RESUMO
The laminar tissue of bovine laminitis may undergo energy failure. The expression of glucose transport protein-1 (GLUT-1) and 5'-adenosine monophosphate-activated protein kinase (AMPK) affects the energy metabolism of digital laminar tissue. This study aimed to determine the expression of glucose uptake and AMPK in laminar wall corium of Holstein heifer claw by oral administration of oligofructose. A total of twelve clinically healthy Holstein heifers were selected and divided into two groups, including control (CON, n = 6) and experimental (OF, n = 6) groups. The heifers of OF group were given 17 g/kg BW oligofructose dissolved in water (20 mL/kg BW) and the heifers of CON group were given water only (20 mL/kg BW). The laminar tissues were collected after euthanasia. The amount of protein and transcript expression of AMPK and GLUT-1 were determined by western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), respectively. Expressions of phosphoenolpyruvate carboxy-kinase (PEPCK), receptor-c coactivator1-α (PGC-1α) and peroxisome proliferator-activated receptor-γ (PPAR-γ) were determined by qRT-PCR. The heifers of OF group showed no significant change in the expression and concentration of AMPK. The phosphor-(Thr172) AMPK and GLUT-1 were significantly decreased, while the gene contents of PPAR-γ and PGC-1α were significantly increased. The activation of AMPK and GLUT-1 in digital laminar tissues of heifers was inhibited, which may contribute to digital laminar tissue damage.
Assuntos
Proteínas Quinases Ativadas por AMP , Casco e Garras/enzimologia , Oligossacarídeos , Proteínas Quinases Ativadas por AMP/genética , Monofosfato de Adenosina , Animais , Bovinos , Feminino , Glucose , Oligossacarídeos/farmacologiaRESUMO
The Hall and Seebeck effects of the stripe-ordered superconductor La(1.6-x)Nd(0.4)Sr(x)CuO(4) single crystals (x = 0.10, 0.12 and 0.15) were investigated systematically. The sign change of Hall and Seebeck coefficients (R(H) and S) from positive to negative with decreasing temperature suggests the presence of electron pockets in the Fermi surface due to the stripe ordering. We successfully tune this behavior through an epitaxial strain induced by the mismatch between the thin film and the substrate. The negative R(H) disappears in the thinner film in which the static charge stripe is greatly suppressed by the strong epitaxial strain, and for a strain released thicker film the negative R(H) recovers. These results indicate the possibility of Fermi surface reconstruction caused by the static charge stripe order in the system.
RESUMO
The field and temperature dependencies of the spin structures in the normal states of La(1.6-x)Nd(0.4)Sr(x)CuO(4) (x = 0.10, 0.12, and 0.15) single crystals have been studied by measuring the magnetoresistance and susceptibility. A negative magnetoresistance appears just below the spin-ordering temperature for the magnetic fields parallel to the CuO(2) plane, which can be attributed to the spin-flop transition of the special spin structure in the normal state of the system. The anisotropic variations of susceptibilities with temperature for all the three specimens can be described in the framework of the crystal-field theory. The well fitted broad peaks of the in-plane susceptibilities χ(ab) for the specimens suggest that the susceptibilities are dominated by Nd(3+), and thus the spin reorientation of Cu(2+) in the CuO(2) plane can not be observed from the study of the susceptibility.
Assuntos
Cobre/química , Lantânio/química , Óxidos/química , Estrôncio/química , Anisotropia , Cristalização , Magnetismo , TemperaturaRESUMO
Molecular cloning of cardiac troponin I-interacting kinase (TNNI3K), a novel cardiac-specific protein kinase containing seven N-terminal ankyrin (ANK) repeats followed by a protein kinase domain and a C-terminal Ser-rich domain, has previously been reported. In the present study, we show that the C-terminal functional region of TNNI3K negatively regulates the kinase activity, and the N-terminal ANK domain is necessary for autophosphorylation. An in vitro kinase assay shows that TNNI3K exhibits dual-specific kinase activity and forms dimers or oligomers that may be necessary for its activation.
Assuntos
MAP Quinase Quinase Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Motivos de Aminoácidos , Linhagem Celular Transformada , Clonagem Molecular , Dimerização , Ativação Enzimática , Regulação da Expressão Gênica , Humanos , MAP Quinase Quinase Quinases/química , Miócitos Cardíacos/enzimologia , Fosforilação , Multimerização Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/metabolismo , Serina/química , Serina/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
OBJECTIVE: To investigate the different killing effect to human pulmonary adenocarcinoma cell line cells GLC-82 with coexpressed double suicide genes compared with single gene. METHODS: Recombinant expression vectors containing CD (cytosine deaminase) and/or TK (thymidine kinase) gene under CMV promoter were constructed successfully. The vectors were transfected to GLC-82 tumor cell lines by use of lipofectamine. The clones were picked out after G418 selection. Extraneous gene integration and expression were confirmed by PCR and semi-quantitative RT-PCR. The cytotoxicity to these transgenic cells under treatment with 5-Fc and GCV were measured by MTT assays. RESULTS: Double and single suicide gene transfer were both stably expressed in GLC-82 cells. The cytotoxic effects of co-expressed TK-CD genes were superior than that of the single gene. CONCLUSION: The CD + TK/5-Fc + GCV co-expression system is more effective for killing effect of tumor cells than CD/5-Fc or TK/GCV system alone.
Assuntos
Citosina Desaminase/genética , Terapia Genética , Vetores Genéticos , Neoplasias Pulmonares/terapia , Timidina Quinase/genética , Linhagem Celular Tumoral , Flucitosina/farmacologia , Ganciclovir/farmacologia , Humanos , Neoplasias Pulmonares/patologia , TransfecçãoRESUMO
In this report, reporter gene beta-galactosidase (LacZ) was chosen to compare two different intramuscular gene transfer methods, direct injection and gene suture. Evidence showed that gene suture can produce a higher foreign gene express efficiency in skeletal muscle compared with the direct injection method. The highly efficient eukaryotic expressing vectors of human atrial natriuretic factor (ANF) were constructed (pcD2/pAdVAntage/hANF and pcDNA3/hANF), and in vivo ANF gene delivery was performed by intramuscular gene suture. The effects of ANF gene transfer on blood pressure and renal sodium and water excretion were studied in three models of hypertensive animals. Results showed that a marked decrease of mean arterial pressure (MAP) and a significant increase of urine volume and urinary sodium excretion was produced in rats receiving the hANF construct due to the local expression of ANF and its secretion into plasma. Taken together, these results indicate that gene suture may represent a novel gene delivery modality in gene therapy.
Assuntos
Terapia Genética/métodos , Hipertensão/terapia , Óperon Lac/genética , Músculo Esquelético/metabolismo , Animais , Fator Natriurético Atrial/genética , Pressão Sanguínea , DNA/administração & dosagem , DNA/genética , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Hipertensão/genética , Imuno-Histoquímica , Injeções Intramusculares , Masculino , Plasmídeos/genética , Ratos , Ratos Endogâmicos SHR , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sódio/urina , beta-Galactosidase/genéticaRESUMO
In the present study, the angiotensin II receptor subtype I-a (AT1a) and I-b (AT1b) mRNA levels in aortic smooth muscle (ASM), ventricular myocardium (VM) and adrenal from 12-week-old stroke-prone spontaneously hypertensive rats (SHRsp) and age-matched Wistar-Kyoto (WKY) rats with normal diet (control) and high salt-loading were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that: (1) The AT1a and AT1b mRNA levels in ASM and VM from SHRsp were lower than those from WKY rats (in ASM, 10% and 23%, while in VM, 23% and 40% lower, respectively). In contrast, both AT1a and AT1b mRNA levels in adrenal from SHRsp were higher (176% and 157%, respectively). (2) In the WKY rats with high salt-loading, the AT1a and AT1b mRNA levels in adrenal, as well as AT1b mRNA level in VM, increased significantly, as compared with the control (in adrenal, 167% and 401%, while in VM, 62%). However, the AT1a and AT1b mRNA levels in ASM, as well as AT1a mRNA level in VM, showed no obvious change. (3) In SHRsp with high salt-loading, the AT1b mRNA level in ASM, as well as AT1a and AT1b mRNA levels in VM, increased markedly (in ASM, 90%, while in VM, 590% and 200%); whereas the AT1a mRNA level in adrenal decreased significantly (58%). There was little influence on the regulation of AT1a (in ASM) and AT1b (in adrenal) receptor gene expression after high salt-loading. The results suggest that AT1a and AT1b receptors may be involved in the pathogenesis of salt-induced hypertension. The up-regulation of AT1b receptors in ASM may induce the remodeling of arterial wall, while that of AT1a and AT1b receptors in VM might contribute to ventricular hypertrophy in hypertension. Furthermore, there are certain differences between SHRsp and WKY rats with respect to the regulation of AT1a and AT1b receptor gene expression with or without external stimulation.
Assuntos
Receptores de Angiotensina/biossíntese , Sódio na Dieta/farmacologia , Glândulas Suprarrenais/metabolismo , Animais , Masculino , Músculo Liso Vascular/metabolismo , Miocárdio/metabolismo , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/biossíntese , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptor Tipo 1 de AngiotensinaRESUMO
The specific binding sites for atrial natriuretic peptide (ANP) were present in cultured vascular smooth muscle cells (VSMC) from stroke-prone spontaneously hypertensive (SHRsp) and Wistar-Kyoto(WKY) rats with a Bmax of 3.65 +/- 0.13 and 1.89 +/- 0.09 pmol/mg pr. and a Kd of 72.0 +/- 10.2 and 42.1 +/- 4.8 x 10(-12) mol/L, respectively. The basal levels of cGMP of the two strains showed no statistical difference. After treatment with ANP (1.67 x 10(-7) mol/L) for 5 min, the cGMP levels of VSMC were increased by 139 folds in SHRsp and 271 folds in WKY rats, i.e., cGMP levels were significantly lower in the former (P less than 0.01). Therefore, the cultured VSMC of SHRsp had higher ANP receptor density but lower affinity and responsiveness to ANP than that of WKY rats. After incubation of VSMC in the medium containing high NaCl (2-folds of normal) at 37 degrees C for 24 h, the number of ANP binding sites decreased to 34.8 +/- 8.2% in SHRsp and to 38.6 +/- 9.4% in WKY rats (P less than 0.01) with a parallel decrease of cGMP, while the affinity of ANP receptor did not change. It is suggested that the lower responsiveness of ANP receptor to ANP in SHRsp might result in a diminution of vasorelaxation to ANP and thus an increase of arterial pressure. In addition, the more down-regulation of ANP receptor by high NaCl in cultured VSMC from SHRsp implicates that it is one of the mechanisms that high dietary intake of NaCl might enhance high blood pressure.
Assuntos
Fator Natriurético Atrial/fisiologia , Músculo Liso Vascular/metabolismo , Receptores de Superfície Celular/fisiologia , Animais , Aorta , Células Cultivadas , GMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Masculino , Músculo Liso Vascular/citologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Cloreto de Sódio/farmacologiaRESUMO
In order to study whether atrial natriuretic factor (ANF) is involved in the depressor effect of clonidine, microinjection of the latter into nucleus tractus solitarii (NTS) was carried out in anesthetized stroke-prone spontaneously hypertensive rats (SHRsp) and normotensive Wistar-Kyoto (WKY) rats. Each strain was randomly divided into three groups by injecting: (1) clonidine (1.0 microgram/0.2 microliter); (2) yohimbine (3.3 micrograms/0.2 microliter) followed by (1); (3) artificial cerebral spinal fluid (ACSF, 0.2 microliter) as control. A decrease of blood pressure and heart rate and a suppression of ANF release elicited by clonidine were significantly greater in SHRsp than in WKY rats. After blockade of alpha 2-receptor with yohimbine, the hypotensive effect of clonidine was blocked completely in WKY rats, but only partially in SHRsp, while the suppression effect on ANF release was eliminated in both strains. In addition, the decrease of plasma catecholamine produced by clonidine could also be blocked after yohimbine. The results suggest that ANF probably does not contribute to the depressor effect of centrally administered clonidine, while in SHRsp the decrease of plasma ANF might be a blood pressure-dependent compensatory response.
Assuntos
Fator Natriurético Atrial/efeitos dos fármacos , Clonidina/farmacologia , Animais , Fator Natriurético Atrial/metabolismo , Fator Natriurético Atrial/fisiologia , Pressão Sanguínea/efeitos dos fármacos , Bulbo/metabolismo , Microinjeções , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
In order to study the mechanism of pressor response to central osmotic stimulation, rats were administered with hypertonic artificial cerebrospinal fluid (ACSF) intracerebroventricularly. Carotid arterial pressure and heart rate were recorded. Ten minutes after administration, blood samples, hypothalamus and hypophysis were taken for the determination of atrial natriuretic peptide (ANP) and vasopressin (AVP) by radioimmunoassay. The results showed that after central administration of hypertonic ACSF, the plasma level of AVP increased significantly with no apparent change in ANP. In hypothalamus and hypophysis, the content of ANP was increased while that of AVP decreased.
Assuntos
Fator Natriurético Atrial/metabolismo , Solução Salina Hipertônica/farmacologia , Vasopressinas/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Hipotálamo/metabolismo , Injeções Intraventriculares , Masculino , Pressão Osmótica , Hipófise/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Mouse phagocytic glycoprotein-1 (Pgp-1; Ly-24) is a 95-kDa glycoprotein of unknown function that has served as an important T cell/leukocyte differentiation marker. Recent work has suggested that it may be related to a human 85- to 95-kDa glycoprotein (termed variously the Hermes Ag/lymphocyte homing receptor, ECMRIII, P80, and CD44) that is involved in lymphocyte binding to high endothelial venules in the process of lymphocyte homing, and has been implicated in other cell adhesion events. The widespread expression of this molecular class in diverse organ systems suggests a broad role in cellular adhesion, and has led to the unifying designation homing-cellular adhesion molecule (H-CAM). By using human H-CAM cDNA probes, we have isolated a full-length cDNA for the mouse homolog. Comparison of the human and mouse sequences reveals that an N-terminal domain homologous to cartilage proteoglycan core and link proteins, as well as the C-terminal transmembrane and cytoplasmic sequences, are highly conserved (89% and 86% identity, respectively). In contrast, a proximal extracellular domain thought to serve as a target for O-glycosylation and chondroitin sulfate attachment has undergone substantial divergence (only 42% identity). Transient expression of the cDNA in CHO cells followed by immunologic staining confirms that this mouse H-CAM cDNA encodes Pgp-1.1, one of two known Pgp-1 alloantigens.
Assuntos
Sequência de Bases , Moléculas de Adesão Celular/genética , Receptores Imunológicos/genética , Homologia de Sequência do Ácido Nucleico , Sequência de Aminoácidos , Animais , Antígenos Ly/genética , Antígenos Ly/isolamento & purificação , Western Blotting , Moléculas de Adesão Celular/isolamento & purificação , Linhagem Celular , Clonagem Molecular , Cricetinae , Cricetulus , DNA/isolamento & purificação , Feminino , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Ovário , Receptores de Retorno de LinfócitosRESUMO
Lymphocyte interactions with high endothelial venules (HEV) during extravasation into lymphoid tissues involve an 85-95 kd class of lymphocyte surface glycoprotein(s), gp90Hermes (CD44). We report here the cloning of cDNA for gp90Hermes expressed in a mucosal HEV-binding B lymphoblastoid cell line, KCA. Northern hybridization revealed the presence of three invariant RNA bands at 1.5, 2.2, and 4.5 kb in mucosal HEV-, lymph node HEV-, or dual-binding cells. The deduced amino acid sequence predicts a mature protein with a C-terminal cytoplasmic tail, a hydrophobic transmembrane domain of 23 amino acids, and an N-terminal extracellular region of 248 amino acids. A proximal extracellular domain is the probable region of O-glycosylation and chondroitin sulfate linkage and displays at least two of the three immunodominant epitope clusters of native gp90Hermes. A distal region contains the majority of potential N-glycosylation sites and cysteines, and exhibits a striking homology to tandemly repeated domains of the cartilage link and proteoglycan core proteins. No significant similarities were found to the immunoglobulin, integrin, or cadherin gene families. Thus gp90Hermes represents a novel class of integral membrane protein involved in lymphocyte-endothelial cell interactions and lymphocyte homing.
Assuntos
Proteínas da Matriz Extracelular , Glicoproteínas/análise , Proteínas/análise , Proteoglicanas , Receptores Imunológicos/análise , Agrecanas , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Adesão Celular , Linhagem Celular , Clonagem Molecular , DNA/isolamento & purificação , Regulação da Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/fisiologia , Humanos , Lectinas Tipo C , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Proteínas/genética , Proteínas/fisiologia , RNA/análise , RNA/genética , Receptores Imunológicos/genética , Receptores Imunológicos/fisiologia , Receptores de Retorno de Linfócitos , Homologia de Sequência do Ácido NucleicoRESUMO
In the hope to find new superagonists of LHRH, here we report the synthesis of six analogs by changing residue 5 of [D-Trp6 (or D-Arg6), des-Gly10]-LHRH-EA. For the convenience of comparison, the known compound [D-Trp6, desGly10]-LHRH-EA was also synthesized.
Assuntos
Hormônio Liberador de Gonadotropina/análogos & derivados , Animais , Células Cultivadas , Feminino , Hormônio Liberador de Gonadotropina/síntese química , Hormônio Liberador de Gonadotropina/farmacologia , Células Lúteas/efeitos dos fármacos , Progesterona/metabolismo , RatosRESUMO
The plasma and cardiac levels of immunoreactive (IR) atrial natriuretic factor (ANF) were measured during the entire lifespan of cardiomyopathic hamsters, which eventually develop spontaneous congestive heart failure, and were correlated with immunohistochemical, ultrastructural, and immunocytochemical changes in the secretory apparatus of atrial and ventricular cardiocytes. Plasma IR-ANF rose in the early stages of the disease, reached a maximum in moderate heart failure, and declined thereafter but remained above control values. The peptide decreased constantly in the atria during the evolution of the disease but increased markedly in the ventricles. Its highest levels were found in the inner half of the left ventricle. In atrial cardiocytes, the size and complexity of the Golgi complex increased with the progression of the disease, whereas the number, size, and IR-ANF content (as assessed by the immunogold technique) of secretory granules decreased constantly. In ventricular cardiocytes, the size of the Golgi complex increased, and typical secretory granules were present in approximately 20% of these cells, regardless of their localization in the myocardium. The results suggest that stimulation of ANF secretion in atrial cardiocytes leads to a dissociation between synthesis and release, the latter being maximal according to ultrastructural and immunocytochemical criteria. In ventricular cardiocytes, the same stimulation culminates in increased synthesis and the possibility of release via two pathways: one constitutive, the other regulated. Thus, the elevated plasma levels of IR-ANF in congestive heart failure may be derived from secretion by both atrial and ventricular cardiocytes.
Assuntos
Fator Natriurético Atrial/análise , Insuficiência Cardíaca/patologia , Miocárdio/análise , Animais , Cricetinae , GMP Cíclico/sangue , Átrios do Coração/patologia , Insuficiência Cardíaca/sangue , Ventrículos do Coração/patologia , Imuno-Histoquímica , Microscopia Eletrônica , NecroseAssuntos
Fator Natriurético Atrial/análise , Sistema de Condução Cardíaco/análise , Animais , Fator Natriurético Atrial/genética , Nó Atrioventricular/análise , Nó Atrioventricular/ultraestrutura , Endocárdio/análise , Endocárdio/ultraestrutura , Feminino , Ventrículos do Coração/análise , Ventrículos do Coração/ultraestrutura , Imuno-Histoquímica , Ramos Subendocárdicos/análise , Ramos Subendocárdicos/ultraestrutura , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos , Nó Sinoatrial/análise , Nó Sinoatrial/ultraestrutura , Veia Cava Inferior/análise , Veia Cava Inferior/ultraestrutura , Veia Cava Superior/análise , Veia Cava Superior/ultraestruturaRESUMO
The relative contribution of atria and ventricles in the release of immunoreactive (IR-) atrial natriuretic factor (ANF) and in the eventual circulating levels of the peptide was evaluated in several situations. In situ, not only atrial but also ventricular cardiocytes contain IR-ANF although the amount present in ventricles is very low, particularly in the adult rat. In cardiomyopathic hamsters with heart failure, the amount of IR-ANF decreases constantly in the atria and increases in parallel in the ventricles so that the ratio of IR-ANF which is 114:1 in control animals becomes 3.9:1 in hamsters with severe heart failure. With the Langendorff preparation, the whole heart secretes much more IR-ANF than the isolated ventricles. From the latter, IR-ANF may originate from subendocardial cells of the conduction system and, more likely, from all ventricular cardiocytes where the peptide may be secreted by two pathways: one constitutive, the other regulated. These results are in agreement with clinical studies where circulating IR-ANF was measured in the same patients following catheterization of coronary sinus and great cardiac vein (draining the left ventricle). In this situation, it was found that while the levels of IR-ANF in the great cardiac vein are higher than in the general circulation, they are much lower than in the coronary sinus. All these results tend to indicate that IR-ANF is secreted by ventricular cardiocytes in relatively low amounts even in periods of intense stimulation.