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1.
PLoS One ; 12(10): e0186392, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29023605

RESUMO

Aeromonas sobria serine protease (ASP) is an extracellular serine protease secreted by the organism. Here, we identified the amino acid residue of ASP that contributes to substrate specificity by using both synthetic peptides and biological protein components. The results showed that the arginine residue at position 566 (Arg-566) of ASP, which is located in the extra occluding region of ASP close to an entrance of the catalytic cavity, is involved in the substrate specificity. A substitutional point mutation of the Arg-566 residue of ASP to Ala residue (ASP[R566A]) caused a decrease of the proteolytic efficiency for a certain substrate. In addition, ASP lost the ability to recognize the primary substrate by such a point mutation, and ASP[R566A] reacted to a wide range of synthetic substrates. It is likely that Arg-566 causes an interaction with the amino acid residue at position P3 of the substrate, which is the third amino acid residue upstream from the cleavage site. Another study using ORF2 protein, a chaperone protein of ASP, further suggested that Arg-566 could also play an important role in interaction with ORF2. We therefore conclude that the Arg-566 residue of ASP is likely responsible for the selection of substrates.


Assuntos
Aeromonas/enzimologia , Arginina/metabolismo , Proteínas de Bactérias/metabolismo , Serina Proteases/metabolismo , Sequência de Aminoácidos , Arginina/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Fibrinogênio/metabolismo , Humanos , Cininogênios/metabolismo , Chaperonas Moleculares/metabolismo , Mutagênese Sítio-Dirigida , Proteólise , Serina Proteases/química , Serina Proteases/genética , Especificidade por Substrato
2.
J Virol Methods ; 209: 136-42, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25241143

RESUMO

Newcastle disease virus (NDV), belonging to the family Paramixoviridae, causes respiratory and neuronal symptoms in almost all birds. NDV has haemagglutinin-neuraminidase (HN) glycoprotein possessing sialidase activity. HN glycoprotein is highly expressed on the surface of NDV-infected cells, resulting in much higher sialidase activity in NDV-infected cells than in non-infected cells. It was reported that mouse and human cancer cells up-regulating sialidase expression were histochemically stained with a fluorescent sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac), which deposits water-insoluble fluorescent compound BTP3 on locations of sialidase activity. By using the BTP3-Neu5Ac assay, we showed that NDV-infected cells and HN gene-expressing cells could be simply detected at room temperature after only 5min. Infection of the cells with the virus resulted in apparent green fluorescence, which disappeared with addition of a sialidase inhibitor. Cells that were stained in the BTP3-Neu5Ac assay were immunostained with an anti-NDV antibody. Moreover, BTP3-Neu5Ac staining was applied to a virus overlay binding assay with NDV particles. NDV-bound protein bands on guinea pig red blood cells were easily and rapidly detected by the BTP3-Neu5Ac assay after Western blotting. BTP3-Neu5Ac offers an easy and rapid protocol for fluorescent staining of NDV and virus-infected cells without antibodies.


Assuntos
Corantes Fluorescentes/metabolismo , Fluorometria/métodos , Neuraminidase/metabolismo , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/enzimologia , Vírus da Doença de Newcastle/isolamento & purificação , Virologia/métodos , Animais , Aves , Cobaias
3.
Virology ; 464-465: 206-212, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25090482

RESUMO

Sialidases, enzymes that remove terminal sialic acid residues, are pivotal in various biological processes such as malignancy and infection with pathogens. For histochemical staining of sialidase activity, we have developed a new synthetic sialidase substrate, sialic acid-conjugated fluorescent benzothiazolylphenol derivative (BTP3-Neu5Ac), for rapid, sensitive, and specific fluorescent staining of sialidase activity. Here, we showed the usefulness of BTP3-Neu5Ac for histochemical fluorescent staining of cells infected with Sendai virus (SV), which possesses sialidase activity. BTP3-Neu5Ac also visualised SV-infected regions of lung sections from SV-infected mice. We succeeded in histochemical fluorescent staining of SV both in vitro and in vivo. SV has been utilised in many virological and biotechnological studies such as developments of an oncolytic virus, a gene therapy vector, and a vaccine candidate. BTP3-Neu5Ac should contribute to rapid progress of such studies and researches on viral sialidase.


Assuntos
Neuraminidase/química , Infecções por Respirovirus/virologia , Vírus Sendai/enzimologia , Coloração e Rotulagem/métodos , Proteínas Virais/química , Benzotiazóis/química , Benzotiazóis/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/metabolismo , Infecções por Respirovirus/diagnóstico , Vírus Sendai/química , Coloração e Rotulagem/instrumentação , Especificidade por Substrato , Proteínas Virais/metabolismo
4.
J Virol ; 88(15): 8445-56, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24829344

RESUMO

UNLABELLED: Some animal influenza A viruses (IAVs) bind not only to N-acetylneuraminic acid (Neu5Ac) but also to N-glycolylneuraminic acid (Neu5Gc), which has been discussed as a virus receptor. Human cells cannot synthesize Neu5Gc due to dysfunction of the CMP-Neu5Ac hydroxylase (CMAH) gene, which converts CMP-Neu5Ac to CMP-Neu5Gc. However, exogenous Neu5Gc from Neu5Gc-rich dietary sources is able to be metabolically incorporated into surfaces of tissue cells and may be related to enhancement of the infectivity and severity of IAV. Here, we investigated the receptor function of Neu5Gc on IAV infection in Neu5Gc-expressing cells by transfection of the monkey CMAH gene into human cells or by incubation with human cells in the presence of N-glycolylmannosamine. Expression of Neu5Gc on human cells clearly suppressed infectivity of IAVs that possess Neu5Gc binding ability. Furthermore, there was no difference in infectivity of a transfectant virus that included the wild-type HA gene from A/Memphis/1/1971 (H3N2), which shows no Neu5Gc binding, between parent MCF7 cells and cells stably expressing the monkey CMAH gene (CMAH-MCF7 cells). On the other hand, cell entry of the transfectant virus that included the Neu5Gc-binding HA gene with a single mutation to Tyr at position Thr155 was arrested at the stage of internalization from the plasma membrane of the CMAH-MCF7 cells. These results indicate that expression of Neu5Gc on the surface of human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding ability. Neu5Gc is suggested to work as a decoy receptor of Neu5Gc-binding IAVs but not a functional receptor for IAV infection. IMPORTANCE: Influenza A viruses (IAVs) bind to the host cell surfaces through sialic acids at the terminal of glycoconjugates. For IAV binding to sialic acids, some IAVs bind not only to N-acetylneuraminic acid (Neu5Ac) as a receptor but also to N-glycolylneuraminic acid (Neu5Gc). Neu5Gc has been discussed as a receptor of human and animal IAVs. Our results showed that Neu5Gc expression on human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding ability. Neu5Gc is suggested to be a "decoy receptor" of Neu5Gc-binding IAVs but not a functional receptor for IAV infection. Human cells cannot synthesize Neu5Gc because of dysfunction of the CMP-N-acetylneuraminic acid hydroxylase gene but can exogenously and metabolically incorporate Neu5Gc from dietary sources. The expression of Neu5Gc on human epithelial cells by taking in exogenous Neu5Gc from Neu5Gc-rich dietary sources may be related to restriction of the infection of IAVs that have acquired Neu5Gc binding ability.


Assuntos
Membrana Celular/química , Células Epiteliais/química , Células Epiteliais/virologia , Vírus da Influenza A/fisiologia , Ácidos Neuramínicos/análise , Receptores Virais/análise , Internalização do Vírus , Animais , Linhagem Celular , Haplorrinos , Humanos
5.
Biochem Biophys Res Commun ; 449(1): 32-7, 2014 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-24796674

RESUMO

We performed first-principles calculations based on the ab initio fragment molecular orbital method on dengue virus envelope protein with a hydrophobic ligand, octyl-ß-D-glucose to develop an entry inhibitor. As several polar amino acid residues are present at the edge of the pocket, the glucose moiety was chemically modified with hydrophilic groups. Introduction of both sulfated and carboxylated groups on glucose enhanced not only binding affinity to the protein but also inhibition of dengue virus entry. Octyl-2-O-sulfo ß-D-glucuronic acid may serve as a molecular probe to study the dengue virus entry process.


Assuntos
Glucuronatos/química , Glucuronatos/farmacologia , Modelos Químicos , Modelos Moleculares , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/ultraestrutura , Replicação Viral/efeitos dos fármacos , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Desenho de Fármacos , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Ativação Viral/efeitos dos fármacos , Ativação Viral/fisiologia , Replicação Viral/fisiologia
6.
PLoS One ; 9(1): e81941, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24427265

RESUMO

Sialidase removes sialic acid from sialoglycoconjugates and plays crucial roles in many physiological and pathological processes. Various human cancers express an abnormally high level of the plasma membrane-associated sialidase isoform.Visualization of sialidase activity in living mammalian tissues would be useful not only for understanding sialidase functions but also for cancer diagnosis. However, since enzyme activity of mammalian sialidase is remarkably weak compared with that of bacterial and viral sialidases, it has been difficult to detect sialidase activity in mammalian tissues. We synthesized a novel benzothiazolylphenol-based sialic acid derivative (BTP-Neu5Ac) as a fluorescent sialidase substrate. BTP-Neu5Ac can visualize sialidase activities sensitively and selectively in acute rat brain slices. Cancer cells implanted orthotopically in mouse colons and human colon cancers (stages T3-T4) were also clearly detected with BTP-Neu5Ac. The results suggest that BTP-Neu5Ac is useful for histochemical imaging of sialidase activities.


Assuntos
Imagem Molecular/métodos , Neuraminidase/metabolismo , Animais , Bactérias/enzimologia , Encéfalo/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Corantes Fluorescentes/química , Corantes Fluorescentes/toxicidade , Humanos , Hidrólise , Masculino , Mamíferos , Camundongos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Ratos , Especificidade por Substrato
7.
J Pept Sci ; 18(10): 620-5, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22961872

RESUMO

Plasmin is best known as the key molecule in the fibrinolytic system, which is critical for clot lysis and can initiate matrix metalloproteinase (MMP) activation cascade. Along with MMP, plasmin is suggested to be involved in physiological processes that are linked to the risk of carcinoma formation. Plasmin inhibitors could be perceived as a promising new principle in the treatment of diseases triggered by plasmin. On the basis of the peptidic sequence derived from the synthetic plasmin substrate, a series of peptidic plasmin inhibitors possessing nitrile as warhead were prepared and evaluated for their inhibitory activities against plasmin and other serine proteases, plasma kallikrein and urokinase. The most potent peptidic inhibitors with the nitrile warhead exhibit the potency toward plasmin (IC(50) = 7.7-11 µM) and are characterized by their selectivity profile against plasma kallikrein and urokinase. The results and molecular modeling of the peptidic inhibitor complexed with plasmin reveal that the P2 residue makes favorable contacts with the open binding pocket comprising the S2 and S3 subsites of plasmin.


Assuntos
Fibrinolisina/antagonistas & inibidores , Nitrilas/química , Oligopeptídeos/farmacologia , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Calicreína Plasmática/antagonistas & inibidores , Inibidores de Serina Proteinase/síntese química , Relação Estrutura-Atividade , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores
8.
Biochem Biophys Res Commun ; 424(3): 573-8, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22776202

RESUMO

A series of 12 carbohydrate compounds were synthesized by introduction of a sulfated group at specific positions and evaluated for their activities against dengue virus (DENV) infection as well as binding to BHK-21 cells. 3-O-sulfated GlcA was active against DENV infection, whereas 2-O-sulfated GlcA and 3,6-di-O-sulfated Glc showed negligible activity. Persulfated compounds did not inhibit DENV infection. These results provided a rationale for designing sulfated carbohydrate compounds with low molecular mass as anti-DENV agents targeting E protein functions. 3-O-Sulfated GlcA showed no significant cytotoxicity at 1mM. The EC(50) value (120 µM) was lower than that of sucrose octasulfate (SOS), a small molecular weight inhibitor of DENV infection. Two negatively charged groups, 3-O-sulfate and 6-C-carboxylic acid, appear to be essential for anti-DENV activity. We performed docking study to investigate the binding potential of 3-O-sulfated GlcA with respect to DENV E protein. The docking study showed that distance and conformation of these negative charges on the carbohydrate may be suitable for association with three amino acid residues of E protein critically involved in virus adsorption (Lys295, Ser145, and Gly159). This interaction may competitively prevent functional DENV binding to receptor(s) on host cells. In conclusion, 3-O-sulfated GlcA is a chemical probe that may facilitate exploration of the molecular mechanisms underlying manifestations of dengue diseases.


Assuntos
Antivirais/química , Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Glucuronídeos/química , Glucuronídeos/farmacologia , Ésteres do Ácido Sulfúrico/química , Ésteres do Ácido Sulfúrico/farmacologia , Animais , Linhagem Celular , Cricetinae , Vírus da Dengue/fisiologia , Ligação Proteica/efeitos dos fármacos , Proteínas do Envelope Viral/antagonistas & inibidores , Ligação Viral/efeitos dos fármacos
9.
Am J Emerg Med ; 30(7): 1326.e1-3, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21871758

RESUMO

A 42-year-old man noted decreased urine output and visited our emergency department. He said that 3 days previously, he had gotten drunk and fallen down a set of stairs. Blood tests and abdominal contrast-enhanced computed tomography revealed no abnormalities. A serum creatinine level of 5.89 mg/dL led to a diagnosis of acute renal failure and his hospitalization. After admission, his ascitic fluid level gradually increased, suggesting urine leakage into the peritoneal cavity. Microscopic examination of his ascitic fluid sediment revealed the presence of hyaline casts enclosing renal tubular epithelial cells. Cystography demonstrated contrast medium leakage into the peritoneal cavity, which led to a diagnosis of bladder rupture. Examination of ascitic fluid sediment is simple and very useful for diagnosing bladder rupture.


Assuntos
Líquido Ascítico/química , Insuficiência Renal/etiologia , Bexiga Urinária/lesões , Ferimentos não Penetrantes/diagnóstico , Adulto , Líquido Ascítico/citologia , Creatinina/sangue , Serviço Hospitalar de Emergência , Humanos , Masculino , Insuficiência Renal/diagnóstico , Ruptura/diagnóstico , Tomografia Computadorizada por Raios X , Bexiga Urinária/diagnóstico por imagem , Ferimentos não Penetrantes/complicações
10.
J Biochem ; 149(2): 191-202, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21186250

RESUMO

An escape mutant of human parainfluenza virus type 1 (hPIV1), which was selected by serial passage in the presence of a sialidase inhibitor, 4-O-thiocarbamoylmethyl-2-deoxy-2,3-didehydro-N-acetylneur-aminic acid (TCM-Neu5Ac2en), exhibited remarkable syncytium formation and virus-induced cell death in LLC-MK2 cells but no difference in susceptibility for the sialidase inhibitor TCM-Neu5Ac2en from that of wild-type hPIV1 strain C35 (WT). The mutant virus also had higher replication and plaque formation abilities. The mutant virus acquired two amino acid mutations, Glu to Gly at position 170 and Ala to Glu 442 in fusion (F) glycoprotein, but no mutations in haemaggulutinin-neuraminidase (HN) glycoprotein. Using cells co-expressing F and HN genes with site-specific mutagenesis, we demonstrated that a point mutation of Glu to Gly at position 170, which was estimated to be located in hPIV1 F glycoprotein heptad repeat 1, was required for obvious syncytium formation and caspase-3-dependent cell death. In contrast, wild-type F glycoprotein induced no synctium formation or cell death. The findings suggest that a single amino acid mutation of hPIV1 F glycoprotein promotes syncytium formation that is followed by caspase-3-dependent cell death.


Assuntos
Caspase 3/metabolismo , Proteína HN/genética , Vírus da Parainfluenza 1 Humana/genética , Vírus da Parainfluenza 1 Humana/metabolismo , Proteínas Virais de Fusão/genética , Sequência de Aminoácidos , Animais , Caspase 3/genética , Morte Celular , Linhagem Celular , Transformação Celular Viral , Células Gigantes/fisiologia , Proteína HN/metabolismo , Humanos , Cinética , Macaca mulatta , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/farmacologia , Neuraminidase/antagonistas & inibidores , Ligação Proteica/fisiologia , Proteínas Virais de Fusão/metabolismo , Replicação Viral/fisiologia
11.
Biochem Biophys Res Commun ; 382(4): 776-9, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19324010

RESUMO

Leucine-rich alpha(2)-glycoprotein (LRG) is a plasma protein in which leucine-rich repeats (LRRs) were first discovered. Although the physiological function of LRG is not known, increases in the serum level of LRG have been reported in various diseases. In this study, we found that LRG was induced by recombinant human IL-6 in human hepatoma HepG2 cells. The induction of LRG by IL-6 was up-regulated synergistically with either IL-1beta or TNFalpha in a pattern similar to those for type 1 acute-phase proteins. We also found that lipopolysaccharide (LPS) administered intraperitoneally to mice enhanced dose-dependently the expression of LRG mRNA in the liver as well as those for mouse major acute-phase proteins. These results strongly suggest that LRG was a secretory type 1 acute-phase protein whose expression was up-regulated by the mediator of acute-phase response.


Assuntos
Reação de Fase Aguda/metabolismo , Endotoxemia/metabolismo , Glicoproteínas/biossíntese , Hepatócitos/metabolismo , Animais , Linhagem Celular , Citocinas/metabolismo , Citocinas/farmacologia , Modelos Animais de Doenças , Glicoproteínas/genética , Glicoproteínas/fisiologia , Hepatócitos/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos ICR , Regulação para Cima
12.
Bioorg Med Chem Lett ; 15(8): 2141-4, 2005 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-15808485

RESUMO

The sulfur analogue of sphingomyelin was designed and stereoselectively synthesized from S-benzyl-N-Boc-cysteine. The introduction of the phosphoryl choline moiety was successfully achieved by our own method using 2-bromoethyl dimethyl phosphite and carbon tetrabromide followed by a trimethylamine treatment. The synthesized compound proved to be a useful substrate for monitoring the enzyme activity of sphingomyelinase by detecting the liberated thiol group with a thiol-sensitive reagent.


Assuntos
Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/síntese química , Compostos de Enxofre/síntese química , Bacillus cereus/efeitos dos fármacos , Bacillus cereus/enzimologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielinas/metabolismo , Compostos de Enxofre/metabolismo
13.
Chem Pharm Bull (Tokyo) ; 52(12): 1479-82, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15577250

RESUMO

Peptide mimics derived from the first extracellular loop of CCR5 bearing non-peptide spacers in place of Ala-Ala-Ala sequence in the peptide moiety were synthesized, and the effects of these compounds on the inhibition against HIV-1 were examined. Compound 2b having m-aminophenoxyacetic acid derivative as a non-peptide spacer significantly inhibited HIV-1.


Assuntos
Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Peptídeos/farmacologia , Receptores CCR5/química , Sequência de Aminoácidos , Linhagem Celular , Cromatografia em Camada Fina , Humanos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Receptores CCR5/efeitos dos fármacos , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Espectrofotometria Infravermelho
14.
Arch Biochem Biophys ; 424(2): 201-9, 2004 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-15047192

RESUMO

Phosphate analogs have been known to inhibit competitively various phosphatases and phospholipase C and D. We found for the first time that only beryllium fluoride (BeF(x)) among the phosphate analogs studied inhibits Bacillus cereus sphingomyelinase (SMase) activity. The active inhibitory species proved to be not BeF(3)(-) but BeF(2) by the measurement of SMase activity and of (19)F NMR spectroscopy in the presence of a fixed concentration of BeCl(2) and different concentrations of NaF, although both the species have been reported for other kinds of enzymes. The result of kinetic experiment also indicated that the BeF(x) binds in the vicinity of the essential binding site for the substrate and that the Mg(2+) binding to SMase is essential for the binding of BeF(x) to the enzyme.


Assuntos
Bacillus cereus/enzimologia , Berílio/química , Cobalto/química , Inibidores Enzimáticos/química , Fluoretos/química , Manganês/química , Fosfatos/química , Esfingomielina Fosfodiesterase/química , Ativação Enzimática , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/metabolismo
15.
Toxicon ; 42(5): 481-90, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14529729

RESUMO

Snake venoms are a very abundant source of nerve growth factors (NGF). NGFs of Elapidae showing 65% sequence homology with mouse or human NGF, while the Viperidae NGF shows N-glycosylation (Asn-21) typical of these mammalian NGFs. Snake NGF-induced neurite outgrowth (neurotropic activity) was measured in the past by using PC12 cell or dorsal root ganglion bioassays. The present study was aimed at comparing, by dose-response experiments, the neurotropic activity of cobra and vipera versus mammalian NGFs, by using a novel bioassay involving PC12 cells genetically engineered to overexpress NGF-trkA receptors of human origin. These cells respond to NGF by differentiation (morphologically expressed as neurite outgrowth) by a process mediated by NGF-trkA receptors. This process was evaluated by two different criteria: (1) elongation of neurites (E), and (2) Percentage of responsive cells (PRC) determined by digital acquisition of data and computer analysis. We found that snake venom NGFs were less potent than mouse NGF, and that cobra NGF was more potent than vipera NGF. These data indicate the following order of NGF activity towards recombinant human trkA receptors: recombinant human NGF>mouse NGF>cobra NGF>vipera NGF. The neurotropic efficacy of these NGFs was found to be similar, reaching 80-90% of maximal activity obtained with all NGF forms. Interestingly, cobra (but not vipera) NGF demonstrated prolonged neurotropic activity compared with mouse NGF. The results of the present study indicate that cobra and vipera venom NGFs represent natural agonists of human trkA-receptor of a lower potency, but of similar efficacy, compared with mammalian NGFs. These compounds are important pharmacological tools to characterize the trkA receptor structure-function relationship, and to develop novel neurotropic drugs.


Assuntos
Bioensaio/métodos , Venenos Elapídicos/farmacologia , Fatores de Crescimento Neural/efeitos dos fármacos , Receptor trkA/metabolismo , Venenos de Víboras/farmacologia , Animais , Relação Dose-Resposta a Droga , Humanos , Camundongos , Fatores de Crescimento Neural/isolamento & purificação , Neuritos/efeitos dos fármacos , Células PC12 , Ratos , Receptor trkA/genética , Proteínas Recombinantes/efeitos dos fármacos
16.
Histochem Cell Biol ; 117(5): 453-8, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12029493

RESUMO

A novel fluorescent cytochemical method for sialidase activity was developed using 5-bromo-4-chloroindol-3-yl-alpha- D- N-acetylneuraminic acid (X-Neu5Ac) as the substrate. Intact nuclei were isolated from porcine liver and incubated at 37 degrees C for 3 h with 1 mM X-Neu5Ac at pH 4.8. The nuclei were stained with blue color that was derived from the oxidized compound of the reaction product X (5-bromo-4-chloro-3-hydroxyindole). A specific sialidase inhibitor, 2,3-dehydro-2-deoxy- N-acetylneuraminic acid, suppressed the staining in a dose-dependent manner. Despite the specificity of the cytochemical reaction, the staining was too weak to analyze the staining distribution and pattern of individual nuclei. To attain more sensitive detection of sialidase activity, the nuclei were incubated with X-Neu5Ac in the presence of Fast Red Violet LB. Individual nuclei of porcine liver were clearly stained with fluorescence that was produced by the conjugated compound of product X with Fast Red Violet LB. This fluorescent cytochemical method was also employed successfully for detection of sialidase activity of intact GOTO neuroblastoma cells in culture. The present method should provide a useful tool for investigating the localization and stage-specific expression of sialidase activity in tissues and cells.


Assuntos
Histocitoquímica/métodos , Indóis/metabolismo , Ácidos Neuramínicos/metabolismo , Neuraminidase/metabolismo , Compostos Orgânicos , Animais , Núcleo Celular/enzimologia , Corantes , Corantes Fluorescentes/química , Indóis/química , Fígado/enzimologia , Ácidos Neuramínicos/química , Especificidade por Substrato , Suínos , Células Tumorais Cultivadas/enzimologia
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