the levels only in the cytosol. Since rapamycin but not lactacystin enhances insulin-stimulated 2-deoxyglucose (2-DOG) uptake, IRS-1-associated PI 3-kinase localized at the LDM was suggested to be important in the regulation of glucose transport. The amino acid deprivation attenuated and the amino acid excess enhanced insulin-induced Ser/Thr phosphorylation and subcellular redistribution and degradation of IRS-1 in parallel with the effects on phosphorylation of p70 S6 kinase and 4E-BP1. Accordingly, the amino acid deprivation increased and the amino acid excess decreased insulin-stimulated activation of Akt and 2-DOG uptake. Furthermore, 2-DOG uptake was affected by amino acid availability even when the degradation of IRS-1 was inhibited by lactacystin. We propose that subcellular redistribution of IRS-1, regulated by the mTOR-dependent pathway, facilitates proteasomal degradation of IRS-1, thereby down-regulating Akt, and that the pathway also negatively regulates insulin-stimulated glucose transport, probably through the redistribution of IRS-1. This work identifies a novel function of mTOR that integrates nutritional signals and metabolic signals of insulin.


Subject(s)
Acetylcysteine/analogs & derivatives , Insulin/metabolism , Phosphoproteins/metabolism , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Acetylcysteine/pharmacology , Adaptor Proteins, Signal Transducing , Adenoviridae/genetics , Amino Acids/chemistry , Animals , Biological Transport , Carrier Proteins/metabolism , Cell Cycle Proteins , Cell Line , Cysteine Endopeptidases/metabolism , Cytosol/metabolism , Deoxyglucose/pharmacokinetics , Down-Regulation , Enzyme Inhibitors/pharmacology , Eukaryotic Initiation Factors , Glucose/metabolism , Humans , Immunoblotting , Insulin Receptor Substrate Proteins , Mice , Multienzyme Complexes/metabolism , Phosphorylation , Precipitin Tests , Proteasome Endopeptidase Complex , Protein Binding , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Ribosomal Protein S6 Kinases/metabolism , Serine/chemistry , Signal Transduction , Sirolimus/pharmacology , Subcellular Fractions/metabolism , TOR Serine-Threonine Kinases , Threonine/chemistry , Time Factors , Tyrosine/metabolism
9.
Diabetes ; 50(5): 1083-92, 2001 May.
Article in English | MEDLINE | ID: mdl-11334412

ABSTRACT

Tumor necrosis factor (TNF)-alpha is one of the candidate mediators of insulin resistance associated with obesity, a major risk factor for the development of type 2 diabetes. The insulin resistance induced by TNF-alpha is antagonized by thiazolidinediones (TZDs), a new class of insulin-sensitizing drugs. The aim of the current study was to dissect the mechanism whereby pioglitazone, one of the TZDs, ameliorates TNF-alpha-induced insulin resistance in 3T3-L1 adipocytes. Pioglitazone restored insulin-stimulated 2-deoxyglucose (DOG) uptake, which was reduced by TNF-alpha, with concomitant restorations in tyrosine phosphorylation and protein levels of insulin receptor (IR) and insulin receptor substrate (IRS)-1, as well as association of the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase with IRS-1 and PI 3-kinase activity. Adenovirus-mediated gene transfer of either wild-type human peroxisome proliferator-activated receptor (PPAR)-gamma2 or a mutant carrying a replacement at the consensus mitogen-activated protein kinase phosphorylation site (hPPAR-gamma2-S112A) promoted adipogenesis of 3T3-L1 fibroblasts and restored TNF-alpha-induced decrease of triglyceride in adipocytes as effectively as pioglitazone. Overexpression of the PPAR-gamma proteins in TNF-alpha-treated adipocytes restored protein levels of IR/IRS-1, but did not improve insulin-stimulated tyrosine phosphorylation of IR/IRS-1 or insulin-stimulated 2-DOG uptake. These results indicate that the ability of pioglitazone to restore insulin-stimulated tyrosine phosphorylation of IR/IRS-1, which is necessary for amelioration of TNF-alpha-induced insulin resistance, may be independent of the adipogenic activity of PPAR-gamma that regulates protein levels of IR/IRS-1.


Subject(s)
Adipocytes/physiology , Hypoglycemic Agents/pharmacology , Insulin Resistance/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Thiazoles/pharmacology , Thiazolidinediones , Transcription Factors/physiology , Transforming Growth Factor alpha/pharmacology , 3T3 Cells , Adipocytes/cytology , Adipocytes/drug effects , Animals , Cell Differentiation , Cell Line , Deoxyglucose/metabolism , Humans , Insulin/pharmacology , Mice , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Phosphotyrosine/metabolism , Pioglitazone , Receptors, Cytoplasmic and Nuclear/drug effects , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Transcription Factors/drug effects , Transfection , Transforming Growth Factor alpha/antagonists & inhibitors
10.
J Gen Psychol ; 128(1): 57-75, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11277448

ABSTRACT

In the present study, the authors observed the effect of informative and uninformative attentional cueing on visual search for targets that were defined by a simple feature or by conjunctions of features. Three different types of attentional cueing were tested in three experiments: peripheral informative cueing, peripheral uninformative cueing, and central informative cueing. Participants showed a greater effect of cueing in detecting a conjunction of features than in detecting unique features only when attention was oriented by either peripheral or central informative cueing. This differential cueing effect was not observed when attention was oriented by peripheral uninformative cueing. The results suggest that voluntarily oriented attention plays a more important role in feature integration than automatically oriented attention does. The results also pose limits on the generalizability of K. A. Briand's (1998) proposal regarding the role of automatically oriented attention in feature integration.


Subject(s)
Attention , Cues , Form Perception , Adult , Analysis of Variance , Female , Fixation, Ocular , Humans , Male , Reaction Time
11.
J Gen Psychol ; 128(4): 357-83, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11892886

ABSTRACT

The phenomenon referred to as illusory line-motion (ILM; O. Hikosaka, S. Miyauchi, & S. Shimojo, 1993a) has been described as a measure of the local facilitation of attention gradient. However, J. Kawahara, K. Yokosawa, S. Nishida, and T. Sato (1996) have demonstrated a spatially parallel search for an "odd man out" in the ILM direction. Apart from showing preattentive ILM perception in terms of an analogy between line-motion and apparent motion, the authors examined whether ILM perception is possible without attention from another point of view. Four experiments revealed that the ILM target can be detected in parallel without invoking attentional facilitation and invalidated the possible contribution of attentional set in parallel ILM search. Participants were able to correctly detect the ILM target among multiple nontargets, even when the line orientation was changed from trial to trial. The authors' independent-detection model predicted ILM search performance well on several occasions. These findings strongly support a preattentive and stimulus-driven explanation of ILM perception.


Subject(s)
Attention , Motion Perception , Optical Illusions , Orientation , Pattern Recognition, Visual , Discrimination Learning , Humans , Models, Psychological , Psychophysics
12.
Phys Rev Lett ; 84(16): 3674-7, 2000 Apr 17.
Article in English | MEDLINE | ID: mdl-11019174

ABSTRACT

Optical conductivity data of the intermetallic compounds (Fe1-xVx)3Al ( 0

13.
Biochem Biophys Res Commun ; 275(1): 115-20, 2000 Aug 18.
Article in English | MEDLINE | ID: mdl-10944451

ABSTRACT

In the early phase of adipocyte differentiation, transient increase of DNA synthesis, called clonal expansion, and transient hyperphosphorylation of retinoblastoma protein (Rb) are observed. We investigated the role of these phenomena in insulin-induced adipocyte differentiation of 3T3-L1 cells. Insulin-induced clonal expansion, Rb phosphorylation and adipocyte differentiation were all inhibited by the PI 3-kinase inhibitors and rapamycin, but not the MEK inhibitor, whereas the MEK inhibitor, but not PI 3-kinase inhibitors or rapamycin, decreased c-fos induction. We conclude that insulin induces hyperphosphorylation of Rb via PI 3-kinase and mTOR dependent pathway, which promotes clonal expansion and adipocyte differentiation of 3T3-L1 cells.


Subject(s)
Adipocytes/cytology , Cell Differentiation , Phosphatidylinositol 3-Kinases/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Kinases , Retinoblastoma Protein/metabolism , 1-Methyl-3-isobutylxanthine/pharmacology , 3T3 Cells , Adipocytes/drug effects , Adipocytes/enzymology , Adipocytes/metabolism , Androstadienes/pharmacology , Animals , Cell Differentiation/drug effects , Clone Cells/cytology , Clone Cells/drug effects , Clone Cells/enzymology , Clone Cells/metabolism , Dexamethasone/pharmacology , Flavonoids/pharmacology , Insulin/pharmacology , Insulin Antagonists/pharmacology , MAP Kinase Kinase 1 , Mice , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-fos/antagonists & inhibitors , Proto-Oncogene Proteins c-fos/metabolism , Signal Transduction/drug effects , Sirolimus/pharmacology , TOR Serine-Threonine Kinases , Wortmannin
14.
Mol Endocrinol ; 14(6): 783-94, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10847581

ABSTRACT

Insulin receptor substrate-1 (IRS-1) is a major substrate of the insulin receptor and acts as a docking protein for Src homology 2 domain containing signaling molecules that mediate many of the pleiotropic actions of insulin. Insulin stimulation elicits serine/threonine phosphorylation of IRS-1, which produces a mobility shift on SDS-PAGE, followed by degradation of IRS-1 after prolonged stimulation. We investigated the molecular mechanisms and the functional consequences of these phenomena in 3T3-L1 adipocytes. PI 3-kinase inhibitors or rapamycin, but not the MEK inhibitor, blocked both the insulin-induced electrophoretic mobility shift and degradation of IRS-1. Adenovirus-mediated expression of a membrane-targeted form of the p110 subunit of phosphatidylinositol (PI) 3-kinase (p110CAAX) induced a mobility shift and degradation of IRS-1, both of which were inhibited by rapamycin. Lactacystin, a specific proteasome inhibitor, inhibited insulin-induced degradation of IRS-1 without any effect on its electrophoretic mobility. Inhibition of the mobility shift did not significantly affect tyrosine phosphorylation of IRS-1 or downstream insulin signaling. In contrast, blockade of IRS-1 degradation resulted in sustained activation of Akt, p70 S6 kinase, and mitogen-activated protein (MAP) kinase during prolonged insulin treatment. These results indicate that insulin-induced serine/threonine phosphorylation and degradation of IRS-1 are mediated by a rapamycin-sensitive pathway, which is downstream of PI 3-kinase and independent of ras/MAP kinase. The pathway leads to degradation of IRS-1 by the proteasome, which plays a major role in down-regulation of certain insulin actions during prolonged stimulation.


Subject(s)
Acetylcysteine/analogs & derivatives , Cysteine Endopeptidases/metabolism , Insulin/pharmacology , Multienzyme Complexes/metabolism , Phosphoproteins/metabolism , Signal Transduction/drug effects , Sirolimus/pharmacology , 3T3 Cells , Acetylcysteine/pharmacology , Adenoviridae/genetics , Adipocytes/metabolism , Animals , Cell Line , Deoxyglucose/metabolism , Embryo, Mammalian , Enzyme Inhibitors/pharmacology , Gene Expression , Humans , Insulin Receptor Substrate Proteins , Kidney , Mice , Multienzyme Complexes/antagonists & inhibitors , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Proteasome Endopeptidase Complex , Transfection
15.
J Pharm Pharmacol ; 52(3): 321-5, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10757421

ABSTRACT

We examined whether pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) affected 5-fluorouracil-induced thrombocytopenia without inducing more severe intimal thickening after injury to rat carotid arteries. Rat carotid arteries were injured using a balloon catheter on day 0. 5-Fluorouracil (100 mg kg(-1)) or vehicle was intravenously administered on day 1 in balloon-injured rats. PEG-rHuMGDF (100 microg kg(-1)) or vehicle was intravenously administered once a day on days 1-5 to balloon-injured rats given 5-fluorouracil or vehicle. 5-Fluorouracil (100 mg kg(-1), i.v.) caused a significant decrease in the platelet count from day 3 and peaked on days 7-9 in balloon-injured rats. PEG-rHuMGDF (100 microg kg(-1), i.v.) reduced this decrease on days 9 and 11. The administration of PEG-rHuMGDF did not accelerate the intimal thickening of balloon-injured arteries in rats treated with 5-fluorouracil compared with control balloon-injured rats. PEG-rHuMGDF did not increase plasma tumour growth factor-beta1 (TGF-beta1) from days 0-9 in balloon-injured rats compared with control balloon-injured rats. These results suggest that PEG-rHuMGDF ameliorated 5-fluorouracil-induced thrombocytopenia without accelerating the intimal thickening of balloon-injured arteries.


Subject(s)
Carotid Arteries/drug effects , Fluorouracil/administration & dosage , Polyethylene Glycols/pharmacology , Thrombocytopenia/prevention & control , Thrombopoietin/pharmacology , Animals , Blood Platelets/drug effects , Carotid Arteries/pathology , Catheterization , Fluorouracil/adverse effects , Humans , Male , Rats , Rats, Sprague-Dawley , Recombinant Proteins/pharmacology , Thrombocytopenia/chemically induced , Time Factors , Transforming Growth Factor beta/blood , Transforming Growth Factor beta/drug effects , Tunica Intima/drug effects , Tunica Intima/pathology
16.
J Pharm Pharmacol ; 51(7): 841-6, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10467960

ABSTRACT

Previous studies have shown that pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) at suprapharmacological dose induces a mild transient decrease of red blood cell counts according to thrombopoiesis in normal mice. To unravel the mechanism underlying this mild transient decrease of red blood cells, we have studied the effect of PEG-rHuMGDF on the circulating plasma and blood volume, and the serum biochemical parameters of anaemia and splenectomy. Also, we have performed histological studies of the bone marrow and the spleen of PEG-rHuMGDF-treated rats. PEG-rHuMGDF (300 microg kg(-1)]) or vehicle was subcutaneously administered to rats once a day for up to five days. From day 6 after the start of PEG-rHuMGDF administration, the platelet counts and plateletcrit levels were significantly increased, reaching peak values on day 10, and recovering to normal by day 20. The red blood cell counts and the haematocrit levels were significantly decreased on day 6 to 13. The decreases in red blood cell levels and haematocrit produced by PEG-rHuMGDF treatment were mild and had recovered by day 15. The plasma and blood volumes were significantly increased on day 10 in PEG-rHuMGDF-treated rats. No alteration of the serum biochemical parameters for anaemia, iron or total bilirubin, were observed on day 10. The histological examination on day 10 revealed a marked increase in megakaryocytes and a slight decrease in erythropoiesis in the bone marrow of rats that received PEG-rHuMGDF (300 microg kg(-1)). There was also a slight increase in splenic megakaryocytes and erythropoiesis. The decrease of red blood cells by PEG-rHuMGDF was not affected by splenectomy. These results suggest that the mild transient decrease of red blood cells induced by PEG-rHuMGDF treatment for up to five days is based mainly on the increases in the plasma and blood volume. These events are secondary changes due to the regulation of the excess production of megakaryocytes in the marrow and the peripheral platelets.


Subject(s)
Polyethylene Glycols/pharmacology , Thrombopoietin/pharmacology , Anemia/blood , Anemia/chemically induced , Anemia/physiopathology , Animals , Blood Platelets/drug effects , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Dose-Response Relationship, Drug , Erythrocyte Count/drug effects , Erythroid Precursor Cells/cytology , Erythroid Precursor Cells/drug effects , Hematocrit , Humans , Megakaryocytes/cytology , Megakaryocytes/drug effects , Plasma Volume/drug effects , Platelet Count/drug effects , Polyethylene Glycols/adverse effects , Rats , Recombinant Proteins/adverse effects , Recombinant Proteins/pharmacology , Spleen/cytology , Spleen/drug effects , Splenectomy , Thrombopoietin/adverse effects
17.
J Cardiovasc Pharmacol ; 33(2): 292-4, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10028939

ABSTRACT

The antihypertensive action of KRN4884 (5-amino-N-[2-(2-chlorophenyl)ethyl]-N'-cyano-3-pyridinecarboxamidine ), a newly synthesized 3-pyridine derivative was examined in conscious spontaneously hypertensive rats (SHRs). A single administration of KRN4884 (0.5, 1.5 mg/kg, p.o.) produced a dose-dependent and long-lasting antihypertensive effect. The 7-day repeated administration of KRN4884 (0.5, 1.5 mg/kg, p.o.) did not diminish antihypertensive activity during the treatment period or induce rebound hypertension after the discontinuation of treatment. To examine the mechanism of the antihypertensive effect of KRN4884, we studied its vasorelaxing effects in rat isolated aortae precontracted with 25 mM KCl. Single application of KRN4884 showed a slower onset of inhibitory action than that of levcromakalim. KRN4884 was approximately 26-fold more potent than levcromakalim and 10-fold less potent than nilvadipine. KRN4884- and levcromakalim-induced vasorelaxation were antagonized by glibenclamide. Furthermore, we observed the recovery of the contraction inhibited by these drugs after repeated washing. The inhibitory effect of KRN4884 was restored only after four washes, whereas that of levcromakalim was completely restored after one wash. The nilvadipine-induced inhibitory effect was the most resistant to washing among these drugs. These results suggest that KRN4884 shows a long-lasting antihypertensive effect based on its potent potassium channel-opening action. The long-lasting action may be due to a slow association/dissociation with/from the binding sites on vascular smooth muscle.


Subject(s)
Antihypertensive Agents/pharmacology , Cromakalim/pharmacology , Nifedipine/analogs & derivatives , Pyridines/pharmacology , Vasodilation/drug effects , Animals , Aorta, Thoracic/drug effects , Blood Pressure/drug effects , Calcium Channel Blockers/pharmacology , Dose-Response Relationship, Drug , Hypertension/pathology , In Vitro Techniques , Male , Nifedipine/pharmacology , Rats , Rats, Inbred SHR , Rats, Wistar , Time Factors
18.
Jpn J Pharmacol ; 78(3): 261-8, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9869259

ABSTRACT

The effects of KRN4884 (5-amino-N-[2-(2-chrolophenyl)ethyl]-N'-cyano-3-pyridinecarboxa midine), a novel K+ channel opener, on the electrocardiogram changes caused by the intracoronary administration of endothelin-1 (ET-1) were studied in anesthetized rats and compared with the effects of levcromakalim, a K+ channel opener; nilvadipine, a Ca2+ antagonist; and propranolol, a beta-adrenoceptor antagonist. KRN4884 (50 microg/kg, i.v.) and levcromakalim (300 microg/kg, i.v.) inhibited the ST segment elevation and the development of arrhythmias induced by ET-1 (5 microg, i.c.) and decreased the incidence of death. Nilvadipine (300 microg/kg, i.v.) and propranolol (1000 and 3000 microg/kg, i.v.) each prevented the ST segment elevation, but the suppressions of the occurrence of arrhythmias produced by nilvadipine and propranolol were less than that shown by KRN4884. KRN4884 (30 and 50 microg/kg, i.v.), levcromakalim (100 and 300 microg/kg, i.v.) and nilvadipine (100 and 300 microg/kg, i.v.) significantly decreased the mean blood pressure in a dose-dependent manner, but propranolol did not. The heart rate was decreased by nilvadipine (100 and 300 microg/kg, i.v.) and propranolol (1000 and 3000 microg/kg, i.v.), but was not affected by KRN4884 (30 and 50 microg/kg, i.v.) or levcromakalim (100 and 300 microg/kg, i.v.). These results suggest that pretreatments with KRN4884 and levcromakalim are more effective on ET-1-induced electrocardiogram changes than those with nilvadipine and propranolol.


Subject(s)
Antihypertensive Agents/pharmacology , Endothelin-1/adverse effects , Heart Diseases/prevention & control , Anesthesia , Animals , Antihypertensive Agents/therapeutic use , Blood Pressure/drug effects , Bronchoconstrictor Agents/pharmacology , Cromakalim/pharmacology , Cromakalim/therapeutic use , Electrocardiography/drug effects , Endothelin-1/pharmacology , Heart Diseases/chemically induced , Heart Rate/drug effects , Injections, Intra-Arterial , Male , Methacholine Chloride/pharmacology , Nifedipine/analogs & derivatives , Nifedipine/pharmacology , Nifedipine/therapeutic use , Potassium Channels/drug effects , Propranolol/pharmacology , Propranolol/therapeutic use , Pyridines/pharmacology , Pyridines/therapeutic use , Rats , Rats, Wistar
19.
J Biol Chem ; 273(28): 17726-31, 1998 Jul 10.
Article in English | MEDLINE | ID: mdl-9651372

ABSTRACT

When the genome of the thermophilic archaeon Pyrococcus horikoshii was sequenced, a gene homologous to the mammalian gene for an acylamino acid-releasing enzyme (EC 3.4.19.1) was found in which the enzyme's proposed active residues were conserved. The P. horikoshii gene comprised an open reading frame of 1,896 base pairs with an ATG initiation codon and a TAG termination codon, encoding a 72,390-Da protein of 632 amino acid residues. This gene was overexpressed in Escherichia coli with the pET vector system, and the resulting enzyme showed the anticipated amino-terminal sequence and high hydrolytic activity for acylpeptides. This enzyme was concluded to be the first acylamino acid-releasing enzyme from an organism other than a eukaryotic cell. The existence of the enzyme in archaea suggests that the mechanisms of protein degradation or initiation of protein synthesis or both in archaea may be similar to those in eukaryotes. The enzyme was stable at 90 degreesC, with its optimum temperature over 90 degreesC. The specific activity of the enzyme increased 7-14-fold with heat treatment, suggesting the modification of the enzyme's structure for optimal hydrolytic activity by heating. This enzyme is expected to be useful for the removal of Nalpha-acylated residues in short peptide sequence analysis at high temperatures.


Subject(s)
Peptide Hydrolases/genetics , Pyrococcus/enzymology , Amino Acid Sequence , Calorimetry, Differential Scanning , Cloning, Molecular , DNA Primers , Enzyme Activation , Enzyme Stability , Escherichia coli/genetics , Hot Temperature , Molecular Sequence Data , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , Pyrococcus/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Substrate Specificity
20.
J Cardiovasc Pharmacol ; 29(6): 814-9, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9234664

ABSTRACT

We examined the antihypertensive effects of KRN4884, 5-amino-N-[2-(2-chlorophenyl)ethyl]-N'-cyano-3-pyridinecarbocamidine+ ++, in normotensive dogs, a high-renin model acute renal hypertensive dog (RHD), and a low-renin model chronic RHD in the conscious state, compared with levcromakalim and nilvadipine. KRN4884 decreased mean blood pressure (MBP) at a dose of 0.1 mg/kg p.o. in normotensive dogs and both RHDs. The decrease in MBP was greater in both RHDs than in normotensive dogs, and there were no significant differences between the two RHDs. A transient increase in heart rate (HR) accompanied the increase in MBP in all three types of dogs. In the chronic RHD, KRN4884 at doses of 0.05, 0.1, and 0.2 mg/kg produced a dose-dependent decrease in MBP. The antihypertensive effect of KRN4884 (0.1 mg/kg) was similar to those of levcromakalim (0.05 mg/kg) and nilvadipine (1.0 mg/kg) in magnitude and more prolonged than those of the compounds. The tachycardia induced by KRN4884 was similar to that induced by levcromakalim and was stronger than that induced by nilvadipine. In the 15-day repeated oral-administration study, KRN4884 (0.1 mg/kg) induced sustained hypotensive effects and transient increases in HR and plasma renin activity. No tolerance to the antihypertensive effect of KRN4884 was observed during a 15-day repeated dosing period. After withdrawal of KRN4884, no rebound phenomena in MBP and HR were observed. Neither the maximal concentration nor area under the curve (AUC) of KRN4884 in plasma were changed at days 1, 8, and 15. These data indicate that KRN4884 produces a strong and persistent antihypertensive response in both low-renin and high-renin models of RHD in a conscious state, which suggests that KRN4884 may be useful as an antihypertensive agent.


Subject(s)
Antihypertensive Agents/pharmacology , Hypertension, Renal/physiopathology , Potassium Channels/agonists , Pyridines/pharmacology , Animals , Antihypertensive Agents/therapeutic use , Benzopyrans/pharmacology , Calcium Channel Blockers/pharmacology , Cromakalim , Dogs , Female , Hypertension, Renal/drug therapy , Male , Nifedipine/analogs & derivatives , Nifedipine/pharmacology , Potassium Channels/drug effects , Pyridines/therapeutic use , Pyrroles/pharmacology
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