RESUMO
OBJECTIVE: We examined whether aldosterone/Rho/Rho-kinase pathway contributed to obesity-associated nephropathy. SUBJECTS: C57BL/6J mice were fed a high fat or low fat diet, and mice on a high fat diet were treated with a mineralocorticoid receptor antagonist, eplerenone. RESULTS: The mice on a high fat diet not only developed obesity, but also manifested renal histological changes, including glomerular hypercellularity and increased mesangial matrix, which paralleled the increase in albuminuria. Furthermore, enhanced Rho-kinase activity was noted in kidneys from high fat diet-fed mice, as well as increased expressions of inflammatory chemokines. All of these changes were attenuated by eplerenone. In high fat diet-fed mice, mineralocorticoid receptor protein levels in the nuclear fraction and SGK1, an effector of aldosterone, were upregulated in kidneys, although serum aldosterone levels were unaltered. Furthermore, aldosterone and 3ß-hydroxysteroid dehydrogenase in renal tissues were upregulated in high fat diet-fed mice. Finally, in cultured mesangial cells, stimulation with aldosterone enhanced Rho-kinase activity, and pre-incubation with eplerenone prevented the aldosterone-induced activation of Rho kinase. CONCLUSION: Excess fat intake causes obesity and renal injury in C57BL/6J mice, and these changes are mediated by an enhanced mineralocorticoid receptor/Rho/Rho-kinase pathway and inflammatory process. Mineralocorticoid receptor activation in the kidney tissue and the subsequent Rho-kinase stimulation are likely to participate in the development of obesity-associated nephropathy without elevation in serum aldosterone levels.
Assuntos
Rim/patologia , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Obesidade/patologia , Espironolactona/análogos & derivados , Quinases Associadas a rho/efeitos dos fármacos , Animais , Quimiocina CCL2/metabolismo , Dieta com Restrição de Gorduras , Dieta Hiperlipídica , Eplerenona , Regulação da Expressão Gênica , Imuno-Histoquímica , Rim/lesões , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Transdução de Sinais , Espironolactona/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Quinases Associadas a rho/genéticaRESUMO
OBJECTIVE: Plasma triglyceride (TG) levels were reported to be high in chronic kidney disease (CKD) patients undergoing haemodialysis (HD) treatment. One of the atherogenic causes of hypertriglyceridemia is the increase in TG-rich lipoprotein remnants, which are equivalent to remnant-like particle cholesterol (RLP-C). Here, we compared the plasma levels of TG, a representative indicator of TG-rich lipoproteins and RLP-C, as well as the TG/RLP-C ratio between CKD patients undergoing HD and controls, in an effort to elucidate the atherogenicity of TG-rich lipoproteins in CKD patients on HD. MATERIALS AND METHODS: Plasma lipid and apo(lipo)protein levels and the TG/RLP-C ratio were compared between 49 CKD patients undergoing HD and 627 controls. Blood sampling for lipid and apoprotein analysis was performed in a 12-h fasting state. Controls were divided into four subgroups according to TG level (from highest to lowest). RLP-C and apo(lipo)proteins were measured using the immunoprecipitation method and turbidimetric immunoassay, respectively. In addition, a comparison between HD patients and age-, gender-, and plasma TG level-matched controls was performed. RESULTS: Plasma TG levels were 107 ± 70 (mean ± SD) mg/dl in HD patients and 115 ± 72 mg/dl in controls. Plasma RLP-C levels were 6.7 ± 4.5 mg/dl in HD patients and 4.6 ± 3.5 mg/dl in the controls (p < 0.0001). RLP-C levels decreased in descending order from the highest to the lowest TG group in controls. RLP-C levels were higher in HD patients than in controls with plasma TG levels of < 150 mg/dl (p < 0.0001). TG/RLP-C ratios were 19.0 ± 12.0 in HD patients and 25.9 ± 9.5 in controls (p < 0.0001). This ratio was significantly lower in HD patients than in all four TG subgroups. The comparison between HD patients and age-, gender-, plasma TG-matched controls revealed identical results. CONCLUSION: Plasma RLP-C levels were high, and the TG/RLP-C ratio was low in CKD patients undergoing HD treatment. These findings indicate that total plasma TG-rich lipoprotein levels were not increased, but the distribution of plasma TG-rich lipoproteins were skewed towards remnant fractions in CKD patients undergoing HD treatment; these plasma TG-rich lipoproteins appear to be more atherogenic than those in controls.
Assuntos
Hipertrigliceridemia/etiologia , Falência Renal Crônica/sangue , Lipoproteínas/metabolismo , Diálise Renal , Triglicerídeos/metabolismo , Idoso , Apolipoproteínas/metabolismo , Estudos de Casos e Controles , Colesterol/metabolismo , Feminino , Humanos , Hipertrigliceridemia/sangue , Falência Renal Crônica/terapia , Metabolismo dos Lipídeos/fisiologia , Masculino , Pessoa de Meia-IdadeRESUMO
In blinded Japanese quail (Coturnix coturnix japonica) encephalic photoreception of the stimulus from long photoperiods is sufficient to induce and maintain normal gonadal function in females (egg laying) and in males (enlargement of the cloacal gland). However, the termination of sexual activity by short days is dependent on these birds having experienced long days at the time of blinding.
RESUMO
T-type calcium channel blockers have been previously shown to protect glomeruli from hypertension by regulating renal arteriolar tone. To examine whether blockade of these channels has a role in protection against tubulointerstitial damage, we used a stereo-selective T-type calcium channel blocker R(-)-efonidipine and studied its effect on the progression of this type of renal injury in spontaneously hypertensive rats that had undergone subtotal nephrectomy. Treatment with racemic efonidipine for 7 weeks significantly reduced systolic blood pressure and proteinuria. The R(-)-enantiomer, however, had no effect on blood pressure but significantly reduced proteinuria compared to vehicle-treated rats. Both agents blunted the increase in tubulointerstitial fibrosis, renal expression of alpha-smooth muscle actin and vimentin along with transforming growth factor-beta (TGF-beta)-induced renal Rho-kinase activity seen in the control group. Subtotal nephrectomy enhanced renal T-type calcium channel alpha1G subunit expression mimicked in angiotensin II-stimulated mesangial cells or TGF-beta-stimulated proximal tubular cells. Our study shows that T-type calcium channel blockade has renal protective actions that depend not only on hemodynamic effects but also pertain to Rho-kinase activity, tubulointerstitial fibrosis, and epithelial-mesenchymal transitions.
Assuntos
Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo T/efeitos dos fármacos , Di-Hidropiridinas/uso terapêutico , Nefropatias/prevenção & controle , Nitrofenóis/uso terapêutico , Animais , Doença Crônica , Hipertensão/complicações , Hipertensão/tratamento farmacológico , Nefropatias/etiologia , Masculino , Nefrectomia/métodos , Compostos Organofosforados/uso terapêutico , Ratos , Ratos Endogâmicos SHRRESUMO
This study elucidates the nature of melanogenesis in B16 and Harding-Passey (HP) mouse melanomas producing melanin and melanosomes of different color and fine structure, i.e., brown-black eumelanosome-like B16 granules and reddish brown pheomelanosome-like HP granules, and compares them with "typical" 3,4-dihydroxyphenylalanine (DOPA) and sepia eumelanins and sepia eumelanosomes. The melanin content of B16 melanosomes was more than three times higher than that of HP melanosomes. The content of free and protein-bound DOPA and 5-S-cysteinyldopa varied greatly in B16, HP, and sepia melanosomes and was unrelated to melanin content. Chemical analysis of the eumelanin: pheomelanin ratio in melanosomes and elemental analysis of isolated melanin showed that B16 and HP melanins are primarily eumelanic, with a higher ratio of pheomelanic component in HP melanin. The spectra of electron spin resonance and IR and X-ray small-angle scattering of B16 and HP melanins were basically similar to those of sepia and DOPA melanins. B16, HP, and DOPA melanins were dissolved in aqueous NH3, while sepia melanin was dissolved to a far lesser extent. It was concluded that both B16 and HP melanomas are primarily involved in eumelanogenesis, although the fine structure of their melanosomes is entirely different, and that the marked color difference in the two melanosomes is related to a difference in the absolute content of eumelanin, the presence of a small amount of pheomelanin, and the mode of chemical bindings of melanin to structural proteins. In contrast to normal skin and hair, melanosome morphogenesis may not directly correspond to melanogenesis type in malignant melanoma.
Assuntos
Melaninas/biossíntese , Melanócitos/ultraestrutura , Melanoma/ultraestrutura , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Melaninas/isolamento & purificação , Melanócitos/metabolismo , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Microscopia Eletrônica , Espectrofotometria Infravermelho , Difração de Raios XRESUMO
A size filtration method to synchronize cultures of the dinoflagellate Gonyaulax polyedra to the beginning of the G1 phase has been developed. This technique selects newly born cells by two sequential filtrations, based on the fact that cell division is restricted to the beginning of the day, so that a decrease in cell volume occurs at this time. The fraction of synchronized cells immediately after the second filtration is about 90%; the procedures do not alter the free-running period or phase of glow rhythm, and the selected cells divide again in a few days. Applying this method, we have found that the generation times of this species in a light-dark cycle (LD 12:12) are indeed quantized to multiples of 24 hr, but are variable from generation to generation.
Assuntos
Ciclo Celular/fisiologia , Dinoflagellida/fisiologia , Animais , Dinoflagellida/crescimento & desenvolvimento , FiltraçãoRESUMO
Filial imprinting in precocial birds is a useful model for studying early learning and cognitive development, as it is characterized by a well-defined sensitive or critical period. We recently showed that the thyroid hormone 3,5,3'-triiodothyronine (T3) determines the onset of the sensitive period. Moreover, exogenous injection of T3 into the intermediate medial mesopallium (IMM) region (analogous to the associative cortex in mammals) enables imprinting even on post-hatch day 4 or 6 when the sensitive period has been terminated. However, the neural mechanisms downstream from T3 action in the IMM region remain elusive. Here, we analyzed the functional involvement of the intermediate hyperpallium apicale (IMHA) in T3 action. Bilateral excitotoxic ablation of the IMHA prevented imprinting in newly hatched chicks, and also suppressed the recovery of the sensitive period by systemic intra-venous or localized intra-IMM injection of T3 in day-4 chicks. In contrast to the effect in the IMM, direct injection of T3 into the IMHA did not enable imprinting in day-4 chicks. Moreover, bilateral ablation of IMHA after imprinting training impaired recall. These results suggest that the IMHA is critical for memory acquisition downstream following T3 action in the IMM and further, that it receives and retains information stored in the IMM for recall. Furthermore, both an avian adeno-associated viral construct containing an anterograde tracer (wheat-germ agglutinin) and a retrograde tracer (cholera toxin subunit B) revealed neural connections from the IMM to the IMHA. Taken together, our findings suggest that hierarchical processes from the primary area (IMM) to the secondary area (IMHA) are required for imprinting.
Assuntos
Comportamento Animal/fisiologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Fixação Psicológica Instintiva/fisiologia , Animais , Encéfalo/fisiopatologia , Galinhas , Período Crítico Psicológico , Ácido Ibotênico , Immunoblotting , Rememoração Mental/fisiologia , Modelos Animais , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/fisiologia , Vias Neurais/fisiopatologia , Técnicas de Rastreamento NeuroanatômicoRESUMO
This study analyzed the free and protein-bound forms of dopa, 5-S-cysteinyldopa (5-S-CD), and 5-S-glutathionedopa (5-S-GD) in B16 and Harding-Passey (HP) mouse melanomas to investigate the role of these catechols for melanogenesis and melanosome morphogenesis, inasmuch as these tumors produce melanosomes different in color and ultrastructure, i.e., eumelanosome type in B16 and pheomelanosome type in HP. Between B16 and HP mouse melanomas, however, we found (a) no significant difference in the level of free dopa and 5-S-CD in melanosomes and tumors, although the levels of these catechols reflected well the type of melanogenesis in control hair of normal mice, (b) a significant difference in free 5-S-GD level, which might, in part, reflect the observed difference in melanogenesis, and (c) no apparent difference in the level of bound dopa and 5-S-CD in either melanosomes or tumors. Thus, the striking difference in the color of melanosomes between B16 and HP melanomas seems to be related primarily to the content--not the type--of melanin pigments.
Assuntos
Di-Hidroxifenilalanina/análise , Melanócitos/análise , Melanoma/análise , Animais , Cisteinildopa/análise , Cabelo/análise , Camundongos , Neoplasias Experimentais/análise , Ligação ProteicaRESUMO
Addition of antibodies against sapecin to the culture medium of NIH-Sape-4 cells derived from a Sarcophaga embryo greatly inhibited cell proliferation, whereas addition of sapecin stimulated cell proliferation. These results suggest that sapecin is involved in the proliferation of embryonic cells of Sarcophaga. Sapecin is known to have potent antibacterial activity, so it seems to have two different biological functions: i.e. protection against bacterial infection and stimulation of embryonic cell proliferation.
Assuntos
Divisão Celular/efeitos dos fármacos , Dípteros/embriologia , Hormônios de Inseto/farmacologia , Proteínas de Insetos , Animais , Linhagem Celular , Embrião não Mamífero , Imunoglobulina G , Hormônios de Inseto/imunologia , CinéticaRESUMO
We have reported a novel serine protease produced by Sarcophaga peregrina (Nakajima et al., J. Biol. Chem. 272 (1997) 23805-23810). This 26-kDa protease showed antibacterial activity against several bacteria. This activity was an intrinsic characteristic of the enzyme protein and not directly related to its protease activity, because treating the 26-kDa protease with diisopropyl fluorophosphate had no appreciable effect on its antibacterial activity. Unlike bovine trypsin, the 26-kDa protease interacted with acidic phospholipids, suggesting that its antibacterial activity is attributable to interaction with bacterial membranes.
Assuntos
Anti-Infecciosos/farmacologia , Dípteros/enzimologia , Larva/enzimologia , Serina Endopeptidases/farmacologia , Animais , Antibacterianos , Anti-Infecciosos/isolamento & purificação , Bacillus subtilis/efeitos dos fármacos , Candida/efeitos dos fármacos , Corynebacterium/efeitos dos fármacos , Dípteros/crescimento & desenvolvimento , Escherichia coli/efeitos dos fármacos , Metamorfose Biológica , Testes de Sensibilidade Microbiana , Salmonella typhi/efeitos dos fármacos , Serina Endopeptidases/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacosRESUMO
The chemotherapeutic activity of three synthetic antibacterial peptides was investigated. KLKLLLLLKLK-NH2 and its D-enantiomer showed significant chemotherapeutic activity in MRSA-infected mice, whereas KLKLLLKLK-NH2, which showed the highest antibacterial activity among them in vitro, was found to have almost no ability to prevent MRSA infection. These results suggest that the antibacterial activity of peptides assessed in vitro does not necessarily correlate with their chemotherapeutic activity. We found that KLKLLLLLKLK-NH2 and its D-enantiomer, but not KLKLLLKLK-NH2, have the ability to activate human neutrophils to produce superoxide, suggesting that the prevention of MRSA infection by these peptides is not simply due to their direct bactericidal activity but to augmentation of the systemic defense mechanism mediated by neutrophils.
Assuntos
Antibacterianos/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Oligopeptídeos/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Animais , Grupo dos Citocromos c/metabolismo , Humanos , Masculino , Resistência a Meticilina , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Oligopeptídeos/síntese química , Oligopeptídeos/uso terapêutico , Oxirredução , Infecções Estafilocócicas/microbiologia , Estereoisomerismo , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
To characterize the proteins involved in cell clump/cell adhesion of insect cellular defense reactions, we induced the cell clump/cell adhesion reaction in vitro with the hemolymph of larvae of the coleopteran insect, Tenebrio molitor. The 72 kDa protein was specifically enriched in the residues of cell clump/cell adhesion and was purified to homogeneity. A cDNA clone for the 72 kDa protein was isolated. We found that the 72 kDa protein was an activated phenoloxidase from Tenebrio pro-phenoloxidase. We suggest that activated phenoloxidase is involved in the cell clump/cell adhesion reaction as well as in the synthesis of melanin.
Assuntos
Catecol Oxidase/genética , Adesão Celular/genética , Ativação Enzimática , Precursores Enzimáticos/genética , Tenebrio/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Catecol Oxidase/química , Agregação Celular/genética , Clonagem Molecular , Cobre/metabolismo , Indução Enzimática/genética , Precursores Enzimáticos/química , Hemolinfa/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/genética , Isopropiltiogalactosídeo/farmacologia , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Tenebrio/embriologiaRESUMO
Recently, we reported two novel early-staged encapsulation-relating proteins (56 kDa and 48 kDa ERPs) isolated from the hemolymph of coleopteran insect, Tenebrio molitor larvae [Cho et al. (1999) Eur. J. Biochem. (in press)]. Here, a cDNA clone for another early-staged encapsulation-relating protein (86 kDa) was isolated. We found that the 86 kDa protein shows high homology with insect diapause protein 1. The 86 kDa protein was localized in the fat body and hemolymph, but not hemocyte lysate. A significant level of 86 kDa protein was detected in pre-pupae stage, but it decreased rapidly at late larvae and pupae, and no protein was found in embryo, early larvae and adult stages. This diapause protein 1-like protein is likely to be a component of early-staged encapsulation-relating proteins in the insect cellular defense reaction.
Assuntos
Genes de Insetos , Proteínas de Insetos/genética , Tenebrio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas de Insetos/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência , Tenebrio/metabolismoRESUMO
A series of N-alkyl- and N,N-dialkyl-4-[alpha-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl] benzyl]-benzamides were synthesized and evaluated for binding affinities at mu, delta, and kappa opioid receptor subtypes. Several compounds (2e,f,h,i,m) strongly bound to the delta receptor with IC50 values in the nanomolar range. On the other hand, the binding affinities of these compounds for the mu and kappa receptors were in the micromolar or greater range indicating excellent delta opioid receptor subtype selectivities. In this series, two important structure-activity relationships were found for the delta receptor binding affinity. First, the spatial orientation of the alpha-benzylic position influenced the affinities with the alpha R derivatives 2a-n generally showing more than 10-fold greater affinity than the alpha S derivatives 3a-n. Second, the binding affinities were strongly influenced by the number of alkyl substituents on the amide nitrogen. N-Monoalkylbenzamide derivatives 2b-d showed lower affinity than N,N-dialkylbenzamide derivatives 2e-n, and the N-unsubstituted benzamide derivative 2a had the lowest affinity for the delta receptor in the series. The dramatic effect of the amide group substitution pattern on the binding affinity for the delta receptor strongly suggests that the amide function is an important structural element in the interaction of this series of compounds at the delta receptor. Selective compounds in this series were examined for binding affinity in cloned human mu and delta receptors. The results obtained generally paralleled those from the rat brain binding assay. Compounds 2e,f with potent delta binding affinities and high delta selectivities were shown to be delta agonists with high selectivity by studies in the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. Compound 2f was the most selective compound in the rat brain and GPI/MVD assays with 1755- and 958-fold delta vs mu selectivity, respectively.
Assuntos
Benzamidas/síntese química , Entorpecentes/agonistas , Entorpecentes/síntese química , Piperazinas/síntese química , Receptores Opioides delta/agonistas , Alquilação , Animais , Benzamidas/química , Benzamidas/farmacologia , Encéfalo/metabolismo , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cristalografia por Raios X , Cobaias , Humanos , Cinética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Entorpecentes/química , Entorpecentes/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Ensaio Radioligante , Ratos , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Relação Estrutura-Atividade , Transfecção , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/fisiologiaRESUMO
To investigate a physiological role of glutathione in the horizontal cells of carp retina, the gap junctional intercellular communication between horizontal cells was studied using the techniques of intracellular recording of light-induced responses and coupling of the fluorescence dye Lucifer Yellow. Intravitreal injection of 2.5 micromol L-buthionine sulfoximine, an inhibitor of glutathione synthesis, induced a dramatic reduction (20% of control) of retinal glutathione level two days after treatment. The low level of glutathione continued for a further four to five days, and thereafter gradually recovered to about 40% (20 days after injection) and 70% (50 days after injection) of the control level. The spatial properties of the photopic L-type horizontal cell response were examined by enlarging the diameter of the central spot and peripheral annulus over the recording point. In normal retinas, the response amplitude of horizontal cells was monotonically enhanced as the diameter of the spot increased (0.5-4.0 mm) and correspondingly the dye diffusion area was wide, as the injected Lucifer Yellow normally diffused to several neighboring cells. Treatment with L-buthionine sulfoximine significantly altered the spatial properties of horizontal cells by increasing the response amplitude to central spots and slightly decreasing that to peripheral annuli, which were observed by four days after injection. It also restricted intracellular Lucifer Yellow to one or two cells. Accompanying the recovery of the cellular level of glutathione, the spatial properties and dye coupling of horizontal cells were restored to normal. A time lag (two days) of initiation in retinal glutathione depletion and alteration of spatial or dye coupling properties of horizontal cells is discussed, together with reactive oxygen species accumulation.
Assuntos
Glutationa/deficiência , Estresse Oxidativo/fisiologia , Retina/citologia , Retina/metabolismo , Animais , Butionina Sulfoximina/farmacologia , Carpas , Comunicação Celular/fisiologia , Difusão , Corantes Fluorescentes/farmacocinética , Glutationa/antagonistas & inibidores , Glutationa/metabolismo , Isoquinolinas/farmacocinética , Retina/fisiologiaRESUMO
Space-occupying lesions (SOL) and irregular intensity distribution are usually observed in the radioisotope images of human liver diseases, hepatic cancer, etc. This paper describes a new image processing method for evaluating such SOL by using a computer. This method analyzes quantitatively the convex and the concave structure of the contour line. From the processed results, the contour lines of radioisotope images of liver diseases are more ragged than those of normal ones; the region of SOL is extracted by the abnormal raggedness in the contour structure.
Assuntos
Hepatopatias/diagnóstico por imagem , Fígado/diagnóstico por imagem , Humanos , Aumento da Imagem/métodos , Modelos Estruturais , Cintilografia , Tecnologia RadiológicaRESUMO
U937 cells were found to be activated by an antibacterial peptide, KLKLLLLLKLK-NH2 (L5), to generate superoxide anion (O2-)-like peripheral neutrophils. However, the state of cell surface calreticulin, a possible receptor for L5, was suggested to differ between neutrophils and U937 cells. Unlike the former, the latter ones were activated by anti-C-domain peptide antibody of calreticulin even in the absence of L5 and generated O2- in a GTP-binding protein (G-protein)-dependent manner.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Chaperonas Moleculares/metabolismo , Monócitos/imunologia , Ribonucleoproteínas/metabolismo , Animais , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Anticorpos/imunologia , Anticorpos/isolamento & purificação , Western Blotting , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/imunologia , Calreticulina , Membrana Celular/metabolismo , Feminino , Imunofluorescência , Humanos , Ativação de Macrófagos , Chaperonas Moleculares/química , Chaperonas Moleculares/imunologia , Monócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Oligopeptídeos/farmacologia , Oxigênio/metabolismo , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Estrutura Terciária de Proteína , Coelhos , Ribonucleoproteínas/química , Ribonucleoproteínas/imunologia , Tretinoína/farmacologia , Células U937RESUMO
A cDNA for prolyl endopeptidase (PEP) of Sarcophaga peregrina (flesh fly) was cloned and its sequence determined. The overall amino acid sequence identity between Sarcophaga and mammalian PEPs was 53%, indicating that these enzymes are structurally very similar. Northern blot hybridization revealed that the Sarcophaga PEP gene was activated significantly at the eversion stage of imaginal disc differentiation. We obtained recombinant PEP by expressing the cDNA in Escherichia coli. The recombinant and authentic enzymes showed almost identical characteristics, in terms of substrate specificities and sensitivities to inhibitors.
Assuntos
Dípteros/enzimologia , Serina Endopeptidases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Dípteros/genética , Escherichia coli , Expressão Gênica , Proteínas de Insetos/genética , Dados de Sequência Molecular , Prolil Oligopeptidases , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência de AminoácidosRESUMO
Myosin has three highly-conserved, unique loops [B (320-327), M (677-689), and N (127-136)] at the entrance of the ATP binding cleft, and we previously showed that the effects of actin are mediated by a conformational change in loop M [Maruta and Homma (1998) J. Biochem. 124, 528-533]. In the present study, loops M and N were photolabeled respectively with fluorescent probes Mant-8-N(3)-ADP and Mant-2-N(3)-ADP in order to study conformational changes in the loops related to energy transduction. The effect of actin on the conformation of loop N was examined by analyzing fluorescence polarization and acrylamide quenching; the results were then compared with those previously reported for loop M. In contrast to loop M, the fluorescence polarization and the value of K(sv) of the Mant-groups crosslinked to loop N were slightly affected by actin binding. To study conformational changes in loops M and N during the ATPase cycle, FRET was analyzed using TNP-ADP.BeFn and TNP-ADP. AlF(4)(-) as FRET acceptors of Mant fluorescence. The resultant estimated distances between loop M and the active site differed for the Mant-S1.TNP-ADP.BeFn and Mant-S1.TNP-ADP.AlF(4)(-) complexes, whereas the distances between loop N and the active site differed slightly. These findings indicate that the conformation of loop M changes during the ATPase cycle, suggesting that Loop M acts as a signal transducer mediating communication between the ATP- and actin-binding sites. Loop N, by contrast, is not significantly flexible.
Assuntos
Trifosfato de Adenosina/metabolismo , Transferência de Energia , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Acrilamida/química , Actinas/metabolismo , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Animais , Sítios de Ligação , Galinhas , Polarização de Fluorescência , Corantes Fluorescentes , Modelos Moleculares , Estrutura Molecular , Músculo Esquelético/química , Marcadores de Fotoafinidade , Ligação Proteica , Conformação Proteica , Espectrometria de Fluorescência , Espectrofotometria , ortoaminobenzoatos/metabolismoRESUMO
Actin binding to skeletal muscle myosin subfragment-1 (S1) increases the dissociation rate of reaction products from the myosin ATPase site; conversely, ATP binding facilitates dissociation of complexed acto-S1. However, details of the molecular mechanism by which the ATP- and actin-binding sites communicate with each other is still obscure. We present evidence that the effect of actin is mediated by a conformational change in the loop containing amino acids from 677 to 689 [loop M (677-689)], a segment of the 20-kDa tryptic fragment that contributes to the structure of the ATP-binding cleft. Initially, a fluorescent ADP analogue, methylanthranyloyl-8-azido-ADP (Mant-8-N3-ADP), was covalently crosslinked to loop M (Mant-S1), perhaps at Lys 681. Actin-activated Mg2+-ATP hydrolysis by Mant-S1 was accelerated approximately 6 times over that by unmodified S1, suggesting that the ATPase site is not blocked by the ADP analogue crosslinked in the loop M (677-689). Nevertheless, analysis of Mant-group fluorescence polarization and acrylamide-induced quenching showed the crosslinked probe to be entrapped within the ATP-binding cleft at a location where Mant-group rotational mobility was hindered, and where it was relatively inaccessible to the solvent. Exposing Mant-S1 to Mg2+-ATP and/or actin elicited similar decreases in fluorescence polarization, indicating increased rotational mobility of the Mant-group and movement of crosslinked Mant-8-N3-ADP to a less hindered position. Stern-Volmer quench curves showed that Mant-8-N3-ADP was translocated to a site where it was more accessible to dissolved quencher, perhaps outside the ATP-binding cleft. Since actin does not bind to the ATPase site, actin-induced translocation of Mant-8-N3-ADP crosslinked to loop M (677-689) probably results from a conformational change in loop M (677-689). These results suggest that loop M acts as a signal transducer mediating communication between the ATP- and actin-binding sites.