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1.
J Appl Microbiol ; 122(4): 893-899, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28035713

RESUMO

AIMS: Quaternary ammonium compounds (QACs), including benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC) are cationic surfactants and have been used widely as general disinfectants in the medical field due to their strong antibacterial effects and low cytotoxicity to human cells. 4,4'-(α,ω-hexametylenedithio) bis (1-octylpyridinium bromide) (4DTBP-6,8) is one of the potent bis-QACs synthesized to improve the antimicrobial activities of mono-QACs such as BAC. This study aimed to assess the effectiveness of 4DTBP-6,8 against Pseudomonas aeruginosa, a prevalent hospital pathogen. METHODS AND RESULTS: The minimum inhibitory concentrations of 4DTBP-6,8, CPC and BAC against P. aeruginosa were measured. 4DTBP-6,8 exhibited strong antibacterial activity. We assessed the bactericidal effects of QACs against P. aeruginosa under certain conditions and their cytotoxicities in human epithelial cells using lactate dehydrogenase (LDH) release. 4DTBP-6,8 exerted excellent bactericidal effects against high concentrations of bacteria, biofilm cells and even in the presence of contaminated proteins. Cellular LDH was not released by the treatment with 4DTBP-6,8. CONCLUSIONS: 4DTBP-6,8 exhibited the strongest bactericidal activity against P. aeruginosa among the three QACs tested without any cytotoxicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The potent bis-QAC, 4DTBP-6,8 has the potential to be an effective disinfectant in preventing hospital infections caused by P. aeruginosa.


Assuntos
Antibacterianos/farmacologia , Desinfetantes/farmacologia , Niacinamida/análogos & derivados , Pseudomonas aeruginosa/efeitos dos fármacos , Compostos de Piridínio/farmacologia , Biofilmes/efeitos dos fármacos , Linhagem Celular , Humanos , Niacinamida/farmacologia
2.
Infect Immun ; 69(10): 6102-9, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11553548

RESUMO

Streptococcus intermedius is a member of the normal flora of the mouth but is also an opportunistic pathogen associated with purulent infections at oral and nonoral sites. Intermedilysin (ILY) has been shown to be a cytolysin capable of generating pores in the cell membrane of erythrocytes demonstrable by electron microscopy. This effect has been shown to be specific for human cells. Since polymorphonuclear cells (PMNs) are the main cell involved in innate immunity we investigated the effect of purified intermedilysin from Streptococcus intermedius on PMN function. Active ILY at a concentration of 40 ng/microl caused a significant decrease in the number of intact PMNs after 60 min. The active cytolysin, when compared with heat-inactivated ILY, did not appear to be chemotactic for the PMNs but did cause an increase in intracellular calcium, with increased cell surface CD11b expression, metabolic burst, and phagocytosis of Staphylococcus aureus. These findings may have implications for the role of ILY in deep-seated abscesses.


Assuntos
Proteínas de Bactérias , Toxinas Bacterianas/farmacologia , Citotoxinas/farmacologia , Neutrófilos/efeitos dos fármacos , Streptococcus , Bacteriocinas , Cálcio/metabolismo , Fusão Celular , Permeabilidade da Membrana Celular , Tamanho Celular , Quimiotaxia de Leucócito/imunologia , Citometria de Fluxo/métodos , Hemólise , Humanos , Líquido Intracelular/metabolismo , Contagem de Leucócitos , Antígeno de Macrófago 1/biossíntese , Neutrófilos/citologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagocitose/imunologia , Staphylococcus aureus/imunologia
3.
Toxicol In Vitro ; 14(2): 139-47, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10793292

RESUMO

The cytotoxic effects of newly synthesized bis-quaternary ammonium compounds (bis-QACs) and benzalkonium chloride were investigated on skin and blood cells, namely normal human epidermal keratinocytes of neonatal foreskin, a normal human skin fibroblast cell line NB1RGB, erythrocytes and a lymphoma cell line JM. The bis-QACs tested were 4, 4'-(1,6-hexamethylenedithio)bis(1-octylpyridinium iodide) (4DTBP-6, 8), N,N'-tetramethylenebis(1-dodecyl-4-carbamoylpyridinium iodide) (4BCAP-4,12), N,N'-hexamethylenebis(1-decyl-4-carbamoylpyridinium iodide) (4BCAP-6,10), 4,4'-(1,4-tetramethylenedicarbonyldiamine) bis (1-decylpyridinium iodide) (4DCABP-4,10), 4,4'-(1, 4-tetramethylenedicarbonyldiamine) bis (1-dodecylpyridinium iodide) (4DCABP-4,12), 4,4'-(1, 4-phenyldicarbonyldiamine)bis(1-dodecylpyridinium iodide) (4DCABP-P, 12), and 4,4'-(1, 6-hexamethylenedioxydicarbonyl)bis(1-dodecylpyridinium iodide) (4DOCBP-6,12). All bis-QACs consisted of two identical alkylpyridinium rings and bridge structure linking rings to each other have a methylene bridge but only 4DCABP-P,12 has a phenyl ring as a bridge. Most of the LD(50) values in acute cytotoxic assays of these bis-QACs tended to be lower than those of benzalkonium chloride. However, the comparison of the antibacterial activity against cytotoxic effects on several human cells revealed that bis-QACs, especially 4DTBP-6,8, have wide concentration ranges showing sufficient antibacterial activity and lower cytotoxic effect on human cells (except for 4DOCBP-6,12), although benzalkonium chloride caused significant human cell damage at the concentrations necessary for antibacterial activity. Moreover, judging from the LD(50) value of 4DTBP-6,8 [67 microM, a 6.7-fold higher concentration than the upper value of MIC] obtained in an artificial human skin model TESTSKIN, 4DTBP-6,8 is thought to be a more effective and safer antiseptic reagent for application to the skin surface than benzalkonium chloride. Taken together, bis-QACs are expected to be a promising new generation of antimicrobial QACs.


Assuntos
Antibacterianos/toxicidade , Compostos de Amônio Quaternário/toxicidade , Antibacterianos/química , Bactérias/efeitos dos fármacos , Compostos de Benzalcônio/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Fibroblastos , Humanos , Indicadores e Reagentes , Queratinócitos/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfoma de Células T/patologia , Testes de Sensibilidade Microbiana , Conservantes Farmacêuticos/toxicidade , Compostos de Amônio Quaternário/química
4.
J Clin Microbiol ; 38(1): 220-6, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10618091

RESUMO

The distribution of intermedilysin, a human-specific cytolysin, among the anginosus group streptococci and the correlation of toxin production and infection by Streptococcus intermedius were investigated. PCR and Southern hybridization specific for the intermedilysin gene revealed that the toxin gene exists only in S. intermedius and no homologue to the toxin gene is distributed in S. anginosus and S. constellatus. Thus, the intermedilysin gene is useful as a marker gene of S. intermedius. Moreover, a human-specific hemolysis assay and Western blotting with intermedilysin-specific antibodies clearly demonstrated that the intermedilysin production level in isolates from deep-seated infections, such as brain and liver abscesses, is higher (6.2- to 10.2-fold, respectively) than in strains from normal habitats, such as dental plaque, or from peripheral infection sites. However, other candidate virulence factors of S. intermedius, such as chondroitin sulfate depolymerase, hyaluronidase, and sialidase activities, did not show such a clear correlation between enzymatic activity and isolation sites or disease severity. From these results, intermedilysin is likely to be the pathogenic or triggering factor of significance in inducing deep-seated infections with S. intermedius.


Assuntos
Genes Bacterianos , Infecções Estreptocócicas/microbiologia , Streptococcus/genética , Streptococcus/patogenicidade , Expressão Gênica , Glicosídeo Hidrolases/análise , Humanos , Boca/microbiologia , Reação em Cadeia da Polimerase , Streptococcus/classificação
5.
Chem Pharm Bull (Tokyo) ; 47(7): 1020-3, 1999 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10434404

RESUMO

Potent new biocides against both bacteria and fungi, 4,4'-(1,6-hexamethylenedioxydicarbonyl)bis(1-alkylpyridinium iodide)s (4DOCBP-6,n) (alkyl chain length, n = 8, 10, 12, 14, 16 and 18) were synthesized, 4DOCBP-6,n is a bis-quaternary ammonium compound (bis-QAC) and has a symmetrical dimeric structure which is composed of two alkylpyridinium iodides connected with a hexamethylenedioxydicarbonyl chain. 4DOCBP-6,10 and 4DOCBP-6,12 exhibited wide and effective antimicrobial spectra, compared with those of typical bactericides, N-dodecylpyridinium iodides (P-12) and benzyldimethyldodecylammonium chloride (BAC), or a popularly-used fungicide, 2-(4-thiazolyl)benzimidazole (TBZ). Their superior properties would be due to the unique dimeric structure which contains two active moieties in a molecule.


Assuntos
Anti-Infecciosos/síntese química , Antifúngicos/síntese química , Iodetos/síntese química , Piridinas/síntese química , Antibacterianos , Anti-Infecciosos/farmacologia , Antifúngicos/farmacologia , Bactérias/efeitos dos fármacos , Fenômenos Químicos , Físico-Química , Fibroblastos , Fungos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Iodetos/farmacologia , Testes de Sensibilidade Microbiana , Piridinas/farmacologia , Relação Estrutura-Atividade
6.
Biol Pharm Bull ; 21(10): 1057-61, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9821809

RESUMO

Bactericidal action of novel bis-quaternary ammonium compounds (bis-QACs), 4,4'-(alpha,omega-polymethylenedithio)bis(1-alkylpyridinium iodide)s (4DTBP-m,n) was studied. The bactericidal activity of 4DTBP-m,n in water was not affected by the molecular hydrophobicity unlike general mono-QAC, N-dodecylpyridinium iodide (P-12), while the bacteriostatic activity in the medium was reduced with their hydrophobicity. This result suggested that the hydrophobic materials in the medium interact with 4DTBP-m,n and cover their active moiety. Since the bactericidal activity using the measurement system supplemented with peptone was influenced by the molecular hydrophobicity, this speculation was supported. The plots of the bacteriostatic activities of 4DTBP-m,n against the surface hydrophobicities of various bacteria accord to the straight line as in the case of P-12. The slope of the line of 4DTBP-6,12 was comparatively smaller than that of 4DTBP-6,8, indicating that the compounds having longer alkyl group tend to reduce their activities against the bacteria with hydrophobic cell surface because of their interaction with the hydrophobic materials. The novel bis-QACs have an ability to liberate rapidly and abundantly the turbid materials from cells, that is, a bacterioclastic activity. The bacterioclastic activity of P-n was influenced by the length of alkyl group, while 4DTBP-6,n had almost the same activity regardless of its length. Observation by scanning electron microscope (SEM) revealed that 4DTBP-6,8 fatally damaged Escherichia coli cells, and that the morphological alteration of the cells caused by the bis-QAC was greatly different from that of the usual QAC. Therefore, the effective bacterioclastic action, and excellent bactericidal action is due to the unique dimeric structure of 4DTBP-m,n.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Compostos de Piridínio/química , Compostos de Piridínio/farmacologia , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Espectrofotometria , Relação Estrutura-Atividade
7.
Histochem Cell Biol ; 108(2): 95-103, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9272428

RESUMO

PACE4 is a mammalian Kexin family protease that is involved in the maturation of precursor proteins. Four PACE4 isoforms have been identified. We identified a novel PACE4 isoform, PACE4E, from a human cerebellum cDNA library, which possesses a hydrophobic cluster in its C-terminus participating in membrane association. The size of PACE4E mRNA from adult rat brain was estimated by Northern blotting to be 4.4 kb. In situ hybridization histochemistry revealed that the highest level of PACE4E mRNA was expressed in the mitral cells of the adult rat olfactory bulb (OB). The OB is a unique sensory organ in that it has a lifelong regenerating capacity and it affects brain development. We further analyzed the expression of PACE4E mRNA in the developing olfactory system. On day 13.5 of gestation, PACE4E mRNA was expressed at high levels in the neuroepithelium of the forebrain vesicle (FV), olfactory epithelium, and cells in the fiber bundles projecting to the FV. As development proceeded, PACE4E mRNA was expressed in developing mitral cells but decreased in the olfactory epithelium. In the newborn, its expression was confined to the mitral cells in both the main and accessory OB and in some periglomerular cells, as shown in adult rats. The spatio-temporal expression of PACE4E suggests that it plays a role in the establishment and maintenance of the olfactory receptor system.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Bulbo Olfatório/enzimologia , Condutos Olfatórios/enzimologia , Serina Endopeptidases/biossíntese , Animais , Especificidade de Anticorpos , Northern Blotting , Desenvolvimento Embrionário e Fetal/fisiologia , Feminino , Humanos , Bulbo Olfatório/embriologia , Bulbo Olfatório/crescimento & desenvolvimento , Condutos Olfatórios/embriologia , Condutos Olfatórios/crescimento & desenvolvimento , Pró-Proteína Convertases , RNA Complementar , Ratos , Ratos Sprague-Dawley , Serina Endopeptidases/genética
8.
J Biochem ; 121(5): 941-8, 1997 May.
Artigo em Inglês | MEDLINE | ID: mdl-9192737

RESUMO

PACE4 is a processing protease which processes the precursor protein to the mature protein. Currently, four PACE4 isoforms have been reported [Tsuji, A. et al. (1994) Biochem. Biophys. Res. Commun. 200, 943 950]. In this study, we have cloned cDNA encoding a novel isoform, PACE4E, by screening the human brain cerebellum cDNA library and reverse transcriptase polymerase chain reaction analysis of total RNA from human hepatoma HepG2 cells. The PACE4E cDNA encoded an amino acid sequence of 975 residues. The sequence from the amino terminus to Arg900 of PACE4E was identical to the corresponding sequence of PACE4A, but the carboxy terminal sequence (75 residues) was unique and contained a hydrophobic cluster (Leu952-Gly968). PACE4E cDNA was transiently transfected in COS-1 cells, and the expressed proteins were a 112-kDa precursor form and a 105-kDa mature form. They were secreted into the culture medium, but their secretion was retarded compared with that of PACE4A. The expression of a mutant of PACE4E truncated up to the hydrophobic cluster from the carboxy terminus resulted in a remarkable increase in secretion level, suggesting that PACE4E tends to be retained intracellularly due to interaction with the membrane through the hydrophobic cluster. On the contrary, the transient expression experiment of PACE4C showed that only 68-kDa protein (precursor form) was detected in the cell and not secreted into the medium. In addition, coexpression experiment revealed that PACE4E was able to process the precursor form of von Willebrand factor to the mature form, but PACE4C did not process it.


Assuntos
Isoenzimas/genética , Serina Endopeptidases/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Análise Mutacional de DNA , DNA Complementar/genética , Deleção de Genes , Humanos , Dados de Sequência Molecular , Pró-Proteína Convertases , Conformação Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Serina Endopeptidases/biossíntese , Serina Endopeptidases/química
11.
Chem Pharm Bull (Tokyo) ; 45(12): 1970-4, 1997 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9433766

RESUMO

Bis-quaternary ammonium compounds (bis-QACs), 4,4'-(alpha,omega-polymethylenedithio)bis(1-alkylpyridinium iodide)s (4DTBP-m,n), which have 3 to 10 carbon atoms in the connecting methylene chain (m) and 8 to 18 carbon atoms of the N-alkyl chain (n), were synthesized. 4DTBP-6,12 exhibited a wide antimicrobial spectrum against gram-positive and gram-negative bacteria and fungi. The activity was stronger than those of N-dodecylpyridinium iodide (P-12), benzyldodecyldimethylammonium chloride and 2-(4-thiazolyl)benzimidazole. The bactericidal activities of 4DTBP-m,n were scarcely affected by the lengths of the alkyl chain and methylene chain. The bis-QAC that showed the highest activity was 4DTBP-6,8 (minimum inhibitory concentration (MIC) = 1.6 microM, minimum bactericidal concentration (MBC) = 2.6 microM), and its activity was about 10 times that of N-hexadecylpyridinium iodide (P-16), which was the most active in the P-n series. In addition, 4DTBP-6,12 showed a high bactericidal activity in the ranges of pH 5 to 8.5 and 10 to 40 degrees C, in contrast to mono-QACs. The bis-QACs synthesized in this study have excellent bactericidal properties.


Assuntos
Antibacterianos/síntese química , Compostos de Piridínio/síntese química , Sulfetos/síntese química , Antibacterianos/farmacologia , Antifúngicos/síntese química , Antifúngicos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Compostos de Piridínio/farmacologia , Relação Estrutura-Atividade , Sulfetos/farmacologia , Temperatura
12.
Infect Immun ; 64(8): 3093-100, 1996 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8757839

RESUMO

A novel cytotoxin (intermedilysin) specific for human cells was identified as a cytolytic factor of Streptococcus intermedius UNS46 isolated from a human liver abscess. Intermedilysin caused human cell death with membrane blebs. Intermedilysin was purified from UNS46 culture medium by means of gel filtration and hydrophobic chromatography. The purified toxin was resolved into major and minor bands of 54 and 53 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These proteins reacted with an antibody against intermedilysin. Five internal peptide fragments of intermedilysin were sequenced and found to have 42 to 71% homology with the thiol-activated cytotoxin pneumolysin. However, the action of intermedilysin differed from that of thiol-activated cytotoxins, especially in terms of a lack of activation by dithiothreitol and resistance to treatments with N-ethylmaleimide and 5,5'-dithio-bis-(2-nitrobenzoic acid), although cholesterol inhibited the toxin activity. Intermedilysin was potently hemolytic on human erythrocytes but was 100-fold less effective on chimpanzee and cynomolgus monkey erythrocytes. Intermedilysin was not hemolytic in nine other animal species tested. Since human erythrocytes treated with trypsin were far less sensitive to intermedilysin than were the intact cells, a cell membrane protein(s) may participate in the intermedilysin action. These data demonstrated that intermedilysin is distinguishable from all known bacterial cytolysins.


Assuntos
Proteínas de Bactérias , Toxinas Bacterianas/toxicidade , Citocinas/toxicidade , Citotoxinas/toxicidade , Streptococcus/química , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/isolamento & purificação , Bacteriocinas , Gatos , Bovinos , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Galinhas , Citocinas/isolamento & purificação , Citocinas/metabolismo , Citotoxinas/isolamento & purificação , Citotoxinas/metabolismo , Cães , Relação Dose-Resposta a Droga , Humanos , Concentração de Íons de Hidrogênio , Abscesso Hepático/microbiologia , Macaca fascicularis , Camundongos , Dados de Sequência Molecular , Pan troglodytes , Ratos , Sais/farmacologia , Análise de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Streptococcus/isolamento & purificação , Streptococcus/metabolismo , Streptococcus/patogenicidade , Reagentes de Sulfidrila , Temperatura , Tripsina
14.
J Biochem ; 119(1): 100-5, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8907182

RESUMO

A novel trypsin-like serine proteinase was purified to homogeneity from the bovine pancreas microsome fraction. The enzyme was solubilized with 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), and purified by a series of column chromatographic steps on Ultrogel AcA-34, trypsin inhibitor-Sepharose 4B, and arginine-Sepharose 4B. The molecular mass of this pancreas trypsin-like proteinase (bPTLP) was estimated to be 29.5 kDa by SDS-PAGE under reducing conditions. The NH2-terminal sequence of bPTLP is very homologous, but not identical to those of other serine proteinases, especially such as elastases IV, II, and III. Substrate specificity studies involving a synthetic substrate and glucagon indicated that the enzyme hydrolyzes Arg-X, Lys-X, and Leu-X bonds. The best synthetic substrate for bPTLP was t-butyloxycarbonyl Gln-Arg-Arg-4-methylcoumaryl 7-amide. The enzyme failed to hydrolyze the substrate for chymotrypsin and elastase. The enzyme activity was inhibited by diisopropyl fluorophosphate, p-amidinophenylmethane sulfonylfluoride, and leupeptin, indicating that it is a serine-proteinase. These findings show that bPTLP is a novel serine-proteinase which differs from all known proteinases. The physiological function of the enzyme has yet to be determined.


Assuntos
Microssomos/enzimologia , Pâncreas/enzimologia , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Bovinos , Fracionamento Químico , Estabilidade Enzimática , Glucagon/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Dados de Sequência Molecular , Pâncreas/química , Pâncreas/ultraestrutura , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/isolamento & purificação , Inibidores de Serina Proteinase/farmacologia , Especificidade por Substrato
15.
Endocrinology ; 136(1): 357-60, 1995 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7828552

RESUMO

The distribution of Kexin family proteases in adult rat pancreatic islets was investigated by immunohistochemical means using a series of specific antibodies specific for PC1, PC2, PC6, Furin, PACE4A and a recently identified member of the Kexin family, PACE4C. PACE4C expression was limited to B cells of the pancreatic islets. PC2 was found in A and in some D cells more than in B cells and PC1 was evident only in B cells. Furin and PC6 were weakly and evenly expressed in the entire islet. PACE4A was hardly found in the islets. These findings indicated that individual Kexin family proteases are uniquely distributed in the islets and suggested that these proteases share roles in these cells as follows: PC2 is involved in the peptide hormone precursor processing in A cells and in D cells, and PACE4C, PC1 and PC2 (mainly PACE4C and PC1) are responsible for the processing event(s) specific to B cells.


Assuntos
Ilhotas Pancreáticas/metabolismo , Pró-Proteína Convertases , Proteínas de Saccharomyces cerevisiae , Subtilisinas/metabolismo , Animais , Feminino , Imuno-Histoquímica , Ratos , Ratos Sprague-Dawley , Subtilisinas/classificação , Distribuição Tecidual
17.
Biochem Biophys Res Commun ; 202(3): 1215-21, 1994 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-8060295

RESUMO

In the previous study [Biochem. Biophys. Res. Commun. (1994) 200, 943-950] we identified two novel cDNAs (PACE4C and PACE4D) encoding human Kexin-like protease, the PACE4 isoforms. In this study, we examined the expression of PACE4 isoform transcripts in various rat tissues. To detect very low levels and to distinguish among these isoforms, we used the reverse transcriptase-polymerase chain reaction (RT-PCR). PACE4C and PACE4D transcripts were detected in most tissues like PACE4A transcripts, however their tissue distribution profiles and the extent of expression differ. PACE4C and PACE4D transcripts are expressed at a much higher level than PACE4A transcript. These results indicate that PACE4C and PACE4D mRNAs are major transcripts of PACE4.


Assuntos
Pró-Proteína Convertases , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae , Subtilisinas/genética , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Primers do DNA , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Ratos , Ratos Wistar , Distribuição Tecidual
18.
Biochem Biophys Res Commun ; 202(3): 1452-9, 1994 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-8060327

RESUMO

By polymerase chain reaction (PCR) with primers corresponding to the sequences of catalytic domain conserved among the mammalian kexin-like protease family, a cDNA fragment encoding a novel member of the family was obtained from rat pituitary. A cDNA for the novel protease was obtained from three overlapping clones isolated from a rat pituitary cDNA library using the PCR product as a screening probe. The protein, designated as PC7, encoded by this cDNA contained a putative activation site with the sequence RXKR, subtilisin-like catalytic domain and a homo B region which are typical of mammalian kexin-like proteases, and short cysteine-rich region at the carboxyl-terminal end. It exhibited surprising sequence similarity to PACE4A. The PC7 transcript was expressed at high levels in the brain. However it was undetectable in the liver, kidney, heart, and spleen. The presence of PC7 isoforms (PC7A and PC7B) was also shown.


Assuntos
Encéfalo/enzimologia , Pró-Proteína Convertases , Proteínas de Saccharomyces cerevisiae , Serina Endopeptidases/genética , Subtilisinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de Aminoácidos
19.
J Biochem ; 115(5): 937-44, 1994 May.
Artigo em Inglês | MEDLINE | ID: mdl-7525546

RESUMO

Improvement of the delivery of exogenous enzymes is essential to achieve effective enzyme replacement therapy in lysosomal storage diseases. To test whether alpha 2-macroglobulin, an endogenous plasma protein, could serve as a transport vehicle of therapeutic agents to cells, alpha 2-macroglobulin and acid alpha-glucosidase or alpha-galactosidase A were coupled using two heterobifunctional cross-linking reagents. The alpha-glucosidase-alpha 2-macroglobulin conjugate was internalized and transported into lysosomes of acid alpha-glucosidase-deficient fibroblasts. The enzyme activity was stable after being taken up by the cells. Uptake of the conjugate resulted in the degradation of glycogen accumulated in lysosomes. The alpha-galactosidase A-alpha 2-macroglobulin conjugate was also internalized into the lysosomes of alpha-galactosidase A-deficient fibroblasts. Internalized alpha-galactosidase A-conjugate degraded globotriaosylceramide accumulated in lysosomes. The endocytosis of both conjugate was inhibited by alpha 2-macroglobulin-trypsin complex, indicating that the conjugates were endocytosed by an alpha 2-macroglobulin receptor system. These results showed the usefulness of alpha 2-macroglobulin as a transport vehicle of lysosomal enzymes for effective enzyme replacement.


Assuntos
Proteínas de Transporte/metabolismo , Lisossomos/enzimologia , alfa-Galactosidase/metabolismo , alfa-Glucosidases/metabolismo , alfa-Macroglobulinas/metabolismo , Animais , Transporte Biológico , Endocitose/fisiologia , Fibroblastos/metabolismo , Glicogênio/metabolismo , Doença de Depósito de Glicogênio Tipo II/metabolismo , Doença de Depósito de Glicogênio Tipo II/terapia , Hidrólise , Frações Subcelulares/enzimologia , Suínos , alfa-Galactosidase/química , alfa-Glucosidases/química
20.
Biochem Biophys Res Commun ; 200(2): 943-50, 1994 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-8179631

RESUMO

PACE4 has been identified as a second human subtilisin-like protease by Keifer et al. [DNA and Cell Biology (1991) 10, 757-769]. In this study, we isolated two novel cDNAs coding for PACE4 isoforms (PACE4C and PACE4D) from a human placenta cDNA library. The deduced PACE4C protein sequence (652 amino acids) lacks the cysteine-rich region located at the carboxy terminus of PACE4. The DNA sequence of the 3'-untranslated region (1 kb) of PACE4C is not homologous to the PACE4 sequence. The protein (497 amino acids) encoded by PACE4D cDNA lacks a signal peptide, a propeptide and the cysteine-rich region. These isoform cDNAs were also isolated from rat pituitary cDNA library.


Assuntos
DNA Complementar/genética , Isoenzimas/genética , Serina Endopeptidases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA/genética , Feminino , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Adeno-Hipófise/enzimologia , Placenta/enzimologia , Gravidez , Pró-Proteína Convertases , Ratos
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