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1.
Phys Rev E ; 105(4-2): 045003, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35590559

RESUMO

In amorphous solids, plastic flow is prone to localization into shear bands via an avalanche of shear-transformation (ST) rearrangements of constituent atoms or particles. However, such banding instability still remains a lack of direct experimental evidence. Using a real 3D colloidal glass under shear as proof of principle, we study STs' avalanches into shear banding that is controlled by strain rates. We demonstrate that, accompanying the emergent shear banding, the elastic response fields of the system, typical of a quadrupole for shear and a centrosymmetry for dilatation, lose the Eshelby-type spatial symmetry; instead, a strong correlation appears preferentially along the banding direction. By quantifying the fields' spatial decay, we identify an elastic criterion for the shear-banding instability, that is, the strongly correlated length of dilatation is smaller than the full length of shear correlation. Specifically, ST-induced free volume has to be confined within the elastic shear domain of ST so that those STs can self-organize to trigger shear banding. This physical picture is directly visualized by tracing the real-space evolution of local dilatation and ST particles. The present work unites the two classical mechanisms: free volume and STs, for the fundamental understanding of shear banding in amorphous solids.

2.
Zhonghua Yan Ke Za Zhi ; 58(3): 231-235, 2022 Mar 11.
Artigo em Chinês | MEDLINE | ID: mdl-35280035

RESUMO

Multiple sclerosis (MS) is an inflammatory reactive autoimmune disease characterized by diffuse axonal degeneration of the central nervous system. The exact etiology and pathogenesis remain unclear. Optical coherence tomography angiography is a new technology of angiography, which can obtain the images of each layer of blood vessels in the scanning area. It is non-invasive, fast and quantifiable. Observation of retinal vasculopathy may assist in the evaluation of brain diseases. This article reviews the previous research results at home and abroad of ocular blood flow changes observed by optical coherence tomography angiography in MS in recent years, in order to provide reference for the study of the underlying pathogenesis of MS and clinical evaluation.


Assuntos
Esclerose Múltipla , Tomografia de Coerência Óptica , Angiofluoresceinografia/métodos , Humanos , Esclerose Múltipla/diagnóstico por imagem , Tomografia de Coerência Óptica/métodos
3.
Zhonghua Fu Chan Ke Za Zhi ; 51(8): 576-80, 2016 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-27561935

RESUMO

OBJECTIVE: To investigate the factors affecting the vaginal birth after cesarean (VBAC). METHODS: Totaly 298 women who underwent trial of labor after cesarean section (TOLAC) from Jan 2015 to Dec 2015 were recruited from Beijing Obstetrics and Gynecology Hospital, FuXing Hospital, Tongzhou Maternal and Child Health Hospital of Beijing, the Second Affiliated Hospital of Chongqing Medical University and the People's Hospital of Chengyang District of Qingdao. The maternal age, the interval from the last cesarean section, the body mass index (BMI) before pregnancy, the weight gain during pregnancy, the way into labor, the Bishop score before labor, the gestational age and the birth weight of the neonate were recorded in a self-made form. The factors affecting VBAC were analyzed by univariate analysis and multivariable logistic regression. RESULTS: (1)The incidence of VBAC, uterine rupture, postpartum hemorrhage and neonatal asphyxia were 70.5% (210/298), 2.7% (8/298), 9.4% (28/298) and 1.3% (4/298), respectively. No maternal death and perinatal death occurred. (2)The univariate analysis suggested that the maternal age, the BMI before pregnancy, the Bishop score before labor, the labor induction, the gestational age at delivery and the neonatal weight were factors affecting VBAC. The maternal age and the Bishop score before labor were significantly higher in the VBAC group than in the unsuccessful TOLAC group(P<0.05). While the BMI before pregnancy, the induction rate, the gestational weeks at delivery and the birth weight of the neonate were significantly lower in the VBAC group than in the unsuccessful TOLAC group (P<0.05). Multivariable logistic regression analysis showed that successful VBAC was affected by the maternal age, the BMI before pregnancy, the Bishop score before labor and the birth weight of the neonates(P<0.05). CONCLUSION: The maternal age, the BMI before pregnancy, the Bishop score before labor and the birth weight of neonate are the main factors affecting VBAC.


Assuntos
Índice de Massa Corporal , Cesárea/estatística & dados numéricos , Idade Materna , Prova de Trabalho de Parto , Nascimento Vaginal Após Cesárea/estatística & dados numéricos , Peso ao Nascer , China/epidemiologia , Feminino , Idade Gestacional , Humanos , Recém-Nascido , Trabalho de Parto Induzido , Modelos Logísticos , Obesidade/complicações , Hemorragia Pós-Parto , Gravidez , Resultado da Gravidez , Estudos Retrospectivos , Ruptura Uterina , Nascimento Vaginal Após Cesárea/efeitos adversos , Aumento de Peso , Adulto Jovem
4.
Zhonghua Yi Xue Za Zhi ; 96(31): 2502-6, 2016 Aug 16.
Artigo em Chinês | MEDLINE | ID: mdl-27562051

RESUMO

OBJECTIVE: To investigate the effect and possible mechanisms of olmesartan medoxomil on atherosclerosis of apolipoprotein E knockout (ApoE(-/-)) mice. METHODS: Sixteen 6-week-old male ApoE(-/-) mice were randomly divided into atherosclerosis model group fed with high fat diet, and olmesartan medoxomil intervention group fed with high fat diet and olmesartan medoxomil (10 mg·kg(-1)·day(-1,) per gavage). Eight C57BL/6 mice were fed with normal diet, and treated for 12 weeks.The blood pressure and the serum level of lipid were detected; the aorta were removed, oil red staining for plaque area, Hematoxylin and eosin (HE) staining for plaque morphology, Elastica van Gieson (EVG) staining for elastin, and picrosirius red (PSR) for collagen respectively; and the expression of cathepsin S (Cat S), smooth action protein (ASMA) and macrophage surface molecule-3 (Mac-3) were detected by immunohistochemisty analysis. RESULTS: Serum cholesterol and low density lipoprotein levels were significantly higher in atherosclerosis model group than in control group[(15.08±1.64) vs (2.06±0.15) mmol/L, (15.60±1.05) vs (0.00±0.00) mmol/L] (all P<0.01), while triglyceride level was similar between the two group.In contrast to model group, the mice in intervention group showed no statistical difference in blood pressure and plasma lipid levels, while the plaque areas in the aorta were significantly decreased (P<0.05) as well as the expression of Cat S and Mac-3[(2.4±1.2) vs (8.8±3.2)%, (2.2±1.2) vs (7.2±2.8)%] (all P<0.01). In addition, the elastin levels, collagen contents, and the expression of ASMA remained significantly higher compared with model group (P<0.05). CONCLUSION: Olmesartan medoxomil could slow down the atherosclerosis process, the possible mechanism was implicated with the suppression of Cat S and decreased inflammatory responses alongside the increased elastin and collagen contents.


Assuntos
Aterosclerose , Animais , Aorta , Apolipoproteínas E , Catepsinas , Dieta Hiperlipídica , Lipídeos , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Olmesartana Medoxomila , Placa Aterosclerótica
5.
Eur Rev Med Pharmacol Sci ; 20(24): 5041-5048, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-28051268

RESUMO

OBJECTIVE: To evaluate the immune activity of bone marrow mesenchymal stem cells (BMSCs), and explore the biological characteristics and capabilities of BMSCs and the potential to be differentiated into neuronal cells in vitro. MATERIALS AND METHODS: The BMSCs were isolated and proliferated in vitro to generate the xenogeneic mixed lymphocyte reaction. Moreover, peripheral BMSCs (pBMSCs) were added according to different ratios, which methods were stated as follows: 1: Dulbecco's Modified Eagle Medium (DMEM) + 10% Fetal Bovine Serum (FBS) + 1 µmol/L all-trans-retinoic acid (ATRA) + 20 µg/L basic fibroblast growth factor (bFGF) + 20 µg/L epidermal growth factor (EGF); 2: DMEM + 2% dimethyl sulfoxide (DMSO) + 100 µmol/L butylated hydroxyanisole (BHA). The immunofluorescence and immunohistochemical staining were finally used to evaluate the differentiation capabilities of human BMSCs (hBMSCs) induced in neuronal cells. RESULTS: hBMSCs inhibited the lymphocyte proliferation in the mixed lymphocyte reaction (MLR) system at a proportional inhibition rate with additional numbers of stem cells. At hour 2 after culture with method 1, the plasma of hBMSCs shrank to nuclei and perinuclear bodies and was visualized under the light microscope. At hours 3-5, most of the hBMSCs formed neuron-like cells with total cell number unchanged. Afterward, the hBMSCs turned into bipolar or multipolar shaped cells and interconnected into a large network at Day 3. With immunofluorescence and immunohistochemical staining, 60-70% of the hBMSCs showed neurospecific enolase (NSE) positive and 45-50% glial fibrillary acidic protein (GFAP) positive while the Nestin-positive cells decreased to 3.4%. However, when cultured 2 hours with method 2, the most of the hBMSCs formed bipolar or multipolar shaped cells, then died after 48 hours. 40-50% NSE and 35-40% GFAP were positively expressed. Significantly, the rate of Nestin-positive cells decreased from 63% to 1.6% from hour 2 after culture to hour 48. CONCLUSIONS: hBMSCs may be effective for cell therapy and tissue engineering for the capability of differentiating into neuronal-like cells, as well as the capability of inhibiting lymphocyte proliferation in MLR system.


Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Proliferação de Células , Células Cultivadas , Humanos , Neurônios , Células-Tronco/citologia , Tretinoína
6.
J Ethnopharmacol ; 169: 287-94, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25910534

RESUMO

AIM: Clematis terniflora DC. has been widely used as a traditional Chinese medicine for the treatment of tonsillitis, rheumatoid arthritis, and prostatitis. Despite its widespread use in China, there are currently no studies systematically examined its therapeutic effects and mechanism of action. As such, the present study was conducted to evaluate the anti-inflammatory, antinociceptive, and immunomodulatory effects of C. terniflora DC. using rodent and cellular models. METHODS: The anti-inflammatory properties of the 70% ethanol eluted fraction of the 70% ethanol extract of C. terniflora DC. (EECTD) were evaluated using the xylene-induced ear swelling test, the carrageenan-induced edema model, and the cotton pellet granuloma method. Its antinociceptive activities were determined using both the acetic acid-induced writhing test and the hot plate assay. In parallel, we conducted an in vitro assay in LPS-induced RAW264.7 cells to examine the anti-inflammatory effects of EECTD and its purified form, aurantiamide acetate (AA) on inhibition of nitric oxide (NO) and prostaglandin E2 (PGE2) release. RESULTS: EECTD (300mg/kg) significantly reduced the number of writhing, extended the pain response latency, and suppressed xylene-induced ear swelling. Each EECTD treatment group also had significant inhibition of cotton granulation formation in addition to reduced carrageenan-induced paw edema. EECTD was also shown to alleviate signs of inflammation in histopathological paw sections. However, it had a less noticeable effect on mouse ear swelling in the delayed type hypersensitivity test. A purified compound was isolated from EECTD and its structure was identified as AA. In vitro experimental results showed that both EECTD and AA were able to significantly inhibit the release of pro-inflammatory cytokines NO and PGE2 on LPS-induced RAW264.7 cells. CONCLUSION: These results suggest that EECTD has significant anti-inflammatory and antinociceptive activities, partially related to one of the active substances identified as AA. We hypothesize that these effects are related to its ability to inhibit the production of cytokines NO and PGE2. However, further work will be needed to determine its exact mechanism of action.


Assuntos
Analgésicos/farmacologia , Anti-Inflamatórios/farmacologia , Clematis/química , Dipeptídeos/farmacologia , Imunomodulação/efeitos dos fármacos , Dor/tratamento farmacológico , Fitoterapia , Analgésicos/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Células Cultivadas , Dinoprostona/metabolismo , Dipeptídeos/isolamento & purificação , Dipeptídeos/uso terapêutico , Modelos Animais de Doenças , Edema/tratamento farmacológico , Feminino , Masculino , Camundongos , Óxido Nítrico/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia
7.
Cell Death Differ ; 22(10): 1590-604, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25633197

RESUMO

Colorectal carcinoma (CRC) is characterized by unlimited proliferation and suppression of apoptosis, selective advantages for tumor survival, and chemoresistance. Lipopolysaccharide (LPS) signaling is involved in both epithelial homeostasis and tumorigenesis, but the relative roles had by LPS receptor subunits CD14 and Toll-like receptor 4 (TLR4) are poorly understood. Our study showed that normal human colonocytes were CD14(+)TLR4(-), whereas cancerous tissues were CD14(+)TLR4(+), by immunofluorescent staining. Using a chemical-induced CRC model, increased epithelial apoptosis and decreased tumor multiplicity and sizes were observed in TLR4-mutant mice compared with wild-type (WT) mice with CD14(+)TLR4(+) colonocytes. WT mice intracolonically administered a TLR4 antagonist displayed tumor reduction associated with enhanced apoptosis in cancerous tissues. Mucosa-associated LPS content was elevated in response to CRC induction. Epithelial apoptosis induced by LPS hypersensitivity in TLR4-mutant mice was prevented by intracolonic administration of neutralizing anti-CD14. Moreover, LPS-induced apoptosis was observed in primary colonic organoid cultures derived from TLR4 mutant but not WT murine crypts. Gene silencing of TLR4 increased cell apoptosis in WT organoids, whereas knockdown of CD14 ablated cell death in TLR4-mutant organoids. In vitro studies showed that LPS challenge caused apoptosis in Caco-2 cells (CD14(+)TLR4(-)) in a CD14-, phosphatidylcholine-specific phospholipase C-, sphingomyelinase-, and protein kinase C-ζ-dependent manner. Conversely, expression of functional but not mutant TLR4 (Asp299Gly, Thr399Ile, and Pro714His) rescued cells from LPS/CD14-induced apoptosis. In summary, CD14-mediated lipid signaling induced epithelial apoptosis, whereas TLR4 antagonistically promoted cell survival and cancer development. Our findings indicate that dysfunction in the CD14/TLR4 antagonism may contribute to normal epithelial transition to carcinogenesis, and provide novel strategies for intervention against colorectal cancer.


Assuntos
Apoptose , Carcinogênese , Neoplasias Colorretais/metabolismo , Células Epiteliais/fisiologia , Receptores de Lipopolissacarídeos/fisiologia , Receptor 4 Toll-Like/fisiologia , Animais , Células CACO-2 , Colo/metabolismo , Colo/fisiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células Epiteliais/metabolismo , Humanos , Camundongos , Transdução de Sinais
8.
Sci Total Environ ; 466-467: 777-87, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23973544

RESUMO

Since the 1960s, the marshes in the Sanjiang Plain, Northeast China, which are an important reservoir for dissolved carbon, have undergone long-term reclamation to farmland, resulting in elevated marsh loss and degradation on a large scale. This study compared the concentrations of dissolved carbon, as well as the chemical characteristics of dissolved organic carbon (DOC), in natural marshes, a degraded marsh, and drainage ditches sampled during the growing seasons between 2008 and 2010 to clarify the temporal-spatial variability of the dissolved carbon in the fluvial system influenced by the long-term reclamation. The results show that the average concentrations of total dissolved carbon (TDC) and DOC are considerably greater in the natural marshes than in the degraded marsh and drainage ditches. The average DOC concentration for the natural marshes, approximately 35.53 ± 5.15 mg L(-1), is approximately 2.39 times that in the degraded marsh (14.84 ± 4.21 mg L(-1)) and 2.77 times the average value in the ditches (12.84 ± 4.49 mg L(-1)). The dissolved inorganic carbon (DIC) exhibits increased trends in the drainage ditches compared with the natural marshes, whereas the hydrophobic fraction of DOC is present at lower concentrations in the degraded marsh and ditches. Fluorescence indices also indicate that the DOC in the degraded marsh and ditches has a simpler humification structure. In total, the long-term reclamation has led to great variability in the DOC concentration and chemical characteristics in the fluvial system. Changes in the DOC production potential and hydrological regimes due to sustained reclamation are deemed the predominant causes of this effect. The continuously decreased DOC concentration and high variability of DOC in the surface fluvial systems are inevitable if reclamation continues in the Sanjiang Plain. More importantly, the presence of tyrosine and tryptophan-like substances in the ditches indicates that there has been extensive agricultural organic pollution in the fluvial systems. This pollution could lead to more unexpected impacts on the downriver aquatic system. To avoid a destructive ecological crisis in the future, adjustment of the reclamation policy and agricultural management measures in the Sanjiang Plain is urgently needed.

9.
Artigo em Chinês | MEDLINE | ID: mdl-21207703

RESUMO

AIM: To investigate whether renal ischemic preconditioning could protect myocardium from ischemic-reperfusion injury. METHODS: Thirty-two adult white rabbits were randomly divided into four groups: group sham operation (SO), myocardial ischemic-reperfusion (I/R), classical ischemic preconditioning (CIPC) and renal ischemic preconditioning (RIPC). Changes of left ventricular functions, myocardial infarct size, myocardial ultrastructure and cardiac arrhythmia were observed. RESULTS: RIPC and CIPC could reduce the myocardial infarct size and the attack rate of arrhythmia (P < 0.01 vs I/R); improve the left ventricular functions (P < 0.01 vs I/R) and minimized the ultrastructure injury of I/R myocardium. CONCLUSION: The RIPC might elicit a myocardial protection against ischemic-reperfusion injury, which is similar to the effect of CIPC.


Assuntos
Precondicionamento Isquêmico/métodos , Rim/irrigação sanguínea , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Coelhos , Distribuição Aleatória
10.
IEEE Trans Neural Netw ; 8(6): 1328-37, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-18255735

RESUMO

This study presents the development and industrial application of an integrated neural system in coating weight control for a modern hot dip coating line (HDCL) in a steel mill. The neural system consists of two multilayered feedforward neural networks and a neural adaptive controller. They perform coating weight real-time prediction, feedforward control (FFC), and adaptive feedback control (FBC), respectively. The production line analysis, neural system architecture, learning, associative memories, generalization and real-time applications are addressed in this paper. This integrated neural system has been successfully implemented and applied to an HDCL at Burns Harbor Division, Bethlehem Steel Co., Chesterton, IN. The industrial application results have shown significant improvements in reduction of coating weight transitional footage, variation of the error between the target and actual coating weight, and the coating material used. Some practical aspects for applying a neural system to industrial control are discussed as concluding remarks.

11.
Ann Nucl Med ; 10(1): 13-8, 1996 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8814716

RESUMO

Tc-99m MIBI myocardial SPECT has shown promise for evaluation of coronary artery disease. But its role in predicting myocardial viability is still under investigation. The purpose of this study was to evaluate the value of Tc-99m MIBI myocardial SPECT during isosorbide dinitrate (ISDN) infusion in the assessment of myocardial viability. Thirty-seven patients with previous myocardial infarction (the infarct age ranged from < or = 30 days to 900 days) were studied, of them 13 patients had Tc-99m MIBI studies before and after coronary artery bypass grafting (CABG). The results showed that out of 134 segments with hypoperfusion at resting SPECT, 56 segments (41.8%) had an increase in Tc-99m MIBI uptake during ISDN infusion. Among them, 17 segments (30.4%) were normalized, 6 segments (10.7%) were significantly improved and 33 segments (58.9%) were improved. The degree of improvement in perfusion was related to the age of the myocardial infarction. In 13 patients with CABG, of 31 segments with improvement in perfusion post CABG, 25 segments (80.6%) showed perfusion improvement during ISDN infusion, and of 28 segments with improved wall motion post CABG, 23 segments (82.1%) showed improvement in perfusion during ISDN infusion. Tc-99m MIBI SPECT during ISDN infusion may therefore be a useful approach for assessing myocardial viability.


Assuntos
Infarto do Miocárdio/diagnóstico por imagem , Tecnécio Tc 99m Sestamibi , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Adulto , Idoso , Ponte de Artéria Coronária , Feminino , Coração/diagnóstico por imagem , Humanos , Infusões Parenterais , Dinitrato de Isossorbida/administração & dosagem , Masculino , Pessoa de Meia-Idade , Fatores de Tempo
12.
Biochemistry ; 33(17): 5056-62, 1994 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-8172881

RESUMO

We investigated the effects of the nonionic detergent octaethylene glycol monododecyl ether (C12E8) on the sarcoplasmic reticulum calcium pump in cardiac microsomes in view of its specific effects on different ATP-accelerated steps in the catalytic cycle of the Ca-ATPase in leaky fast skeletal muscle microsomes. At low concentrations of MgATP2- (< 2.5 microM), a nonsolubilizing concentration of added C12E8 (15 microM) increased apparent Vmax(MgATP) of oxalate-facilitated calcium uptake associated with MgATP2- binding to the high affinity catalytic site. An ATP induced acceleration of calcium uptake, attributable to regulatory nucleotide binding, was seen between 2 and 3 microM MgATP2- in both C12E8-treated and control microsomes. These effects of C12E8 are similar to those seen previously with trypsin treatment of microsomes [Lu, Y.-Z., Xu, Z.-C., & Kirchberger, M.A. (1993) Biochemistry 32, 3105-3111]. However, at a saturating Ca2+ between 3 and 10 microM MgATP2-, C12E8 produced a greater reduction in the magnitude of the ATP-induced acceleration of calcium uptake seen with trypsin. At 1 mM MgATP2-, C12E8 and trypsin as well as protein kinase A-catalyzed microsomal phosphorylation all increased the Ca2+ affinity of the pump, but only the latter two treatments significantly increased apparent Vmax(Ca). In fact in trypsin-treated and phosphorylated microsomes, C12E8 reduced Vmax(Ca) to close to the control values; it reduced Vmax(Ca) only slightly in control microsomes. Under our experimental conditions, comparable effects of 15 microM C12E8 on calcium uptake were absent in fast skeletal muscle microsomes, which lack phospholamban.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Cálcio/metabolismo , Detergentes/farmacologia , Microssomos/metabolismo , Miocárdio/metabolismo , Polietilenoglicóis/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Bovinos , Proteínas Quinases Dependentes de AMP Cíclico/isolamento & purificação , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cinética , Microssomos/efeitos dos fármacos , Miocárdio/enzimologia , Fosforilação , Coelhos , Tripsina/farmacologia
13.
Science ; 261(5119): 334-7, 1993 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-17836844

RESUMO

Pulsed laser ablation of graphite targets combined with an intense, atomic nitrogen source has been used to prepare C-N thin film materials. The average nitrogen content in the films was systematically varied by controlling atomic nitrogen flux. Rutherford backscattering measurements show that up to 40 percent nitrogen can be incorporated on average into these solids under the present reaction conditions. Photoelectron spectroscopy further indicates that carbon and nitrogen form an unpolarized covalent bond in these C-N materials. Qualitative tests indicate that the C-N solids are thermally robust and hard. In addition, strong electron diffraction is observed from crystallites within the films. Notably, analysis of these diffraction data show that the only viable structure for the C-N crystallites is that of beta-C(3)N(4), a material predicted theoretically to exhibit superhardness. The experimental synthesis of this new C-N material offers exciting prospects for both basic research and engineering applications.

14.
Biochemistry ; 32(12): 3105-11, 1993 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-8384487

RESUMO

The purpose of this study was to investigate the functional relationship between phospholamban and the nucleotide site of the calcium pump protein of the cardiac sarcoplasmic reticulum. We used control and trypsin-treated cardiac microsomes in which cleavage of the inhibitory cytoplasmic domain of phospholamban is associated with an activation of the calcium pump similar to that produced by protein kinase A catalyzed phospholamban phosphorylation. Phenylglyoxal was shown to inactivate the calcium pump in a pseudo-first-order reaction by binding to a single Arg at the nucleotide binding site. No differences upon trypsin treatment of microsomes were observed in the kinetics of phenylglyoxal inactivation or the ability of millimolar ATP to protect against inactivation. In subsequent kinetic studies, Ca-uptake rates measured at saturating Ca2+ and 5 microM-1 mM MgATP2- were increased 15-32% by trypsin treatment in each of three different microsome preparations. Double-reciprocal plots of the data showed marked downward curvature indicating an acceleratory effect associated with ligand binding to a lower affinity site. At 0.32 microM Ca2+, Ca-uptake rates were lower than at 11 microM Ca2+ but were stimulated to a greater extent by trypsin treatment; control microsomes showed reduced evidence of apparent negative cooperativity. At 0-2 microM MgATP2- and saturating Ca2+, there was a 50% increase in Vmax(app) when the Hill coefficient (N) was 1. At 0-10 microM MgATP2-, second-site binding was evident. At both 0-10 microM and 5 microM-1 mM MgATP2-, trypsin-treated microsomes showed greater activation of Ca uptake attributable to second-site binding than did control microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Trifosfato de Adenosina/farmacologia , Proteínas de Ligação ao Cálcio/farmacologia , ATPases Transportadoras de Cálcio , Cálcio/metabolismo , Miocárdio/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Arginina/metabolismo , Sítios de Ligação , ATPase de Ca(2+) e Mg(2+)/metabolismo , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Cães , Cinética , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Miocárdio/ultraestrutura , Fenilglioxal/metabolismo , Fenilglioxal/farmacologia , Retículo Sarcoplasmático/efeitos dos fármacos , Tripsina/farmacologia
16.
Biochemistry ; 23(26): 6870-6, 1984 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-6085013

RESUMO

Thymidylate synthase has been purified 1700-fold from fetal pig livers by using chromatography on Affigel-Blue, DEAE-52, and hydroxylapatite. Steady-state kinetic measurements indicate that catalysis proceeds via an ordered sequential mechanism. When 5,10-methylenetetrahydro-pteroylmonoglutamate (CH2-H4PteGlu1) is used as the substrate, dUMP is bound prior to CH2-H4PTeGlu1, and 7,8-dihydropteroylmonoglutamate (H2PteGlu1) is released prior to dTMP. Pteroylpolyglutamates (PteGlun) are inhibitors of thymidylate synthase activity and are competitive with respect to CH2-H4PteGlu1 and uncompetitive with respect to dUMP. Inhibition constants (Ki values), which correspond to dissociation constants for the dissociation of PteGlun from the enzyme-dUMP-PteGlun ternary complex, have been determined for PteGlun derivatives with one to seven glutamyl residues: PteGlu1, 10 microM; PteGlu2, 0.3 microM; PteGlu3, 0.2 microM; PteGlu4, 0.06 microM; PteGlu5, 0.10 microM; PteGlu6, 0.12 microM; PteGlu7, 0.15 microM. Thus, thymidylate synthase from fetal pig liver preferentially binds pteroylpolyglutamates with four glutamyl residues, but derivatives with two to seven glutamyl residues all bind at least 30-fold more tightly than the monoglutamate. When CH2-H4PteGlu4 is used as the one carbon donor for thymidylate biosynthesis, the order of substrate binding and product release is reversed, with binding of CH2-H4PteGlu4 preceding that of dUMP and release of dTMP preceding release of H2PteGlu4. Vmax and Km values for dUMP and CH2-H4PteGlun show relatively little change as the polyglutamate chain length of the substrate is varied.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Fígado/enzimologia , Metiltransferases/metabolismo , Timidilato Sintase/metabolismo , Animais , Feto/enzimologia , Ácido Fólico , Cinética , Ácido Poliglutâmico , Especificidade por Substrato , Suínos
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