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ß-delayed one-proton emissions of ^{22}Si, the lightest nucleus with an isospin projection T_{z}=-3, are studied with a silicon array surrounded by high-purity germanium detectors. Properties of ß-decay branches and the reduced transition probabilities for the transitions to the low-lying states of ^{22}Al are determined. Compared to the mirror ß decay of ^{22}O, the largest value of mirror asymmetry in low-lying states by far, with δ=209(96), is found in the transition to the first 1^{+} excited state. Shell-model calculation with isospin-nonconserving forces, including the T=1, J=2, 3 interaction related to the s_{1/2} orbit that introduces explicitly the isospin-symmetry breaking force and describes the loosely bound nature of the wave functions of the s_{1/2} orbit, can reproduce the observed data well and consistently explain the observation that a large δ value occurs for the first but not for the second 1^{+} excited state of ^{22}Al. Our results, while supporting the proton-halo structure in ^{22}Al, might provide another means to identify halo nuclei.
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Objective: To explore the feasibility of dynamic-enhanced magnetic resonance imaging (DCE-MRI) and blood oxygen level-dependent MRI (BOLD-MRI) in assessing the hemodynamics and tumor aggressiveness during treatment. Methods: The colon cancer xenograft model was established in BALB/C nude mice with HCT116 cell line. Sixteen nude mice were randomly divided into treatment and control groups (aged 6 to 8 weeks, weighted 15 to 18 g, Certificate No. 11400700325797), which were treated with bevacizumab and saline by intraperitoneal injection on the 1st, 4th, 7th, 10th and 13th day. DCE-MRI and BOLD-MRI were performed before and on the 3th, 6th, 9th, 12th, and 15th day after treatment. The vascular maturity and microenvironment hypoxia were confirmed by pathology. Results: The tumor volume of treatment group was significantly smaller than that of control group after 15 days ((712±43) vs (1 051±112) mm(3),P<0.01).The measurements of K(trans) were (0.135±0.005),(0.147±0.006),(0.175±0.009),(0.161±0.006), (0.140±0.005),(0.116±0.008)/min (F=81.386, P<0.01); K(ep) were (0.788±0.030),(0.804±0.036),(0.983±0.059), (1.105±0.091),(0.840±0.047),(0.786±0.041)/min(F=45.901,P<0.01);Ve were (0.652±0.006), (0.559±0.026), (0.466±0.016), (0.286±0.027), (0.363±0.020), (0.246±0.033) (F=384.290, P<0.01) and R2* values were (24.813±0.961), (24.675±1.070), (21.425±1.371), (17.850±0.885), (24.613±0.640), (27.013±0.734)/s (F=89.323, P<0.01) showed different trends with time in the treatment group, and the differences were statistically significant. The K(trans) values and tumor vessel maturity index (VMI) were higher than baseline values during 3-12 d after treatment. CD31 positive staining rate and VMI had the strongest correlations with K(trans) values (r=0.854 and 0.795), followed by AUC(180) (r=0.750 and 0.808), Ve (r=0.744 and 0.712) and K(ep) values (r=0.729 and 0.758), all P<0.05. R2* value positively correlated with the positive staining rate of HIF-1α and fibronectin (r=0.810 and 0.816), all P<0.05. Conclusion: DCE-MRI and BOLD-MRI are adequate to observe the tumor perfusion and hypoxia during anti-vascular treatment, and the R2* value can predict the tumor metastatic potential during the process of vascular normalization.
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Meios de Contraste , Imageamento por Ressonância Magnética , Animais , Xenoenxertos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos NusRESUMO
Objective: To evaluate the feasibility of intravoxel incoherent motion diffusion-weighted magnetic resonance imaging (IVIM-DWI MRI) in the evaluation of tumor vascular normalization in a mouse model of colorectal cancer induced by recombinant human endostatin (rhES). Methods: The CT26 colorectal cancer xenograft model of BALB/c mice were established and divided into rhES group and control group, with 20 mice in each group. The mice of rhES group were intravenously injected with rhES 5 mg·kg(-1)·d(-1) once daily for 12 days, while the mice of the control group were intravenously injected with the same volume of 0.9% saline. 5 mice of rhES group and control group were randomly selected to perform IVIM-DWI MRI as following times: before treatment and four, eight, twelve days after treatment. The parameters of IVIM-DWI were recorded, including true diffusion coefficient(D), pseudo-diffusion coefficient (D(*)) and perfusion fraction (f). Meanwhile, microvessel density (MVD), pericyte coverage and tumor perfusion in tumor tissues were detected by immunofluorescence, respectively. Results: The tumor volumes of control group and rhES group before treatment were (154.42±24.65) mm(3) and (174.24±28.27)mm(3,) respectively, without statistically significant difference (P=0.440). From day 2 to day 12 after treatment, the tumor volume of rhES group was significantly smaller than that of control group (all P<0.05). There were no statistical significances of D value between the rhES group and control group before and after treatment (all P>0.05). The D(*) values of the rhES group were (10.940±2.834)×10(-3)mm(2)/s and (12.940±2.801)×10(-3)mm(2)/s in day 4 and 8 after treatment respectively, significantly higher than (6.980±1.554)×10(-3)mm(2)/s and (7.898±1.603)×10(-3)mm(2)/s of control group (P<0.05). Moreover, compared with control group, the D(*) value of rhES group was significantly lower in day 12 (6.848±1.460)×10(-3)mm(2)/s vs (9.950±2.596)×10(-3)mm(2)/s, (P<0.05). The f value of rhES group in day 8 was (0.226±0.021)%, significantly higher than (0.178±0.016)% of control group (P<0.01). The MVD of rhES group was significantly lower than that of control group (P<0.05), while the pericyte coverage and tumor perfusion of rhES group were significantly higher than those of control group in day 4 and 8 after treatment (all P<0.05). In addition, we found D(*) value of IVIM-DWI in rhES group was significantly related with MVD, pericyte coverage and tumor perfusion (r=-0.354, r=0.555, r=0.559, all P<0.05). Meanwhile, the f value in rhES group was also significantly related with MVD, pericyte coverage and tumor perfusion (r=-0.391, r=0.538, r=0.315, all P<0.05). Conclusions: IVIM-DWI MRI can effectively evaluate the vascular normalization in rhES-induced CT26 colorectal tumor.The parameters D(*) and f are closely related to intratumorally microvessel density, pericyte coverage and perfusion, which can effectively monitor the occurrence of tumor vascular normalization time.
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Neoplasias Colorretais/diagnóstico por imagem , Imagem de Difusão por Ressonância Magnética/métodos , Neovascularização Patológica/diagnóstico por imagem , Animais , Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/induzido quimicamente , Modelos Animais de Doenças , Endostatinas/toxicidade , Estudos de Viabilidade , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/toxicidadeRESUMO
BACKGROUND: Aberrant functional connectivity within the default network is generally assumed to be involved in the pathophysiology of obsessive compulsive disorder (OCD); however, the genetic risk of default network connectivity in OCD remains largely unknown. METHOD: Here, we systematically investigated default network connectivity in 15 OCD patients, 15 paired unaffected siblings and 28 healthy controls. We sought to examine the profiles of default network connectivity in OCD patients and their siblings, exploring the correlation between abnormal default network connectivity and genetic risk for this population. RESULTS: Compared with healthy controls, OCD patients exhibited reduced strength of default network functional connectivity with the posterior cingulate cortex (PCC), and increased functional connectivity in the right inferior frontal lobe, insula, superior parietal cortex and superior temporal cortex, while their unaffected first-degree siblings only showed reduced local connectivity in the PCC. CONCLUSIONS: These findings suggest that the disruptions of default network functional connectivity might be associated with family history of OCD. The decreased default network connectivity in both OCD patients and their unaffected siblings may serve as a potential marker of OCD.
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Córtex Cerebral/fisiopatologia , Conectoma/métodos , Transtorno Obsessivo-Compulsivo/fisiopatologia , Adulto , Biomarcadores , Feminino , Predisposição Genética para Doença , Humanos , Imageamento por Ressonância Magnética , Masculino , Transtorno Obsessivo-Compulsivo/genética , IrmãosRESUMO
In this paper, the application of the strong-form finite block method (FBM) to three-dimensional fracture analysis with functionally graded materials is presented. The main idea of the strong-form FBM is that it transforms the arbitrary physical domain into a normalized domain and utilizes the direct collocation method to form a linear system. Using the mapping technique, partial differential matrices of any order can be constructed directly. Frameworks of the strong-form FBM for three-dimensional problems based on Lagrange polynomial interpolation and Chebyshev polynomial interpolation were developed. As the dominant parameters in linear elastic fracture mechanics, the stress intensity factors with functionally graded materials (FGMs) were determined according to the crack opening displacement criteria. Several numerical examples are presented using a few blocks to demonstrate the accuracy and efficiency of the strong-form FBM.
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To evaluate the stability of salvianolic acid B (Sal B) under ultrasound-assisted extraction in the pharmaceutical industry, degradation of Sal B under ultrasonic irradiation was investigated as the function of buffer concentration, pH, and temperature. With regard to Sal-B concentration, a first-order degradation process was determined, with 10% change in assay from its initial concentration as t90=4.81h, under maximum stability acidic conditions (pH 2.0) and at 25°C. The logkpH-pH profile described by specific acid-base catalysis and water molecules supported the experimental results. Liquid chromatography-mass spectrometry (LC-MS) analyses revealed 7 major degradation products whose structures were characterized by electrospray ionization/mass spectrometry. A primary degradation pathway involved cleavage of the ester bond and ring-opening of benzofuran in Sal B was proposed. The complete degradation pathway of Sal B was also proposed. Results showed that ultrasonic irradiation leads to degradation of Sal B in aqueous solution.
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Benzofuranos/química , Soluções/química , Água/química , Cromatografia Líquida/métodos , Estabilidade de Medicamentos , Concentração de Íons de Hidrogênio , Espectrometria de Massas por Ionização por Electrospray/métodos , Temperatura , Ultrassom/métodosRESUMO
The effect of norepinephrine (NE) upon human chorionic gonadotrophin (hCG) production by 6-8 week gestation placental explants has been investigated. NE (5 micrograms/ml) enhanced hCG secretion significantly from the second day of treatment. The stimulatory effect of NE on hCG secretion could be abolished by the alpha 1-receptor specific antagonist prazosin (10(-4) M) and partly diminished by the beta 1-receptor specific antagonist atenolol (10(-4) M), but was not influenced by the alpha 2-receptor specific antagonist yohimbine (10(-4) M). The involvement of the alpha-receptor in the regulation of hCG secretion was further confirmed by addition of the alpha-receptor agonist clonidine (10(-6) M) which had a similar stimulatory effect on hCG release but the effect was antagonized by both prazosin and yohimbine. Further study showed that NE induced a significant increase in cyclic adenosine monophosphate (cAMP) production by trophoblast tissue. Cyclic AMP secretion in the NE-treated group was fivefold higher than that of the control group. Both the protein kinase C (PKC) specific activator 1-deoyl-2-acetyl-sn-glycerol (OAG) and the PKC non-specific activator phorbol-12-myristate-13-acetate (PMA) had a stimulatory effect on hCG secretion, while the PKC inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H7) diminished the hCG secretion stimulated by NE. The effect of NE was blocked by the voltage-dependent calcium channel blocker nifedipine but not by the voltage-independent calcium channel blocker gadolinium chloride (GdCl3). On the other hand, anti-gonadotrophin releasing hormone (GnRH) IgG and the GnRH antagonist (D-Phe2, D-Trp6)-GnRH did not influence the stimulatory effect of NE on hCG release. The results indicate that NE regulates hCG production in human first trimester trophoblast tissue. The effect of NE was mainly mediated by alpha 1 and partly by beta 1 receptors. Cyclic AMP, the PKC signal transduction pathway and the voltage-dependent calcium channels were involved in NE action.
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Gonadotropina Coriônica/biossíntese , Norepinefrina/farmacologia , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Atenolol/farmacologia , Cálcio/farmacologia , Gonadotropina Coriônica/metabolismo , Clonidina/farmacologia , AMP Cíclico/biossíntese , Feminino , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Humanos , Técnicas In Vitro , Isoquinolinas/farmacologia , Piperazinas/farmacologia , Prazosina/farmacologia , Gravidez , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Ioimbina/farmacologiaRESUMO
Norepinephrine (NE) was investigated for its effect on progesterone secretion from 6-8 week gestation human trophoblast tissue cultured in serum-free medium. Norepinephrine (5 micrograms/ml) enhanced progesterone secretion significantly on the third day of treatment. The stimulatory effect of NE on progesterone production was abolished by the alpha 1-receptor specific antagonist prazosin (10(-4) M) (P < 0.05), but was not influenced by the alpha 2-receptor specific antagonist yohimbine (10(-4) M) and the beta 1-receptor specific antagonist atenolol (10(-4) M). On the other hand, the alpha-receptor agonist clonidine (10(-6) M) had a similar stimulatory effect on progesterone release but the effect was antagonized by both the alpha 1-antagonist prazosin and the alpha 2-antagonist yohimbine. Further study showed that NE induced a significant increase in cyclic adenosine monophosphate (cAMP) production by trophoblast tissue. Cyclic AMP secretion in the NE treated group was fivefold higher than that of the control group. The effect of NE was blocked by the voltage-dependent calcium channel blocker nifedipine (100 microM) but not by the voltage-independent calcium channel blocker gadolinium chloride (GdCl3) (10 microM). In addition, anti-gonadotropin releasing hormone (GnRH) IgG (5 micrograms/ml) and GnRH antagonist, (D-Phe2, D-Trp6)-GnRH (10(-6) M) did not influence the stimulatory effect of NE on progesterone release. The results indicate that NE regulates progesterone production in human first trimester trophoblast tissue. The effect of NE was mediated by the alpha 1 receptors. Cyclic AMP and voltage-dependent calcium channel were involved in its action.
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Norepinefrina/farmacologia , Progesterona/biossíntese , Trofoblastos/metabolismo , Atenolol/farmacologia , Clonidina/antagonistas & inibidores , Clonidina/farmacologia , Meios de Cultura Livres de Soro , AMP Cíclico/metabolismo , Feminino , Gadolínio/farmacologia , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/farmacologia , Humanos , Nifedipino/farmacologia , Norepinefrina/antagonistas & inibidores , Técnicas de Cultura de Órgãos , Prazosina/farmacologia , Gravidez , Primeiro Trimestre da Gravidez , Estimulação Química , Trofoblastos/efeitos dos fármacos , Ioimbina/farmacologiaRESUMO
The negative inotropic effect of Pra-C was supposed to be related to the blocking of extracellular calcium influx but direct evidence is not known. We, therefore, examined the effects of Pra-C on [Ca2+]i in isolated rat ventricular myocytes using the fluorescent Ca(2+)-indicator Fura-2/AM. It was found that Pra-C inhibited the elevation of [Ca2+]i induced by potassium-depolarization, high extracellular calcium and calcium agonist Bay K 8644 in a dose-dependent manner. At 1.0 mumol.L-1, it decreased the [Ca2+]i at the presence of 75 mmol.L-1 KCl, 10 mol.L-1 CaCl and 3 mumol.L-1 Bay K 8644 by 50, 31 and 42% respectively. No significant effect on ouabain evoked [Ca2+]i increase was found, showing that it does not affect sarcolemmal Na(+)-Ca2+ exchange. These results further indicate that Pra-C may decrease the [Ca2+]i of myocyte by blocking voltage-dependent calcium channels.
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Bloqueadores dos Canais de Cálcio/farmacologia , Cálcio/metabolismo , Cumarínicos/farmacologia , Miocárdio/citologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/antagonistas & inibidores , Animais , Cloreto de Cálcio/antagonistas & inibidores , Células Cultivadas , Relação Dose-Resposta a Droga , Miocárdio/metabolismo , Cloreto de Potássio/antagonistas & inibidores , Ratos , Ratos WistarRESUMO
Clausenamide is a compound isolated from Clausena lansium (lour) with the structure similar to piracetam. Pharmacological experiments showed that clausenamide po 100-200 mg/kg prolonged both the duration of gasping after decapitation and the survival time after sc NaNO2 225 mg/kg, clausenamide at the concentration of 10(-5) mol/L inhibited the contraction of basilar artery caused by 5-HT, PGF2 alpha and arachidonic acid, indicating that clausenamide is a cerebral protective agent. In addition, multiple doses clausenamide were shown to inhibit the liver lipid peroxidation caused by 50% alcohol and increase the GSH-peroxidase activity significantly in rat liver and brain cytosol.
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Artéria Basilar/efeitos dos fármacos , Lactamas/farmacologia , Lignanas , Peroxidação de Lipídeos/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasodilatadores , Animais , Encéfalo/metabolismo , Feminino , Glutationa Peroxidase/metabolismo , Hipóxia Encefálica/prevenção & controle , Fígado/metabolismo , Masculino , Malondialdeído/metabolismo , Camundongos , Microssomos Hepáticos/metabolismo , RatosRESUMO
Isolated mouse tail artery strip was used for the study of alpha 1-, alpha 2-adrenoceptor agonists and antagonists. NA (alpha 1 and alpha 2 agonist) was shown to have greater activity in contracting tail artery. Phenylephrine (alpha 1 agonist) and clonidine (alpha 2 agonist) exhibited the same contractile action but much weaker than NA. Prazosin (alpha 1 antagonist) and yohimbine (alpha 2 antagonist) greatly diminished the contraction induced by phenylephrine and clonidine. These results indicate that mouse tail artery is rich in postsynaptic alpha 1- and alpha 2-adrenoceptor. In addition, mouse tail artery preparation was shown to be a useful tool for screening calcium agonists and antagonists. This model has advantages of being simple and easy to prepare, short equilibrium time and more economic in comparison with the helical strips of isolated rat tail artery.
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Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Animais , Artérias/efeitos dos fármacos , Feminino , Masculino , Camundongos , Ratos , Ratos Endogâmicos , Cauda/irrigação sanguíneaRESUMO
Using fluorescent probe Fura-2/AM, ginsenoside Rb1 (10, 50 and 100 mumol.L-1) was shown to dose-dependently reduce intracellular Ca2+ concentration of freshly dissociated brain cells isolated from newborn rats. It was also found to increase the fluidity of synaptosome membrane impaired by FeSO4-Cystein. Rb1 (10 mumol.L-1) shifted the dose-response curve to the right and decreased the maximal response in isolated mouse tail artery. Rb1 (10 and 100 mumol.L-1) was also found to reduce 5-HT-induced contraction of isolated rat basilar artery. With the whole-cell patch clamp technique, Rb1 (100 mumol.L-1) was shown to have no obvious effect on calcium current and potassium current. However, Rb1 was shown to increase Na(+)-K+ ATPase and Ca(2+)-Mg2+ ATPase activities at low dose in rat cerebral cortical synaptosomes. The results indicate that decrease of [Ca2+]i by Rb1 may be attributed to increase of ATPase activity.
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Encéfalo/metabolismo , ATPase de Ca(2+) e Mg(2+)/metabolismo , Cálcio/metabolismo , Fármacos do Sistema Nervoso Central/farmacologia , Saponinas/farmacologia , Animais , Encéfalo/citologia , Ginsenosídeos , Masculino , Fluidez de Membrana/efeitos dos fármacos , Camundongos , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/metabolismo , Membranas Sinápticas/efeitos dos fármacos , Sinaptossomos/enzimologiaRESUMO
Weaning mice were supplied drinking water containing Rb1 or Rg1 0.125 or 0.25 mg.ml-1 for 4 weeks. Rb1 (28.6 and 56.1 mg.kg-1) and Rg1 (27.4 and 53.9 mg.kg-1) were found to accelerate young mice body and brain development as well as facilitate memory acquisition in step down and step through avoidance response tests. With the technique of quantitative measurement synapses, we found for the first time that Rb1 and Rg1 administration for 4 weeks can increase synapse number in the hippocampal CA3 region of mice. This is the morphological basis for explaining Rb1 and Rg1 induced facilitation of learning and memory.
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Aprendizagem da Esquiva/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Saponinas/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Feminino , Ginsenosídeos , CamundongosRESUMO
The effect of hormones on progesterone secretion by 6-8 week human trophoblast tissue cultured in serum-free medium has been investigated. GnRH at low concentration (10(-10)-10(-8) mol/L) stimulated progesterone secretion, while high dose (10(-6)-10(-5) mol/L) produced inhibitory effect. The progesterone secretion could be significantly decreased by addition of anti-hCG antiserum or monoclonal anti-hCG IgG in a dose- and time-dependent manner. Various concentrations of TRH, PGE2, PGF2 alpha, testosterone and estradiol were found to be ineffective. These data indicate clearly that progesterone production by human trophoblast tissue at early gestation stage is under the modulation of GnRH and hCG.
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Hormônio Liberador de Gonadotropina/fisiologia , Progesterona/metabolismo , Trofoblastos/metabolismo , Animais , Células Cultivadas , Gonadotropina Coriônica/imunologia , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Humanos , Soros Imunes , Gravidez , Primeiro Trimestre da Gravidez , RatosRESUMO
To investigate the relationship between left atrial (LA) size, endothelial dysfunction and different markers of target organ damage (TOD), we measured left atrial diameter (LAD) and endothelial function in hypertensive patients with or without TOD. In this study, 197 patients with hypertension were divided into four groups as follows: no TOD (Group I, n=40), one TOD (Group II, n=76), two TOD (Group III, n=46) and ≥3 TOD (Group IV, n=35). Endothelial function was assessed by endothelium-dependent vasodilatation (flow-mediated dilation, FMD) of the brachial artery. We also assessed serum creatinine, the urinary albumin-creatinine ratio (UACR), the intima-media thickness (IMT) of the common carotid, carotid to femoral pulse wave velocity (cf-PWV) and left ventricular mass index (LVMI). Our results were as follows: LA size was increased in 50.8% of patients and was associated with the number of TOD. LAD was larger in the patient groups with ≥3 TOD as compared with patients with two TOD, one TOD and no TOD. FMD was lower in patients with LAD enlargement. LAD exhibited significant relationships with serum creatinine, UACR, cf-PWV, IMT and LVMI. In stepwise multivariate regression analysis, LVMI (ß=0.37, P<0.001), BMI (ß=0.33, P<0.001), duration of hypertension (ß=0.20, P=0.001) and FMD (ß=-0.17, P=0.006) were the independent predictors of LAD. FMD significantly correlated with LAD (ß=-0.26, P=0.001), male sex (ß=-0.23, P=0.004) and pulse pressure (PP) (ß=-0.16, P<0.05). In conclusions, enlargement of LAD may be an important predictor of endothelial dysfunction and may be considered to be an indicator for evaluating TOD in hypertensive patients.
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Povo Asiático , Artéria Braquial/fisiopatologia , Átrios do Coração/patologia , Hipertensão/fisiopatologia , Adulto , Albuminúria/fisiopatologia , Artérias Carótidas/diagnóstico por imagem , Artérias Carótidas/fisiopatologia , Espessura Intima-Media Carotídea , Creatinina/sangue , Creatinina/urina , Endotélio Vascular/diagnóstico por imagem , Endotélio Vascular/fisiopatologia , Feminino , Átrios do Coração/diagnóstico por imagem , Átrios do Coração/fisiopatologia , Humanos , Hipertensão/diagnóstico por imagem , Hipertensão/epidemiologia , Hipertrofia Ventricular Esquerda/diagnóstico por imagem , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Pessoa de Meia-Idade , Tamanho do Órgão , Prevalência , Rigidez Vascular/fisiologia , Vasodilatação/fisiologiaRESUMO
The identification of growth factors and/or receptors produced by mammalian embryos or present in the maternal reproductive tract is of basic interest, as well as having practical application. Early studies established that receptors binding insulin and the insulin-like growth factors (IGFs) are expressed by preimplantation mouse embryos. These studies have been confirmed at the molecular level using RT-PCR techniques. In addition, high resolution electron microscopy has shown that insulin is internalized by the cells of the blastocyst stage mouse embryo, and that immunologically intact insulin is detectable in the cells of the trophectoderm and inner cell mass. Similar studies with gold labelled IGF-I have shown that this ligand is also bound and internalized by mouse blastocysts. However, although all blastocysts express receptors that bind IGF-I on the basolateral cell surface of the trophectoderm, only 30% exhibit apically located receptors. In order to elucidate the functions of IGFs in early mouse development, we are in the process of constructing protein data-bases for embryos at the eight-cell and blastocyst stage. By the use of the database, it should prove possible to elucidate targets of growth factor action.
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Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário e Fetal , Fator de Crescimento Insulin-Like II/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Animais , Blastocisto/metabolismo , Tubas Uterinas/metabolismo , Feminino , Mamíferos , GravidezRESUMO
High-resolution microscopy in conjunction with colloidal gold-labeled insulin-like growth factor I (IGF-I) has been used to provide evidence that the IGF-I receptor is first detected in 8-cell-stage mouse embryos, confirming the results of previous reverse transcriptase polymerase chain reaction (RT-PCR) studies. Specificity for the IGF-I receptor was demonstrated by displacement with unlabeled IGF-I and dual-labeling experiments with colloidal gold-labeled or unlabeled insulin. Labeled IGF-I ligand is internalized by means of receptor-mediated endocytosis following its concentration in coated pits, and it can be visualized within cytoplasmic organelles. Immunocytochemical analyses at the blastocyst stage, using gold-labeled antibodies to the receptor, confirmed the expression of IGF-I receptors on all cells of the embryo. Similar studies with antibodies directed against the ligand demonstrated that IGF-I internalized by the embryo in vivo is maternally derived. Approximately 40% of blastocysts showed apical plasma membrane binding of gold-labeled ligand ("responders"), while approximately 60% did not demonstrate binding ("nonresponders"); however, both classes of embryo expressed receptors on basolateral membranes of trophectoderm cells and on the surface of inner masses. Functional studies show that incubating embryos in physiological levels of IGF-I (40 ng/ml) results in increased numbers of cells in the inner cell mass (p < 0.05), but not the trophectoderm, as compared to controls.
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Blastocisto/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Receptor IGF Tipo 1/metabolismo , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Contagem de Células , Endocitose , Feminino , Técnicas In Vitro , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/farmacologia , Camundongos , Microscopia Imunoeletrônica , GravidezRESUMO
Previously constructed protein databases for two stages of preimplantation mouse embryogenesis, the compacted eight-cell stage and the fully expanded blastocyst stage, have been used to analyze the effects of insulin, IGF-I, and IGF-II on protein synthesis in these developmental stages. Proteins were labeled by placing, for 2 hr, synchronous cohorts of 35-50 embryos into human tubal fluid (HTF) medium containing L-[35S]-methionine (1 mCi/ml) in the presence or absence of one of the growth factors. The embryos were then washed with medium and lysed. Samples were processed for 2-D gel analysis. For each embryonic stage and each growth factor, four or five experimental replicates were done and the gel images were compared using the PDQUEST system. Using the computer-assisted analysis, we were able to identify proteins that showed a statistically significant (P < 0.05) change in synthesis. At the eight-cell stage of development insulin caused increased synthesis of two proteins and decreased synthesis in three proteins. Insulin-treated blastocyst stage embryos exhibited an increased synthesis in eight proteins and decreased synthesis for one protein. The effect of IGF-I at the eight-cell stage of development was mostly inhibitory; the synthesis of only one protein increased and the synthesis of five proteins showed a decrease. Similar results were obtained with blastocyst stage embryos; four proteins demonstrated an increase in synthesis while 14 proteins showed a decrease. Eight-cell stage embryos incubated with IGF-II had seven proteins with a decreased synthesis, although in blastocyst stage embryos, nine proteins showed increased synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Blastocisto/efeitos dos fármacos , Fase de Clivagem do Zigoto/efeitos dos fármacos , Proteínas Fetais/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Insulin-Like II/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Insulina/farmacologia , Animais , Blastocisto/metabolismo , Fase de Clivagem do Zigoto/metabolismo , Eletroforese em Gel Bidimensional , CamundongosRESUMO
High-resolution two-dimensional sodium dodecyl sulfate-polyacrylamide (2D-SDS) gel electrophoresis combined with computerized analysis of gel images was used to construct and analyze protein data-bases for two stages of preimplantation mouse embryogenesis, the compacted eight-cell stage and the fully expanded blastocyst stage. These stages were chosen for their ease in identification of multiple synchronous embryos. Synchronous cohorts of 30-50 embryos were labelled with L-[35S]methionine for 2 hr. The embryos were then lysed in 30 microliters hot SDS sample buffer, and the lysates were stored at -80 degrees C until the gels were run. Five replicates were run for eight-cell embryos, and four for blastocyst-stage embryos. The samples were processed for 2D gel electrophoresis and fluorography; multiple exposures were made. Gel images were analyzed using the PDQUEST system, and databases were constructed. Analysis of the databases for both developmental stages showed high reproducibility of protein spots in multiple gel images. Of 1,674 total spots in eight-cell embryo standards, > 79% of spots had a percentage error (S.E.M./average) < 50%, and > 45% had a percentage error < 30%. Similarly, of 1,653 total spots in blastocyst-stage embryo standards, 74% of spots had a percentage error < 50%, and approximately 47% of spots had a percentage error < 30%. Forty-three spots (approximately 3% of the total spots) were found to be detected only in the eight-cell stage, while 75 spots were detected solely in the blastocyst stage. Sixty-nine proteins showed a greater than threefold increase in isotope incorporation from the eight-cell to the blastocyst stage, with a percentage error < 50% in both the eight-cell and the blastocyst stages. In contrast, 41 of the proteins showed a decrease during this period. Analysis of the protein databases described in this study has allowed us to document the overall quantitative changes in proteins from the compacted eight-cell stage to the blastocyst stage of mouse preimplantation development. These data-bases provide a valuable tool for further detailed quantitative analysis of specific proteins associated with developmental events. In addition they will permit analysis of the effects of environmental factors, such as growth factors, on early embryo development.
Assuntos
Blastocisto/fisiologia , Bases de Dados Factuais , Proteínas/análise , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Divisão Celular , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Feminino , Masculino , Metionina/metabolismo , Camundongos , Camundongos Endogâmicos , Peso Molecular , Biossíntese de Proteínas , Proteínas/isolamento & purificaçãoRESUMO
Insulin and insulin-like growth factors (IGF-I and -II) are members of a family of growth factors which are known to be developmentally regulated during preimplantation mouse embryogenesis. The physiological actions of the insulin family of growth factors are mediated by interactions with specific cell surface receptors that are detectable on the cells of preimplantation mouse embryos. Mouse embryonic stem (ES) cells are totipotent cells derived directly from the inner cell mass of the blastocyst. ES cells have the ability to differentiate into all three germ layers and have unlimited growth potential under certain culture conditions. The great advantage of ES cells is the ability to obtain large amounts of tissue for biochemical studies as compared with preimplantation embryos. To examine in greater detail the biological actions of the insulin family of growth factors, the expression of their cognate receptors on ES cells was examined. ES cells were cultured in DMEM medium supplemented with leukemia inhibitory factor (LIF) to maintain the undifferentiated state. Receptor expression was evaluated at the mRNA level using the reverse transcription polymerase chain reaction (RT-PCR), and at the protein level by radioactive labeled ligand-receptor binding assay. Using RT-PCR, mRNAs of all three growth factor receptors were detected in ES cells. Messenger RNA from ES cells was reverse transcribed into cDNA by AMV reverse transcriptase at 42 degrees C for 1 hr. The reverse transcription reaction was amplified with Taq polymerase and specific primers for insulin, IGF-I, or IGF-II receptors by PCR. RT-PCR and the control plasmid cDNA PCR products were resolved electrophoretically on 3% agarose gels. Each amplified PCR product showed the predicted correct size.(ABSTRACT TRUNCATED AT 250 WORDS)