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Biomedical subjects

J Jin

Publications and source records attributed to J Jin.

At least 91 records · Page 5Linked to original sources
PubMed1999

Protein kinase B/Akt participates in GLUT4 translocation by insulin in L6 myoblasts.

L6 myoblasts stably transfected with a GLUT4 cDNA harboring an exofacial myc epitope tag (L6-GLUT4myc myoblasts) were used to study the role of protein kinase B alpha (PKBalpha)/Akt1 in the insulin-induced translocation of GLUT4 to the cell surface. Surface GLUT4myc was detected by immunofluorescent labeling of the myc epitope in nonpermeabilized cells. Insulin induced a marked translocation of GLUT4myc to the plasma membrane within 20 min. This was prevented by transient transfection of a dominant inhibitory construct of phosphatidylinositol (PI) 3-kinase (Deltap85alpha). Transiently transfected cells were identified by cotransfection of green fluorescent protein. A constitutively active PKBalpha, created by fusion of a viral Gag protein at its N terminus (GagPKB), increased the cell surface density of GLUT4myc compared to that of neighboring nontransfected cells. A kinase-inactive, phosphorylation-deficient PKBalpha/Akt1 construct with the mutations K179A (substitution of alanine for the lysine at position 179), T308A, and S473A (AAA-PKB) behaved as a dominant-negative inhibitor of insulin-dependent activation of cotransfected wild-type hemagglutinin (HA)-tagged PKB. Furthermore, AAA-PKB markedly inhibited the insulin-induced phosphorylation of cotransfected BAD, demonstrating inhibition of the endogenous PKB/Akt. Under the same conditions, AAA-PKB almost entirely blocked the insulin-dependent increase in surface GLUT4myc. PKBalpha with alanine substitutions T308A and S473A (AA-PKB) or K179A (A-PKB) alone was a less potent inhibitor of insulin-dependent activation of wild-type HA-PKB or GLUT4myc translocation than was AAA-PKB. Cotransfection of AAA-PKB with a fourfold DNA excess of HA-PKB rescued insulin-stimulated GLUT4myc translocation. AAA-PKB did not prevent actin bundling (membrane ruffling), though this response was PI 3-kinase dependent. Therefore, it is unlikely that AAA-PKB acted by inhibiting PI 3-kinase signaling. These results outline an important role for PKBalpha/Akt1 in the stimulation of glucose transport by insulin in muscle cells in culture.

Cells, Cultured↗
PubMed1999

Role of intracellular signaling events in ADP-induced platelet aggregation.

Human platelets express two distinct G protein-coupled ADP receptors, one coupled to phospholipase C through Gq, P2Y1, and the other to inhibition of adenylyl cyclase through Gi, P2TAC. We have recently shown that concomitant intracellular signaling from both the P2TAC and P2Y1 receptors is essential for ADP-induced platelet aggregation. Previous studies have tested whether ADP causes a decrease in the basal cAMP level and this reduction promotes platelet aggregation, but did not study the effect of decreased cAMP levels when the Gq pathway is selectively activated. Since we are now aware that platelet aggregation requires activation of two receptors, we investigated whether the function of P2TAC receptor activation, leading to inhibition of platelet adenylyl cyclase, could be replaced by direct inhibition of adenylyl cyclase, when Gq pathway is also activated, a possibility that has not been addressed to date. In the present study, we supplemented the P2Y1 mediated Gq signaling pathway with inhibition of the platelet adenylyl cyclase by using SQ22536 or dideoxyadenosine, or by selective activation of the alpha2A adrenoceptors with epinephrine. Although SQ22536, dideoxyadenosine, and epinephrine reduced the cAMP levels, only epinephrine could mimic the P2TAC receptor mediated signaling events, suggesting that reduction in basal cAMP levels does not directly contribute to ADP-induced platelet activation. Adenosine-5'-phosphate-3'-phosphosulfate, a P2Y1 receptor antagonist, completely blocked ADP-induced inositol 1,4,5-trisphosphate and inositol 1,3.4-trisphosphate formation suggesting that P2TAC-mediated activation of Gi (or other G proteins) does not activate phospholipase C. These results suggest that a signaling event downstream from Gi, independent of the inhibition of platelet adenylyl cyclase, contributes to alphaIIb beta3 activation.

Adenine↗
PubMed1999

New buccinator myomucosal island flap: anatomic study and clinical application.

The authors studied the vascular anatomy of the buccinator muscle by dissecting fresh cadavers. The anatomy of the buccal branches of the facial artery consistently confirmed the existence of a posterior buccal branch, a few inferior buccal branches, and anterior buccal branches to the posterior, inferior, and anterior portions of the buccinator. The buccal artery and posterior buccal branch anastomose to each other and ramify over the muscle. Several veins originate from the lateral aspect of the muscle, converge into the buccal venous plexus, and drain into the facial vein (from two to four tributaries) or into the pterygoid plexus and the internal maxillary vein (from the buccal vein). These vessels and nerves enter the posterior half of the buccinator posterolaterally. The facial artery and vein are located at variable distances from each other around the oral commissure and the nasal base. Two patterns of buccinator musculomucosal island flaps supplied by these buccal arterial branches are proposed in this article. The buccal musculomucosal neurovascular island flap (posteriorly based), supplied by the buccal artery, its posterior buccal branch, and the long buccal nerve, can be passed through a tunnel under the pterygomandibular ligament for closure of mucosal defects in the palate, pharyngeal sites, the alveolus, and the floor of the mouth. The buccal musculomucosal reversed-flow arterial island flap (superiorly based), supplied by the distal portion of the facial artery through the anterior buccal branches, can be used to close mucosal defects in the anterior hard palate, alveolus, maxillary antrum, nasal floor and septum, lip, and orbit. The authors have used the flaps in 12 patients. There has been no flap necrosis, and results have been satisfactory, both aesthetically and functionally.

Adult↗
PubMed1999

[Re-assessment of the inframammary crease ligament].

OBJECTIVE: An anatomical study was carried out to examine the existence of the inframammary crease ligament. METHODS: 18 breasts of preserved female cadavers were dissected and dyed for the fascia. RESULTS: The so-called inframammary crease ligament described by other authors does not exist. CONCLUSION: The reinforced Cooper's ligament supports the inframammary fold in position.

Adult↗
PubMed1999

[Clinical and experimental study on Yinhua mixture aerosol in treating infantile viral pneumonia].

OBJECTIVE: To explore the therapeutic effect of using Yinhua mixture aerosol in treating infantile viral pneumonia and its mechanism. METHODS: Comparing sixty-nine infantile viral pneumonia patients using Yinhua mixture aerosol with 33 infantile viral pneumonia patients using gentamycin, pharmacodynamical animal experiments of anti-tussis and anti-asthma were also observed. RESULTS: The markedly effective rate of Yinhua mixture aerosol and gentamycin aerosol were 58.0% and 27.3% respectively, while the total effective rate of these two drugs was 95.7% and 69.7% respectively. Experimental study revealed that Yinhua mixture was effective in antifebrile, anti-asthma and expectorative effect, which was better than that of gentamycin (P < 0.05). CONCLUSION: Yinhua mixture aerosol is effective in antifebrile, anti-asthma, and expectorative effect.

Administration, Inhalation↗
PubMed1999

[Potentiated effects of total saponins of Panax Ginseng on inhibition of leukemic cells by cytotoxic drugs].

OBJECTIVE: To investigate the potentiated effects of total saponins of Panax Ginseng (TSPG) on inhibition of leukemic progenitor cells by cytotoxic drugs in acute myelocytic leukemia. METHODS: Using bone marrow culture of colony forming unite-acute myeloid leukemia (CFU-AML) method, the sensitivity of leukemic cells obtained from 18 patients to homoharringtonin (HHr), cytarabine (Ara), adriamycin (Adr) and etoposide (VP-16) were detected separately. RESULTS: TSPG alone (20 micrograms/ml) could stimulate proliferation of CFU-AML obviously, and increase the colony numbers by 37.98% over the non-TSPG control (P < 0.01). In the presence of TSPG, the inhibition rates of CFU-AML of HHr, Ara, Adr and VP-16 were 51.2%-62.0% respectively, which were significantly higher than 30.4%-47.4% of non-TSPG control (all P < 0.01). In the combination of TSPG with cytotoxic drugs, the leukemic progenitor cells became more sensitive to cytotoxic drugs, CFU-AML colony numbers at 1.84-2.23 fold as more as those of non-TSPG control were inhibited by HHr, Ara, Adr and VP-16. Sensitivity test of 17 among 72 drugs reversed from resistant (suppression rate less than 30%) to sensitive (suppression rate more than 30%) by TSPG. CONCLUSIONS: TSPG could drive non-cycling leukemic progenitors to enter cell cycle, and thereby enhance their susceptibility to cytotoxic drugs.

Adult↗
PubMed1999

[Experimental study of immunization of mice with hepatitis C virus genetic vaccine constructs].

OBJECTIVE: To inquire into the immune responses to expression protein in mice immunized with genetic vaccine of hepatitis C virus (HCV) and lay a foundation for HCV genetic vaccine development in future. METHODS: The gene fragments coding C and most E regions of HCV-II type were inserted into pCD-SRalpha(1) of eukaryotic expression vector and formed genetic vaccine constructs of pCD-HCV(1) and then was injected into the quadriceps muscles of Balb/c mice. The serum anti-HCV level of mice was tested by ELISA and peripheral blood mononuclear cell (PBMC) proliferative responses to HCV antigens were detected by (3)H-TdR incorporation method (cpm). RESULTS: The serum antibody level reached to 0.71 +/- 0.08 - 0.77 +/- 0.06 (A value, the same below) after genetic vaccine pCD-HCV(1) (100 microg/mouse) were inoculated into the mice (n = 12) three or four times while blank vector pCD-SRalpha(1) could not induce the mice (n = 8) to generate antibody response in same way. After the antibody levels in mice (n = 8) immunized by pCD-HCV(1) had ascended to peak value (0.71), there was no trend of descending during the following 18 weeks of detection (0.68 +/- 0.06 - 0.75 +/- 0.07). Specific fragment of HCV cDNA identified by polymerase chain reaction (PCR) from DNA extracted from the muscles of the mice after pCD-HCV(1) had been inoculated three months. PBMC proliferative responses to HCV synthetic peptides CP(9) and gene recombinant antigens C, E(1) in the mice immunized with pCD-HCV(1) were detected and its stimulation indexes (SI) were 4.07 +/- 1.58, 3.88 +/- 0.70 and 3.69 +/- 1.13 respectively and there was a significant difference (P < 0.001) as compared with that of PBMC in mice immunized with pCD-SRalpha(1). CONCLUSION: These investigations demonstrated that genetic vaccine constructs made of HCV structural region can induce Balb/c mice to generate antibody and PBMC proliferative responses to HCV antigens via DNA immunization.

Animals↗
PubMed1999

[Influence of functional groups of huatanyuxin decoction on Ca2+ transmembrane influx in rat aorta].

OBJECTIVE: To study the Ca2+ antagonistic effect of functional groups of Huatanyuxin decoction. METHOD: The 45Ca transmembrane influx technique was used to measure the Ca2+ blocking effect of leak, receptor-operated Ca2+ channel (ROC) and potential-dependent Ca2+ channel (PDC) in rat aorta. RESULT: ROC and PDC could be blocked when the complex prescription was used at concentrations of 0.5-5 mg/ml. Among the functional groups, Huoxue group (activating blood) and Xinqi group (promoting and normalizing flow of vital energy) showed better Ca2+ antagonistic effect. CONCLUSION: The mechanism of therapeutic effect of Huatanyuxin decoction may be related to its Ca2+ blocking effect similar to verapamil.

Animals↗
PubMed1999

[Molecular cloning of rat OB gene and its expression in Escherichia coli].

In order to provide rat OB gene product for studying the relationship between obesity and noninfectious diseases, rat OB cDNA was amplified by RT-PCR technique. 460 bp fragment of OB cDNA was subcloned into EcoRI/BamHI site of plasmid pUC 19. Sequence analysis of OB cDNA revealed that the translation reading frame was identical with that reported in the literature. Thereby plasmid pBV220-rOB was constructed and the specific expression of OB gene in E. coli identified by SDS-PAGE electrophoresis was obtained.

Amino Acid Sequence↗
PubMed1998

Downregulation of the pro-apoptotic protein Bak is required for the ras-induced transformation of intestinal epithelial cells.

Anoikis is a form of programmed cell death induced in normal epithelial cells by detachment from the extracellular matrix [1] [2] [3]. In epithelial cells of the intestine and other organs, activated rasinduces resistance to anoikis [3] [4], but the actual molecular effectors directly involved in the apoptotic machinery that execute or block anoikis have not yet been identified. Bak, a pro-apoptotic member of the Bcl-2 family, is downregulated in a high proportion of colorectal tumours [5]. In addition, Bak is an important regulator of apoptosis in normal intestinal epithelial cells [6] [7]. Here, we show that activated rasinduces the downregulation of Bak in rat and human intestinal epithelial cells. This ras-induced downregulation of Bak expression could be suppressed by an inhibitor of phosphatidylinositol (PI) 3-kinase, an enzyme already implicated in ras-induced resistance to anoikis [8]. Ectopic expression of Bak in ras-transformed rat intestinal epithelial IEC-18 cells inhibited ras-induced resistance to anoikis and significantly reduced their tumorigenicity. We conclude, therefore, that the ability of rasto downregulate Bak, and the consequent resistance to anoikis, are essential components of the transforming capacity of this oncogene in intestinal epithelial cells.

Animals↗
PubMed1998

An inhibitor of inducible nitric oxide synthase ameliorates experimental autoimmune myocarditis in Lewis rats.

We studied the effect of nitric oxide (NO) on experimental autoimmune myocarditis (EAC) in rats. We examined the role of inducible nitric oxide synthase (iNOS), an enzyme that produces NO, on hearts affected with EAC, by testing the effects of aminoguanidine (AG), a selective iNOS inhibitor, on the course of EAC. Western blotting detected iNOS in the affected cardiac tissues, but not in CFA immunized cases. Immunohistochemically, the majority of ED1+ macrophages in the EAC lesions were positive for iNOS and nitrotyrosine. A high dose of AG (200 mg/kg/day) significantly reduced the incidence of EAC (p < 0.05) and ameliorated the histological score for the cardiac inflammation (p < 0.01) compared with the low dose AG (100 mg/kg/day) and vehicle treated groups. The immunoblot analysis showed that a high dose of AG effectively suppressed iNOS in hearts affected with EAC. An iNOS band was barely detected in the high dose AG (200 mg/kg) treated group, while it was distinctively visualized in the vehicle and low dose AG (100 mg/kg) treated groups. These results suggest that iNOS is upregulated in EAC lesions and increased NO production plays an important role in the development of EAC. In addition, selective iNOS inhibitors may have a therapeutic role in treating certain autoimmune diseases including EAC.

Animals↗
PubMed1998

Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation.

While a plethora of extracellular molecules exist that modulate cellular functions via binding to membrane receptors inside the cell, their actions are mediated by relatively few signalling mechanisms. One of these is activation of phosphatidylinositol 3-kinase (PI-3K), which results in the generation of a membrane-restricted second messenger, polyphosphatidylinositides containing a 3'-phosphate. How these molecules transduced the effects of agonists of PI-3K was unclear until the recent discovery that several protein kinases become activated upon exposure to 3'-phosphorylated inositol lipids. These enzymes include protein kinase B (PKB)/AKT and PtdIns(3,4, 5)P3-dependent kinases 1 and 2, the first two of which interact with 3'-phosphorylated phosphoinositides via pleckstrin homology domains. Once targeted to the membrane by this motif, PKB becomes phosphorylated at two residues, which relieves intermolecular inhibition, allowing the activated complex to dissociate and modify its targets. Identification of these substrates is the subject of intensive research, since at least one must play a key role in suppressing apoptosis, as demonstrated by expression of activated alleles of PKB. The generation of effective transdominant mutants, coupled with genetic analysis of the protein kinase in simpler organisms, should help in elucidating outstanding questions in the functions, targets and regulation of this important mediator of PI-3K signalling.

Animals↗
PubMed1998

Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation.

ADP is an important platelet agonist causing shape change and aggregation required for physiological hemostasis. We recently demonstrated the existence of two distinct G protein-coupled ADP receptors on platelets, one coupled to phospholipase C, P2Y1, and the other to inhibition of adenylyl cyclase, P2TAC. In this study, using specific antagonists for these two receptors, we demonstrated that concomitant intracellular signaling from both the P2TAC and P2Y1 receptors is essential for ADP-induced platelet aggregation. Inhibition of signaling through either receptor, by specific antagonists, is sufficient to block ADP-induced platelet aggregation. Furthermore, signaling through the P2TAC receptor could be replaced by activation of alpha2A-adrenergic receptors. On the other hand, activation of serotonin receptors supplements signaling through the P2Y1 receptor. Moreover, this mechanism of ADP-induced platelet aggregation could be mimicked by coactivation of two non-ADP receptors coupled to Gi and Gq, neither of which can cause platelet aggregation by itself. We propose that platelet aggregation results from concomitant signaling from both the Gi and Gq, a mechanism by which G protein-coupled receptors elicit a physiological response.

Adenosine Diphosphate↗
PubMed1998

Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity.

This study was designed to identify functionally important factor IX (FIX) residues. Using recombinant techniques and cell culture, we produced a mutant FIX with arginine at 338 changed to alanine (R338A-FIX). This molecule had approximately 3 times greater clotting activity than that of wild type FIX (wt-FIX) in the activated partial thromboplastin assay. R338A-FIX reacted normally with a panel of three FIX specific monoclonal antibodies and migrated on sodium dodecyl sulfate-polyacrylamide gels indistinguishably from wt-FIX. Using functional assays, we determined that R338A-FIXa's Kd for factor VIIIa (FVIIIa) was similar to that of wt-FIXa. Our kinetic analysis, using factor X as substrate, indicated that the mutation's major effects were a 3-fold increase in kcat and a 2-fold decrease in Km both manifested only in the presence of FVIIIa. R338A-FIXa's increased catalytic efficiency did not result from ablation of a thrombin sensitive site, reported to occur at arginine 338, since in our assays the thrombin inhibitor, hirudin, had no effect on activity of either wt-FIXa or R338A-FIXa. R338A-FIXa and wt-FIXa had equal activity, with or without FVIIIa, toward the synthetic substrate, methylsulfonyl-D-cyclohexylglycyl-arginine-p-nitroanilide. Interestingly, R338A-FIXa had reduced affinity for heparin. Therefore, we propose that R338A-FIXa's increased activity is not due to an allosteric effect on the active site, but that the Arg-338 residue is part of an exosite that binds both factor X and the mucopolysaccharide, heparin.

Alanine↗
PubMed1998

Mapping of ORF121, a factor that activates baculovirus early gene expression.

The protein product of the 39k gene of Autographa californica nuclear polyhedrosis virus is thought to be important for viral replication because of its association with the virogenic stroma and its role in activation of late gene expression Transient expression assays showed that addition of a DNA fragment encoding a 58-amino-acid polypeptide increased expression of a 39k reporter plasmid. This stimulation was dependent on cotransfection of a plasmid encoding IE1. Cotransfection of this gene, orf121, also stimulated ie1 expression, and the activation of ie1 was even more dramatic in the presence of IE1. These data suggested that ORF-121 stimulated 39k expression by upregulation of IE1 expression. Activation of 39k by ORF121 and the viral transcription factor IE2 was additive, while activation by ORF-121 and the apoptotic suppressor P35 was synergistic. Cotransfection of p39cat and pIE1 with plasmids encoding ORF121, IE2, and P35 stimulated 39cat expression more than 100-fold compared to cells transfected with only p39cat and pIE1. These data suggest that IE2 and ORF121 work by similar mechanisms and indirectly activate p39cat by increasing IE1 expression, while P35 increases 39cat expression by a different mechanism.

Amino Acid Sequence↗
PubMed1998

Genetic analysis of protein kinase B (AKT) in Drosophila.

The decision between survival and death is an important aspect of cellular regulation during development and malignancy. Central to this regulation is the process of apoptosis, which is conserved in multicellular organisms [1]. A variety of signalling cascades have been implicated in modulation of apoptosis, including the phosphatidylinositol (Pl) 3-kinase pathway. Activation of Pl 3-kinase is protective, and inhibition of this lipid kinase enhances cell death under several conditions including deregulated expression of c-Myc, neurotrophin withdrawal and anoikis [2-7]. Recently, the protective effects of Pl 3-kinase have been linked to its activation of the pleckstrin homology (PH)-domain-containing protein kinase B (PKB or AKT) [8]. PKB/AKT was identified from an oncogene, v-akt, found in a rodent T-cell lymphoma [9]. To initiate a genetic analysis of PKB, we have isolated and characterized a Drosophila PKB/AKT mutant (termed Dakt1) that exhibits ectopic apoptosis during embryogenesis as judged by induction of membrane blebbing, DNA fragmentation and macrophage infiltration. Apoptosis caused by loss of Dakt function is rescued by caspase suppression but is distinct from the previously described reaper/grim/hid functions. These data implicate Dakt1 as a cell survival gene in Drosophila, consistent with cell protection studies in mammals.

Animals↗
PubMed1998

Molecular basis for ADP-induced platelet activation. I. Evidence for three distinct ADP receptors on human platelets.

Acting through cell surface receptors, ADP activates platelets resulting in shape change, aggregation, thromboxane A2 production, and release of granule contents. ADP also causes a number of intracellular events including inhibition of adenylyl cyclase, mobilization of calcium from intracellular stores, and rapid calcium influx in platelets. However, the receptors that transduce these events remain unidentified and their molecular mechanisms of action have not been elucidated. The receptor responsible for the actions of ADP on platelets has been designated the P2T receptor. In this study we have used ARL 66096, a potent antagonist of ADP-induced platelet aggregation, and a P2X ionotropic receptor agonist, alpha,beta-methylene adenosine 5'-triphosphate, to distinguish the ADP-induced intracellular events. ARL 66096 blocked ADP-induced inhibition of adenylyl cyclase, but did not affect ADP-mediated intracellular calcium increases or shape change. Both ADP and 2-methylthio-ADP caused a 3-fold increase in the level of inositol 1,4,5-trisphosphate over control levels which peaked in a similar fashion to the Ca2+ transient. The increase in inositol 1,3,4-trisphosphate was of similar magnitude to that of inositol 1,4,5-trisphosphate. alpha,beta-Methylene adenosine 5'-triphosphate did not cause an increase in either of the inositol trisphosphates. These results clearly demonstrate the presence of two distinct platelet ADP receptors in addition to the P2X receptor: one coupled to adenylyl cyclase and the other coupled to mobilization of calcium from intracellular stores through inositol trisphosphates.

Adenosine Diphosphate↗