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Biomedical subjects

Y Ren

Publications and source records attributed to Y Ren.

At least 109 records · Page 6Linked to original sources
PubMed1996

Angiotensin II action in isolated microperfused rabbit afferent arterioles is modulated by flow.

We have recently presented evidence that endogenous nitric oxide (NO) and prostaglandins (PGs) modulate angiotensin II (Ang II) action in microperfused afferent arterioles (Af-Arts). Because flow may be a physiological stimulus of endothelial release of NO and PGs, we tested the hypothesis that flow through the lumen of the Af-Art stimulates the endothelium to produce NO and PGs, which in turn modulate the action of Ang II. We microdissected the terminal segment of an interlobular artery together with two Af-Arts, their glomeruli and efferent arterioles (Ef-Art). The two Af-Arts were perfused simultaneously from the interlobular artery, while one Ef-Art was occluded. Since the arteriolar perfusate contained 5% albumin, oncotic pressure built up in the glomerulus with the occluded Ef-Art and opposed the force of filtration, resulting in little or no flow through the corresponding Af-Art. Thus this preparation allowed us to observe Ang II action in free-flow and non-flow Af-Arts simultaneously. Ang II-induced constriction was weaker in free-flow than non-flow Af-Arts, with the luminal diameter decreasing by 8 +/- 2% and 23 +/- 3% at 10(-9) M, respectively (P < 0.013 free-flow vs. non-flow; N = 9). Disrupting the endothelium augmented Ang II action in free-flow (33 +/- 5.1%; P < 0.01 vs. intact endothelium) but not non-flow Af-Arts (31 +/- 5.3%), thus abolishing the differences between them (N = 8). Pretreatment with an inhibitor of either NO synthase (N-nitro-L-arginine methyl ester) or cyclooxygenase (indomethacin) augmented Ang II action more in free-flow than non-flow Af-Arts, likewise abolishing the differences between them. These results suggest that intraluminal flow modulates the vasoconstrictor action of Ang II in Af-Arts via endothelium-derived NO and PGs. Thus flow may be important in the fine control of glomerular hemodynamics.

Angiotensin II↗
PubMed1996

The intracellular functions of alpha6beta4 integrin are regulated by EGF.

Upon ligand binding, the alpha6beta4 integrin becomes phosphorylated on tyrosine residues and combines sequentially with the adaptor molecules Shc and Grb2, linking to the ras pathway, and with cytoskeletal elements of hemidesmosomes. Since alpha6beta4 is expressed in a variety of tissues regulated by the EGF receptor (EGFR), we have examined the effect of EGF on the cytoskeletal and signaling functions of alpha6beta4. Experiments of immunoblotting with anti-phosphotyrosine antibodies and immunoprecipitation followed by phosphoamino acid analysis and phosphopeptide mapping showed that activation of the EGFR causes phosphorylation of the beta4 subunit at multiple tyrosine residues, and this event requires ligation of the integrin by laminins or specific antibodies. Immunoprecipitation experiments indicated that stimulation with EGF does not result in association of alpha6beta4 with Shc. In contrast, EGF can partially suppress the recruitment of Shc to ligated alpha6beta4. Immunofluorescent analysis revealed that EGF treatment does not induce increased assembly of hemidesmosomes, but instead causes a deterioration of these adhesive structures. Finally, Boyden chamber assays indicated that exposure to EGF results in upregulation of alpha6beta4-mediated cell migration toward laminins. We conclude that EGF-dependent signals suppress the association of activated alpha6beta4 with both signaling and cytoskeletal molecules, but upregulate alpha6beta4-dependent cell migration. The changes in alpha6beta4 function induced by EGF may play a role during wound healing and tumorigenesis.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Platelet-activating factor dilates efferent arterioles through glomerulus-derived nitric oxide.

UNLABELLED: Despite evidence that platelet-activating factor (PAF) is produced by the glomerulus, its direct action on the glomerular microcirculation is poorly understood. It was recently reported that at picomolar concentrations, PAF dilates isolated microperfused afferent arterioles (Af-Art) via nitric oxide (NO). The present study tested the hypothesis that PAF acts on the glomerulus to release NO, which in turn controls the resistance of the efferent arteriole (Ef-Art). Rabbit Ef-Art were perfused from the distal end (retrograde perfusion [RP]) to eliminate the influence of the glomerulus, or through the glomerulus from the end of the Af-Art (orthograde perfusion [OP]) to maintain the influence of the glomerulus. Ef-Art were preconstricted by approximately 40% with norepinephrine and increasing doses of PAF were added to both the arteriolar perfusate and bath. Only with OP did PAF at picomolar concentrations cause significant dilation: at 400 pmol, the diameter increased by 64 +/- 11% from the preconstricted level (N = 6, P < 0.01). This dilation was completely abolished by pretreatment with an NO-synthesis inhibitor. To study its possible constrictor action, PAF was added to nonpreconstricted Ef-Art. At nanomolar concentrations, PAF constricted Ef-Art similarly in both RP and OP: at 40 nM, the diameter decreased by 24 +/- 4% (N = 6, P < 0.01) and 20 +/- 2% (N = 6, P < 0.01), respectively. This constriction was attenuated by pretreatment with indomethacin (Indo) in both RP (14 +/- 2%, N = 7; P < 0.02 versus without Indo) and OP (10 +/- 2%, N = 6; P < 0.02 versus without Indo). IN CONCLUSION: (1) at picomolar concentrations, PAF stimulates the glomerulus to release NO, which in turn dilates the Ef-Art; and (2) at nanomolar concentrations, PAF constricts the Ef-Art partly through release of cyclooxygenase metabolites. Thus, PAF may play a role in glomerular hemodynamics under various physiological and pathological conditions.

Animals↗
PubMed1996

[Detection of konjac glucomannan in seven Amorphophallus Blume species].

Seven Amorphophallus species were detected for konjac glucomananam (KGM). It has been found out that A. albus, A. konjac, A. yuloensis, A. xemengensis and A. dunnii are of higher contents of KGM (30%), but A. sinensis and A. yunnanensis are not as further comfirmed by TLC analysis. It follows that A. albus, A. konjac, A. yuloensis, A. xemengensis and A. dunnii are high quality resources of KGM, but A. sinensis and A. yunanensis cannot be used as substitutes for the above-cited five species.

Chromatography, Thin Layer↗
PubMed1995

Topology of prostaglandin H synthase-1 in the endoplasmic reticulum membrane.

Prostaglandin H synthase-1 is an integral endoplasmic reticulum membrane protein which catalyzes a key control step in prostaglandin biosynthesis. The overall arrangement of the prostaglandin H synthase-1 polypeptide with respect to the endoplasmic reticulum membrane was examined in transiently transfected COS-1 cells, using immunofluorescence microscopy. A bacterial toxin, streptolysin-O, was used for selective plasma membrane permeabilization and a detergent, saponin, for general membrane permeabilization. Treated cells were probed with six antibodies specific for particular prostaglandin H synthase-1 peptide segments and one antibody specific for an inserted viral reporter epitope. Control experiments established that actin, a cytoplasmic marker, was accessible to fluorescein-labeled phalloidin after streptolysin-O treatment, whereas antibodies against protein disulfide isomerase, an endoplasmic reticulum lumenal marker, bound only after saponin treatment, Using this approach to investigate prostaglandin H synthase-1, it was found that streptolysin-O treatment was sufficient to obtain staining of intracellular membranes by antibodies specific for the endogenous C-terminal segment, for the viral reporter inserted at the C-terminus, and for the protease-sensitive region near arg277. In contrast, saponin treatment was necessary for staining by antibodies specific for peptides spanning residues 51-66, 156-170, and 377-390. Antibodies targeted against residues 483-496 did not stain transfected cells even after saponin permeabilization, although they did bind to detergent-solubilized prostaglandin H synthase-1. These results indicate that the C-terminus and arg277 regions of the synthase can be exposed on the cytoplasmic side of the endoplasmic reticulum membrane, whereas regions near N-glycosylation sites are confined to the endoplasmic reticulum lumen and residues 483-496 are inaccessible from either side of the endoplasmic reticulum membrane.

Amino Acid Sequence↗
PubMed1995

Mechanism of metallothionein gene regulation by heme-hemopexin. Roles of protein kinase C, reactive oxygen species, and cis-acting elements.

Heme-hemopexin or cobalt protoporphyrin (CoPP)-hemopexin (a model ligand for hemopexin receptor occupancy) is shown to increase transcription of the metallothionein-1 (MT-1) gene by activation of a signaling pathway. Promoter deletion analysis followed by transient transfection assays show that 110 base pairs (-153 to -43) of 5'-flanking region of the murine MT-1 promoter are sufficient for increasing transcription in response to heme-hemopexin or to CoPP-hemopexin in mouse hepatoma cells. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), prevented the increase in MT-1 transcription by heme-hemopexin, CoPP-hemopexin, or phorbol 12-myristate 13-acetate, but the protein kinase A inhibitor, HA1004, was without effect. N-Acetylcysteine (NAC) and glutathione, as well as superoxide dismutase and catalase, inhibited both the increase in endogenous MT-1 mRNA and the activation of reporter gene activity by heme-hemopexin, CoPP-hemopexin, and phorbol 12-myristate 13-acetate. In sum, these data suggest that reactive oxygen intermediates are generated by heme-hemopexin via events associated with receptor binding, including protein kinase C activation. Induction of heme oxygenase-1 expression, in contrast to MT-1, is significantly less sensitive to NAC. Deletion and mutation analyses of the MT-1 proximal promoter revealed that the sequence 5'-GTGACTATGC-3' (from -98 to -89 base pairs) is, in part, responsible for the hemopexin-mediated regulation of MT-1 which is inhibited by H7. Regulation via this element is also induced by H2O2 showing that it is an antioxidant response element. Heme itself acts via more distal elements on the MT-1 promoter. In contrast to NAC and glutathione, diethyl dithiocarbamate and pyrrolidine dithiocarbamate, which inactivate reactive oxygen intermediates and chelate Zn(II), synergistically augment the induction of MT-1 mRNA levels and reporter gene activity in response to heme-hemopexin via the antioxidant response element by both metal-responsive element-dependent and -independent mechanisms.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

CD36 gene transfer confers capacity for phagocytosis of cells undergoing apoptosis.

Phagocyte recognition and ingestion of intact cells undergoing apoptosis are key events in this generally important program of cell death. Insufficient phagocyte capacity for apoptotic cells can result in failure to clear dying cells before membrane integrity is lost, resulting in leakage of noxious cell contents and severe tissue damage. However, no means has been available to increase phagocytic clearance of apoptotic cells. We now report that transfection of the macrophage adhesion molecule CD36 into human Bowes melanoma cells specifically conferred greatly increased capacity to ingest apoptotic neutrophils, lymphocytes, and fibroblasts, comparable to that exhibited by macrophages. Furthermore, when CD36 was transfected into another cell type with limited capacity to take up apoptotic bodies, the monkey COS-7 cell, similar effects were observed. Therefore, CD36 gene transfer can confer "professional" capacity to ingest apoptotic cells upon "amateur" phagocytes.

Animals↗
PubMed1995

Proinflammatory cytokines potentiate thrombospondin-mediated phagocytosis of neutrophils undergoing apoptosis.

Apoptosis leads to swift recognition, ingestion, and degradation of intact senescent neutrophils by macrophages. This protects tissues from leakage of noxious contents from dying cells and may promote resolution of inflammation. However, little has been known of the mechanisms that regulate macrophage capacity for apoptotic cells during an inflammatory response. We examined whether proinflammatory cytokines modulated phagocytosis of senescent neutrophils undergoing apoptosis by human monocyte-derived macrophages at 4 days maturity (4d M phi), an in vitro model of neutrophil "disposal" by apoptosis. Pretreatment of 4d M phi with granulocyte-macrophage-CSF increased the proportion of 4d M phi taking up apoptotic PMN in a concentration-dependent fashion by up to approximately 240%. This was by a rapid effect detectable by 4 h and exerted on the M phi, not the PMN. Granulocyte-macrophage-CSF also increased the number of apoptotic PMN taken up by each M phi. IFN-gamma, IL-1 beta, TNF-alpha, and TGF-beta 1 also enhanced phagocytosis, but IL-4 and IL-6 had no effect. In each case, the cytokine-expanded phagocytic subpopulation employed the thrombospondin (TSP)-dependent recognition mechanism defined for mature M phi, in which M phi vitronectin receptor and CD36 cooperate. However, commensurate increases in M phi expression of VnR, TSP, or CD36 were not detectable, indicating that TSP-mediated recognition can be recruited by other mechanisms. Cytokines did not recruit phosphatidylserine-dependent recognition, the other major mechanism by which some macrophage populations ingest apoptotic cells. Thus, M phi phagocytosis of apoptotic neutrophils can be potentiated by proinflammatory cytokines, suggesting a mechanism for negative feedback control of neutrophil number at inflamed sites.

Apoptosis↗
PubMed1995

Prostaglandin H synthase-1: evaluation of C-terminus function.

The first committed step in prostaglandin biosynthesis is catalyzed by prostaglandin H synthase, an enzyme localized to the endoplasmic reticulum (ER) membrane in a variety of cells. Several types of C-terminal region peptide sequence motifs have been found to lead to ER retention of other proteins. We have tested the potential role for such signals in the ER localization and catalytic activity of human isoform-1 of the synthase (PGHS-1). PGHS-1 mutants with alterations in the C-terminus designed to disrupt potential retention signals were expressed in transfected COS-1 cells. The mutations included: substitution of valine for the ultimate leucine residue (position 600) to disrupt a KDEL-type signal, substitution of a neutral glutamine for arginine at position 595 to disrupt signals based on positive charge, and deletion of the last six residues, to remove all of the wild-type extreme C-terminus. The subcellular localization of each recombinant PGHS-1 was assessed by differential centrifugation and by immunofluorescence microscopy. None of the mutations led to a significant change in the distribution of PGHS-1 between microsomes and other cellular fractions. Immunostaining of wild-type PGHS-1 and all of the mutants colocalized with that of protein disulfide isomerase, an ER marker protein. However, mutation of the terminal leucine or deletion of the last six residues did lead to loss of cyclooxygenase activity. Mutation of the terminal leucine also altered the pattern of fragments produced by limited proteolysis, indicating that this mutation led to changes in the polypeptide folding which might account for the loss of activity. The results indicate that the extreme C-terminal region is important to the functional integrity of PGHS-1, but it is not an essential part of the intracellular targeting mechanism.

Amino Acid Sequence↗
PubMed1995

A rifampicin-induced hepatic microsomal enzyme system for the generation of cyclosporine metabolites.

A drug-induced rabbit hepatic microsomal enzyme system has been developed to produce milligram quantities of cyclosporine metabolites (CMs). Using a rifampicin-induced microsomal preparation in the presence of a NADPH regenerating system, 60% of the cyclosporine (CsA) was converted to CMs in 2 h. The CMs were recovered by solid phase extraction, and separated by gradient high performance liquid chromatography with two Ultrasphere Ocyl (C8) columns connected in tandem. More than 20 CMs were resolved. The quantities of major CMs produced by 45 mg of microsomal proteins were established by comparing peak areas with known concentrations of authentic CM standards. These major CMs included AM1, AM9, AM19, AM4N, AM1c and the aldehydic isomers (AM1cAL plus AM1AL). Other CMs that were not quantified included AM14N, AM4N9, AM1A, AM1c9, and AM1D1. Several CMs remained to be identified. All CMs were detected by radioimmunoassay using a non-specific CsA antiserum. The purity of the CMs were confirmed by fast atomic bombardment mass spectrometry. Similar findings were observed when erythromycin or trolandomycin was used to induce the hepatic microsomal enzymes. The procedure used to generate CMs was simple. With the enzyme fraction derived from one rabbit liver, 90 to 100 mg of CMs can be obtained. In this study, the metabolite patterns of CsA produced by rabbit liver microsomes were shown to resemble those observed for humans. These results indicate the possibility of using rabbit models to predict CsA biotransformation in man. The CMs generated by this enzyme system can be used to acquire information relevant to the situation in man.

Animals↗
PubMed1995

The orphan receptor hepatic nuclear factor 4 functions as a transcriptional activator for tissue-specific and hypoxia-specific erythropoietin gene expression and is antagonized by EAR3/COUP-TF1.

The erythropoietin (Epo) gene is regulated by hypoxia-inducible cis-acting elements in the promoter and in a 3' enhancer, both of which contain consensus hexanucleotide hormone receptor response elements which are important for function. A group of 11 orphan nuclear receptors, transcribed and translated in vitro, were screened by the electrophoretic mobility shift assay. Of these, hepatic nuclear factor 4 (HNF-4), TR2-11, ROR alpha 1, and EAR3/COUP-TF1 bound specifically to the response elements in the Epo promoter and enhancer and, except for ROR alpha 1, formed DNA-protein complexes that had mobilities similar to those observed in nuclear extracts of the Epo-producing cell line Hep3B. Moreover, both anti-HNF-4 and anti-COUP antibodies were able to supershift complexes in Hep3B nuclear extracts. Like Epo, HNF-4 is expressed in kidney, liver, and Hep3B cells but not in HeLa cells. Transfection of a plasmid expressing HNF-4 into HeLa cells enabled an eightfold increase in the hypoxic induction of a luciferase reporter construct which contains the minimal Epo enhancer and Epo promoter, provided that the nuclear hormone receptor consensus DNA elements in both the promoter and the enhancer were intact. The augmentation by HNF-4 in HeLa cells could be abrogated by cotransfection with HNF-4 delta C, which retains the DNA binding domain of HNF-4 but lacks the C-terminal activation domain. Moreover, the hypoxia-induced expression of the endogenous Epo gene was significantly inhibited in Hep3B cells stably transfected with HNF-4 delta C. On the other hand, cotransfection of EAR3/COUP-TF1 and the Epo reporter either with HNF-4 into HeLa cells or alone into Hep3B cells suppressed the hypoxia induction of the Epo reporter. These electrophoretic mobility shift assay and functional experiments indicate that HNF-4 plays a critical positive role in the tissue-specific and hypoxia-inducible expression of the Epo gene, whereas the COUP family has a negative modulatory role.

Anaerobiosis↗
PubMed1995

[Study of anti-cerebral thromboembolism injection's activity on experimental arterial thrombosis and its therapeutical mechanism].

Anti-Cerebral Thromboembolism Injection (ACTI), applied intravenously in Cattaneo's animal model of arterial thrombosis, could significantly reduce the weight of thrombus, blood viscosities, plasma TXB2 level and delay the prothrombin time, compared to control group (P < 0.05). From this trial, it was indicated that ACTI's reducing thrombosis action was the result of inhibiting aggregation and releasing of platelets through reducing whole blood viscosities and TXB2 level.

Animals↗
PubMed1995

[An experimental study on the integrated effects of heat-cleaning and detoxifying drugs and fire-purging drugs used in combination].

Heat-cleaning and detoxifying drugs and fire-purging drugs used in combination have been proved able to restore the endotoxin-induced deviant of HCT, blood viscositites, PTT and fibrinolytic activity, and reduce the blood content of both endotoxin and PGE2 in rabbits. In addition, the combination can also decrease the pes swelling percentage in rats as well as evans blue extravasation volume in mice. In practice, however, it should be noted that the bitter and cold qualities of these two kinds of drugs may be harmful to health.

Albumins↗