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1.
Anal Biochem ; 607: 113831, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32739346

RESUMO

We developed a new procedure for the comprehensive analysis of metabolites and enzymes involved in polyamine metabolism pathways. The procedure utilizes stable isotope-labeled polyamines and directly and precisely determines labeled products from enzymatic reactions by ESI-Q-TOF-MS. The activity of different enzymes could be determined in essentially the same manner by suitably adjusting the reaction conditions for each individual enzyme. We applied the procedure to extracts of regenerating rat liver and analyzed the changes in polyamine-metabolizing enzymes and polyamine contents during recovery from partial hepatectomy. A general outline of polyamine metabolism and information of polyamine dynamics were obtained. This kind of comprehensive information would be valuable in unifying detailed but fragmentary information obtained through conventional analyses focusing on one or a few enzymes and on a limited aspect of polyamine metabolic pathway.


Assuntos
Enzimas/metabolismo , Poliaminas/análise , Poliaminas/metabolismo , Animais , Isótopos de Carbono/química , Ativação Enzimática , Marcação por Isótopo , Fígado/metabolismo , Masculino , Metionina/química , Ratos , Espectrometria de Massas por Ionização por Electrospray , Espermidina/química , Espermidina/metabolismo , Espermina/química , Espermina/metabolismo
2.
J Plant Res ; 125(2): 311-6, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22246111

RESUMO

Early interactions between invading penetration hyphae of the pathogenic fungus Magnaporthe oryzae and rice cells occur at the apoplast, the free diffusional space outside the plasma membrane of leaves. After initial colonization, intercellular hyphae are again in intimate contact with the rice apoplast. While several studies have looked at proteomics in rice-Magnaporthe interactions, none have focused on apoplast localized proteins. We adjusted a protocol for intercellular washing fluids (IWF) to rice leaves infected with Magnaporthe oryzae for proteomic analysis. In our IWF extract, we identified several proteins associated with compatible or incompatible pathogen interactions. Three DUF26 domain proteins were identified as changing in abundance 12 h after inoculation, confirming DUF26 domain-containing proteins are among early, pathogen stress-responsive proteins induced by infection with Magnaporthe oryzae. A Magnaporthe cyclophilin, previously identified as a virulence factor was also identified in the intercellular washing fluid.


Assuntos
Líquido Extracelular/metabolismo , Proteínas Fúngicas/metabolismo , Magnaporthe/fisiologia , Oryza/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Eletroforese em Gel Bidimensional , Interações Hospedeiro-Patógeno , Espectrometria de Massas , Oryza/microbiologia , Oryza/fisiologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteoma , Proteômica
3.
J Neurochem ; 119(3): 569-78, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21883218

RESUMO

Activation of the Keap1/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway and consequent induction of phase 2 antioxidant enzymes is known to afford neuroprotection. Here, we present a series of novel electrophilic compounds that protect neurons via this pathway. Natural products, such as carnosic acid (CA), are present in high amounts in the herbs rosemary and sage as ortho-dihydroquinones, and have attracted particular attention because they are converted by oxidative stress to their active form (ortho-quinone species) that stimulate the Keap1/Nrf2 transcriptional pathway. Once activated, this pathway leads to the production of a series of antioxidant phase 2 enzymes. Thus, such dihydroquinones function as redox-activated 'pro-electrophiles'. Here, we explored the concept that related para-dihydroquinones represent even more effective bioactive pro-electrophiles for the induction of phase 2 enzymes without producing toxic side effects. We synthesized several novel para-hydroquinone-type pro-electrophilic compounds (designated D1 and D2) to analyze their protective mechanism. DNA microarray, PCR, and western blot analyses showed that compound D1 induced expression of heat-shock proteins (HSPs), including HSP70, HSP27, and DnaJ, in addition to phase 2 enzymes such as hemeoxygenase-1 (HO-1), NADP(H) quinine-oxidoreductase1, and the Na(+)-independent cystine/glutamate exchanger (xCT). Treatment with D1 resulted in activation of Nrf2 and heat-shock transcription factor-1 (HSF-1) transcriptional elements, thus inducing phase 2 enzymes and HSPs, respectively. In this manner, D1 protected neuronal cells from both oxidative and endoplasmic reticulum (ER)-related stress. Additionally, D1 suppressed induction of 78 kDa glucose-regulated protein (GRP78), an ER chaperone protein, and inhibited hyperoxidation of peroxiredoxin 2 (PRX2), a molecule that is in its reduced state can protect from oxidative stress. These results suggest that D1 is a novel pro-electrophilic compound that activates both the Nrf2 and HSF-1 pathways, and may thus offer protection from oxidative and ER stress.


Assuntos
Antioxidantes/metabolismo , Proteínas de Ligação a DNA/fisiologia , Fator 2 Relacionado a NF-E2/fisiologia , Fármacos Neuroprotetores/farmacologia , Quinonas/farmacologia , Epitélio Pigmentado da Retina/enzimologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/fisiologia , Antioxidantes/síntese química , Antioxidantes/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Chaperona BiP do Retículo Endoplasmático , Fatores de Transcrição de Choque Térmico , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , Fármacos Neuroprotetores/síntese química , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Quinonas/síntese química , Epitélio Pigmentado da Retina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo
4.
Vet J ; 190(3): 378-82, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21277238

RESUMO

The carboxylesterase cauxin is a major urinary protein in cats that is also found in seminal fluid (SF). This study investigated cauxin in feline SF including biochemical features, concentration, distribution and gene expression in epididymal tissue, and its reaction with acylglycerol substrates. Monomeric, dimeric, and/or multimeric forms of cauxin carrying N-glycosylations were detected on Western blots of feline SF but most were monomeric. Cauxin concentrations were markedly lower in SF (0.042±0.020 mg/mL) than in urine (∼0.5 mg/mL) and cauxin gene expression was 60-fold lower in the epididymis than in the kidney. Immunohistochemical examination localised cauxin within the stereocilia and cytoplasm of epithelial cells lining the caput and corpus epididymis. Cauxin-positive spermatozoa were detected in the lumen of the cauda epididymis but not in the cytoplasm of the epithelial cell lining. Using an in vitro assay, cauxin hydrolysed saturated 1-mono- but not di- and tri-acylglycerols. The results suggest that cauxin secreted from the caput and corpus epididymis acts as an esterase on lipid within feline SF.


Assuntos
Carboxilesterase/análise , Gatos , Epididimo/enzimologia , Glicerídeos/metabolismo , Sêmen/enzimologia , Animais , Western Blotting/veterinária , Carboxilesterase/genética , Carboxilesterase/urina , Expressão Gênica , Rim/enzimologia , Masculino , RNA Mensageiro/análise , Testículo/enzimologia
5.
Vet J ; 189(3): 312-7, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20920865

RESUMO

Proteinuria is an important indicator of urinary tract disease and urine dipsticks are simple and sensitive tools to screen for this marker. However, the use of dipsticks to screen for proteinuria may not be appropriate in cats, since cauxin, a 70 kDa glycoprotein, is secreted by the kidneys in clinically normal animals of this species. To circumvent this problem, a Lens culinaris agglutinin (LCA) lectin tip was developed to remove cauxin from feline urine, followed by conventional urine dipstick testing for proteinuria. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue R-250 staining indicated that >90% cauxin in the urine of 13 clinically normal cats was trapped by the LCA lectin tip, so that the dipstick protein 'score' changed from 'positive' (≥30 mg/dL) for untreated urine to 'negative' (≤10 mg/dL) for lectin tip-treated urine. In contrast, SDS-PAGE indicated that lectin tip-treated samples from 20 animals with renal disease contained high concentrations of albumin and low-molecular weight proteins; dipstick testing of lectin tip-treated urine resulted in a consistently positive protein score. The accuracy of the dipstick method for detecting cats with abnormal proteinuria is enhanced if dipsticks are used with urine samples that have first been passed through the LCA lectin tip.


Assuntos
Carboxilesterase , Doenças do Gato/urina , Proteinúria/veterinária , Urinálise/métodos , Aglutininas/química , Animais , Doenças do Gato/diagnóstico , Gatos , Eletroforese em Gel de Poliacrilamida/veterinária , Feminino , Lens (Planta)/química , Masculino , Lectinas de Plantas/química , Valor Preditivo dos Testes , Proteinúria/diagnóstico , Proteinúria/urina , Fitas Reagentes
6.
Biosci Biotechnol Biochem ; 74(11): 2293-8, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21071846

RESUMO

Sendai virus (SeV) is an enveloped virus with a non-segmented negative-strand RNA genome. SeV envelope fusion (F) glycoproteins play crucial roles in the viral life cycle in processes such as viral binding, assembly, and budding. In this study, we developed a viable recombinant SeV designated F-EGFP SeV/ΔF, in which the F protein was replaced by an F protein fused to EGFP at the carboxyl terminus. Living infected cells of the recombinant virus were directly visualized by green fluorescence. The addition of EGFP to the F protein maintained the activities of the F protein in terms of intracellular transport to the plasma membrane via the ER and the Golgi apparatus and fusion activity in the infected cells. These results suggest that this fluorescent SeV is a useful tool for studying the viral binding, assembly, and budding mechanisms of F proteins and the SeV life cycle in living infected cells.


Assuntos
Proteínas de Fluorescência Verde/genética , Vírus Sendai/genética , Proteínas do Envelope Viral/genética , Fluorescência , Transporte Proteico , Proteínas Recombinantes de Fusão , Projetos de Pesquisa , Proteínas Virais de Fusão/fisiologia
7.
J Vet Med Sci ; 71(10): 1343-7, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19887741

RESUMO

We have cloned the arginine deiminase (ADI) gene from Mycoplasma hominis PG21 genomic DNA by polymerase chain reaction, and changed four TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The recombinant ADI (rADI) was purified to apparent homogeneity by Ni-affinity chromatography after extraction from inclusion bodies followed by refolding. The rADI expressed in E. coli was estimated to be 50 kDa. Dimeric forms of rADI exerted enzymatic activity. We found that high concentration of potassium dihydrogenphosphate (PDP) and L-arginine addition in refolding reaction increases the enzyme activity. The specific activity of rADl was calculated as 0.618 U/mg. In addition, the enzyme activity of purified rADI remained for at least one month in 100 mM PDP solution (pH 6.5), but diminished within one week in 100 mM PDP solution (pH 7.4). Anti-tumor activity of the purified rADI was estimated to be 0.036 U/ml as 50% growth inhibitory activity against human melanoma cell line G-361.


Assuntos
Hidrolases/metabolismo , Hidrolases/farmacologia , Melanoma/metabolismo , Mycoplasma hominis/enzimologia , Proteínas Recombinantes , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Hidrolases/química , Hidrolases/genética
8.
Res Vet Sci ; 87(3): 408-12, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19386337

RESUMO

Recombinant adeno-associated virus (rAAV) vectors provide excellent gene delivery into the kidney in several mammals. This study evaluated gene delivery into the cat kidney using an rAAV vector. First, infection and reporter gene expression using rAAV vector encoding the enhanced green fluorescent protein gene (rAAV-EGFP) was examined in vitro in epithelial crandell reese feline kidney (CRFK) cells. At 12h after transduction, green fluorescence was detected in cells. Next, the rAAV-EGFP construct was injected into the kidneys of two anesthetized cats via the skin, similar to a renal biopsy. On 3 and 12days after injection, green fluorescence was detected in renal tubules localized near the injected site, but not in glomeruli, blood vessels, or interstitial cells. Finally, the rAAV-EGFP construct was transduced into kidney sections cultured ex vivo. EGFP was expressed in renal tubules between the outer cortex and inner medulla regions. These results demonstrate that rAAV vectors effectively mediate gene delivery into cat renal tubules, and may prove usefulness in gene therapy for cats with renal diseases.


Assuntos
Adenoviridae , Gatos , Terapia Genética/veterinária , Rim/fisiologia , Animais , Linhagem Celular , Proteínas de Fluorescência Verde , Rim/citologia , Masculino , Técnicas de Cultura de Tecidos
9.
J Exp Bot ; 58(10): 2617-25, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17586608

RESUMO

P23k is a monocot-unique protein that is highly expressed in the scutellum of germinating barley seed. Previous expression analyses suggested that P23k is involved in sugar translocation and/or sugar metabolism. However, the role of P23k in barley physiology remains unclear. Here, to elucidate its physiological function, BSMV-based virus-induced gene silencing (VIGS) of P23k in barley leaves was performed. Expression and localization analyses of P23k mRNA in barley leaves showed up-regulation of P23k transcript with increased photosynthetic activity and the localization of these transcripts to the vascular bundles and sclerenchyma, where secondary wall formation is most active. VIGS of the P23k gene led to abnormal leaf development, asymmetric orientation of main veins, and cracked leaf edges caused by mechanical weakness. In addition, histochemical analyses indicated that the distribution of P23k in leaves coincides with the distribution of cell wall polysaccharides. Considering these results together, it is proposed that P23k is involved in the synthesis of cell wall polysaccharides and contributes to secondary wall formation in barley leaves.


Assuntos
Parede Celular/metabolismo , Inativação Gênica , Hordeum/genética , Proteínas de Plantas/genética , Parede Celular/ultraestrutura , Técnicas Genéticas , Hordeum/ultraestrutura , Hordeum/virologia , Folhas de Planta/anatomia & histologia , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Proteínas de Plantas/análise , Polissacarídeos/biossíntese , RNA Mensageiro/análise , RNA Mensageiro/metabolismo
10.
Biosci Biotechnol Biochem ; 71(3): 811-6, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17341822

RESUMO

Cauxin is a carboxylesterase-like glycoprotein excreted as a major component of cat urine. Cauxin contains four putative N-glycosylation sites. We characterized the structure of an N-linked oligosaccharide of cauxin using nano liquid chromatography (LC)-electrospray ionization (ESI) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) and MS/MS, and high-performance liquid chromatography (HPLC) with an octadecylsilica (ODS) column. The structure of the N-linked oligosaccharide of cauxin attached to (83)Asn was a bisecting complex type, Galbeta1-4GlcNAcbeta1-2Manalpha1-3(Galbeta1-4GlcNAcbeta1-2Manalpha1-6)(GlcNAcbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc.


Assuntos
Carboxilesterase/química , Polissacarídeos/química , Sequência de Aminoácidos , Animais , Gatos , Glicosilação , Dados de Sequência Molecular , Nanotecnologia , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
Res Vet Sci ; 82(1): 76-9, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16919690

RESUMO

Cauxin, a member of mammalian carboxylesterases (EC 3.1.1.1), is excreted as a major urinary protein in the domestic cat. Urinary cauxin is derived from the kidney proximal straight tubules. Here, we report changes in the renal expression and urinary excretion of cauxin in cats with tubulointerstitial nephritis (TIN). Immunohistochemistry using anti-cauxin antibody showed fewer cauxin-positive tubules in 15 TIN cases than in normal animals. In areas with tubulointerstitial damage, fibroblasts and inflammatory cells replaced renal tubules, and cauxin-positive tubules consequently disappeared. Urine was analysed in six of the 15 cases. In the two cases with mild tubulointerstitial changes, urinary cauxin was detected using SDS-PAGE with Coomassie staining. In the four cases with severe tubulointerstitial changes, urinary cauxin was below the detection limit using Western blotting. These results indicate that the renal expression and urinary excretion of cauxin decrease with the progression of TIN in cats.


Assuntos
Carboxilesterase/metabolismo , Doenças do Gato/metabolismo , Doenças do Gato/urina , Regulação para Baixo , Rim/metabolismo , Nefrite Intersticial/veterinária , Animais , Gatos , Feminino , Rim/citologia , Masculino , Nefrite Intersticial/metabolismo , Nefrite Intersticial/urina
12.
Chem Biol ; 13(10): 1071-9, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17052611

RESUMO

Domestic cats spray urine with species-specific odor for territorial marking. Felinine (2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid), a putative pheromone precursor, is excreted in cat urine. Here, we report that cauxin, a carboxylesterase excreted as a major urinary component, regulates felinine production. In vitro enzyme assays indicated that cauxin hydrolyzed the felinine precursor 3-methylbutanol-cysteinylglycine to felinine and glycine. Cauxin and felinine were excreted age dependently after 3 months of age. The age-dependent increases in cauxin and felinine excretion were significantly correlated. In mature cats, cauxin and felinine levels were sex-dependently correlated and were higher in males than in females. In headspace gas of cat urine, 3-mercapto-3-methyl-1-butanol, 3-mercapto-3-methylbutyl formate, 3-methyl-3-methylthio-1-butanol, and 3-methyl-3-(2-methyldisulfanyl)-1-butanol were identified as candidates for felinine derivatives. These findings demonstrate that cauxin-dependent felinine production is a cat-specific metabolic pathway, and they provide information for the biosynthetic mechanisms of species-specific molecules in mammals.


Assuntos
Carboxilesterase/metabolismo , Cisteína/análogos & derivados , Dipeptídeos/metabolismo , Feromônios/biossíntese , Animais , Carboxilesterase/urina , Gatos , Cisteína/biossíntese , Cisteína/química , Cisteína/urina , Dipeptídeos/química , Dipeptídeos/urina , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Concentração de Íons de Hidrogênio , Masculino , Dados de Sequência Molecular , Estrutura Molecular , Oligopeptídeos/biossíntese , Oligopeptídeos/química , Oligopeptídeos/urina , Pentanóis/química , Pentanóis/urina , Feromônios/química , Feromônios/urina , Sensibilidade e Especificidade , Maturidade Sexual/fisiologia
14.
Artigo em Inglês | MEDLINE | ID: mdl-17045831

RESUMO

Domestic cats exhibit physiological proteinuria due to the excretion of cauxin, a carboxylesterase, into the urine. In the present report, we demonstrate that cauxin is excreted in a species-, sex-, and age-dependent manner. Although the cauxin gene is conserved in mammals, including human, mouse, and dog, urinary cauxin was found only in member of the genus Felis and lynx (bobcat, and lynx) and not in other Felidae (genus: Panthera and puma) tested. In mature cats, cauxin excretion was higher in intact males than in castrated males or in intact or spayed females. Daily cauxin excretion decreased immediately after castration. Immunohistochemistry confirmed that cauxin expression in the kidney proximal straight tubules was higher in intact males than in castrated males. Urinary cauxin was detectable by Western blotting in cats older than about 3 months, and its excretion increased with age. In a zymographic esterase assay, urine contained a major cauxin band; by contrast, kidney homogenates contained three major bands, comprising two carboxylesterases and an unidentified esterase, and one minor cauxin band. These results suggest that 1. cauxin excretion is regulated by sex hormones, such as testosterone, 2. cauxin functions as an esterase in the urine rather than in kidney cells, and 3. the decomposition products by cauxin are excreted in a species-, sex-, and age-dependent manner, as is cauxin itself.


Assuntos
Carboxilesterase/urina , Gatos/urina , Felidae/urina , Envelhecimento , Animais , Carboxilesterase/metabolismo , Castração , Gatos/metabolismo , Felidae/metabolismo , Feminino , Rim/enzimologia , Masculino , Filogenia , Caracteres Sexuais , Especificidade da Espécie , Distribuição Tecidual
15.
Biosci Biotechnol Biochem ; 70(7): 1717-23, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16861808

RESUMO

Double-stranded RNA-activated protein kinase (PKR) plays an important role in interferon-induced antiviral responses, and is also involved in intracellular signaling pathways, including the apoptosis, proliferation, and transcription pathways. In this study, a new 120-kDa PKR-associated protein designated double-stranded RNA binding protein (DRBP)-120 was identified using co-immunoprecipitation with anti-PKR antiserum and two-dimensional electrophoresis. Furthermore, DRBP-120 is a double-stranded RNA (dsRNA)-binding protein, and it was detected in both the cytoplasm and the nucleus of HeLa cells associated with PKR.


Assuntos
RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo , eIF-2 Quinase/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Células HeLa , Humanos , Proteínas do Fator Nuclear 90/metabolismo , Ligação Proteica
16.
Eur J Biochem ; 271(2): 356-66, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14717703

RESUMO

Silk fibroin of Bombyx mori is secreted from the posterior silk gland (PSG) as a 2.3-MDa elementary unit, consisting of six sets of a disulfide-linked heavy chain (H-chain)-light chain (L-chain) heterodimer and one molecule of fibrohexamerin (fhx)/P25. Fhx/P25, a glycoprotein, associates noncovalently with the H-L heterodimers. The elementary unit was found and purified from the endoplasmic reticulum (ER) extract of PSG cells. A substantial amount of fhx/P25 unassembled into the elementary unit was also present in ER. In normal-level fibroin-producing breeds (J-139 and C108), the elementary unit contained fhx/P25 of either 30 kDa (major) or 27 kDa (minor). The 27-kDa fhx/P25 was produced from the 30-kDa form by digestion with the bacterial alpha1,2-mannosidase in vitro. The elementary unit in the ER extract contained only the 30-kDa fhx/P25, whereas both 30- and 27-kDa forms of fhx/P25 were present in the ER plus Golgi mixed extracts. In naked-pupa mutants [Nd(2), Nd-s and Nd-sD], extremely small amounts of fibroin were produced and they consisted of one molecule of 27-kDa fhx/P25 and six molecules of H-chain but no L-chain. When the Nd-sD mutant was subjected to transgenesis with the normal L-chain gene, the (H-L)6fhx1-type elementary unit containing the 30-kDa fhx/P25, was produced. These results suggest that fhx/P25 in the elementary unit is largely protected from digestion with Golgi alpha1,2-mannosidases when L-chains are present in the unit. Models suggesting a role of L-chain for the protection of alpha1,2-mannose residues of fhx/P25 are presented.


Assuntos
Retículo Endoplasmático/metabolismo , Fibroínas/metabolismo , Fibroínas/fisiologia , Glicoproteínas/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Insetos/fisiologia , Manosidases/metabolismo , Animais , Animais Geneticamente Modificados , Western Blotting , Bombyx/fisiologia , Ensaio de Imunoadsorção Enzimática , Complexo de Golgi/enzimologia , Larva/genética , Larva/metabolismo , Manose/metabolismo , Pupa/genética , Pupa/metabolismo , Seda , Transformação Genética , Transgenes
17.
Biochem Pharmacol ; 66(11): 2213-21, 2003 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-14609746

RESUMO

We reported previously that the protopanaxatriol saponins in Panax ginseng greatly reduce the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh). However, protopanaxadiol saponins showed only slight inhibitory effects. Recent studies have demonstrated that oligosaccharides connected to the hydroxyl groups of the aglycone in ginseng saponins (ginsenosides) are in turn hydrolyzed in the digestive tract and absorbed into the circulation following oral administration of ginseng. Therefore, the present study was performed to investigate the effects of the major ginsenoside metabolites (M1, M2, M3, M4, M5, M11, and M12) on catecholamine secretion. All of these metabolites were shown to be potent inhibitors of ACh-evoked secretion, and M4 was the most effective. M4 blocked not only the ACh-induced Na(+) influx into the chromaffin cells but also the ACh-induced inward current into Xenopus oocytes expressing human alpha 3 beta 4 neuronal nicotinic ACh receptors. M4 reduced the secretion induced by high K(+), an activator of voltage-sensitive Ca(2+) channels, to a much lesser extent than that evoked by ACh. M1, M2, M3, M5, and M12 are protopanaxadiol saponin-derived metabolites. Therefore, these results imply that the protopanaxadiol saponins are prodrugs, and they show more potent inhibitory activity following metabolism in the digestive tract. The results further suggest that the metabolites act on nicotinic ACh receptors, blocking Na(+) influx through the receptors, and consequently reduce the catecholamine secretion from bovine adrenal chromaffin cells. The inhibitory effect of ginsenoside metabolites is probably one of the mechanisms of action responsible for the pharmacological effects of ginseng.


Assuntos
Catecolaminas/antagonistas & inibidores , Catecolaminas/metabolismo , Células Cromafins/efeitos dos fármacos , Células Cromafins/metabolismo , Panax/metabolismo , Saponinas/metabolismo , Animais , Bovinos , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Humanos , Sapogeninas/metabolismo , Sapogeninas/farmacologia , Saponinas/farmacologia , Triterpenos/metabolismo , Triterpenos/farmacologia , Xenopus
18.
Biosci Biotechnol Biochem ; 67(7): 1522-9, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12913296

RESUMO

Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of four subunits: alpha (51 kDa), beta (26 kDa), gamma (49 kDa), and delta (33 kDa). The EF-1alpha subunit catalyzes the binding of aminoacyl-tRNA to the ribosome concomitant with the hydrolysis of GTP. The EF-1alpha-bound GDP is then exchanged for GTP by the EF-1betagammadelta complex. To facilitate analysis of the roles of the individual EF-1beta, gamma, and delta subunits in GDP/GTP exchange on EF-1alpha, we cloned the cDNAs for these subunits and expressed them in Escherichia coli. EF-1beta, EF-1gamma, and the carboxyl-terminal half of EF-1delta were expressed, purified, and examined for protein:protein interactions by gel filtration chromatography and by a quartz-crystal microbalance method. An 80-kDa species containing EF-1beta and gamma subunits in a 1:1 molar ratio was detected by gel filtration. A higher molecular weight species containing an excess of EF-1gamma relative to EF-1beta was also detected. The amino-terminal region of EF-1beta (amino acid residues 1-129) was sufficient for binding to EF-1gamma. The carboxyl-terminal half of EF-1delta did not appear to form a complex with EF-1gamma.


Assuntos
Bombyx/química , Proteínas de Insetos/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia em Gel , Sequência Conservada , Dados de Sequência Molecular , Fator 1 de Elongação de Peptídeos/química , Fator 1 de Elongação de Peptídeos/genética , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Seda , Especificidade da Espécie , Pesos e Medidas
19.
Biosci Biotechnol Biochem ; 67(3): 592-8, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12723608

RESUMO

The roles of N-linked glycosylation in the intracellular transport and biological activities of the Sendai virus hemagglutinin-neuraminidase (HN) protein were studied. The protein contains four potential N-glycosylation sites: N77, N448, N499, and N511. By site-directed mutagenesis of these positions, the mature protein contained three N-linked oligosaccharides attached to N77, N499, and N511. The role of each added oligosaccharide in the structure and functions of the protein was identified by characterization of surface expression, hemadsorption, and neuraminidase activities of the corresponding mutant proteins. Elimination of the sites of N499 and N511 had the most detrimental effect, decreasing surface expression and hemadsorption. Elimination of the sites of N77 and N448 had similar but weaker effects. Mutants missing the sites of N499 and N511 were not able to induce syncytia formation in cells expressing mutant HN proteins and the fusion protein. Therefore, the N-linked oligosaccharides attached to N499 and N511 were important for intracellular transport and for the fusion promotion.


Assuntos
Proteína HN/química , Proteína HN/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Vírus Sendai/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Antígenos de Superfície/análise , Asparagina/genética , Sequência de Bases , Sítios de Ligação , Células COS , Técnica Direta de Fluorescência para Anticorpo , Glicosilação , Proteína HN/genética , Células HeLa , Hemadsorção , Humanos , Mutagênese Sítio-Dirigida , Vírus Sendai/metabolismo , Relação Estrutura-Atividade , Transfecção , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo
20.
Biochem J ; 370(Pt 1): 101-10, 2003 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-12401131

RESUMO

Normal mammals generally excrete only small amounts of protein in the urine, thus avoiding major leakage of proteins from the body. Proteinuria is the most commonly recognized abnormality in renal disease. However, healthy domestic cats ( Felis catus ) excrete proteins at high concentrations (about 0.5 mg/ml) in their urine. We investigated the possible cause of proteinuria in healthy cats, and discovered a 70 kDa glycoprotein, which was excreted as a major urinary protein in cat urine, irrespective of gender. To elucidate the biochemical functions and the excretion mechanism of this protein, we cloned the cDNA for this protein from a cat kidney cDNA library. The deduced amino acid sequence shared 47% identity with the rat liver carboxylesterase (EC 3.1.1.1), and both the serine hydrolase active site and the carboxylesterase-specific sequence were conserved. Therefore we named this protein cauxin (carboxylesterase-like urinary excreted protein). In contrast to the mammalian carboxylesterases, most of which are localized within the cells of various organs, cauxin was expressed specifically in the epithelial cells of the distal tubules, and was secreted efficiently into the urine, probably because it lacked the endoplasmic reticulum retention sequence (HDEL). Based on our finding that cauxin is not expressed in the immature cat kidney, we conclude that cauxin is involved in physiological functions that are specific for mature cats. Recently, cauxin-like cDNAs were found from human brain and teratocarcinoma cells. These data suggest that cauxin and cauxin-like human proteins are categorized as a novel group of carboxylesterase multigene family.


Assuntos
Carboxilesterase/urina , Hidrolases de Éster Carboxílico/urina , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Gatos , Clonagem Molecular , DNA Complementar , Feminino , Imuno-Histoquímica , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
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