RESUMO
Proteolysis of sperm histones in the sea urchin male pronucleus is the consequence of the activation at fertilization of a maternal cysteine protease. We previously showed that this protein is required for male chromatin remodelling and for cell-cycle progression in the newly formed embryos. This enzyme is present in the nucleus of unfertilized eggs and is rapidly recruited to the male pronucleus after insemination. Interestingly, this cysteine-protease remains co-localized with chromatin during S phase of the first cell cycle, migrates to the mitotic spindle in M-phase and is re-located to the nuclei of daughter cells after cytokinesis. Here we identified the protease encoding cDNA and found a high sequence identity to cathepsin proteases of various organisms. A phylogenetical analysis clearly demonstrates that this sperm histone protease (SpHp) belongs to the cathepsin L sub-type. After an initial phase of ubiquitous expression throughout cleavage stages, SpHp gene transcripts become restricted to endomesodermic territories during the blastula stage. The transcripts are localized in the invaginating endoderm during gastrulation and a gut specific pattern continues through the prism and early pluteus stages. In addition, a concomitant expression of SpHp transcripts is detected in cells of the skeletogenic lineage and in accordance a pharmacological disruption of SpHp activity prevents growth of skeletal rods. These results further document the role of this nuclear cathepsin L during development.
Assuntos
Catepsina L/metabolismo , Endopeptidases/metabolismo , Histonas/metabolismo , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Catepsina L/análise , Catepsina L/genética , DNA Complementar/genética , Endopeptidases/análise , Endopeptidases/genética , Fertilização , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Dados de Sequência Molecular , Filogenia , Ouriços-do-Mar/citologia , Ouriços-do-Mar/genética , Alinhamento de Sequência , Espermatozoides/metabolismoRESUMO
Identification of synthetic peptide substrates for novel peptidases is an essential step for their study. With this purpose we synthesized fluorescence resonance energy transfer (FRET) peptide libraries Abz (or MCA)-GXXXXXQ-EDDnp and Abz (or MCA)-GXXZXXQ-EDDnp, where X consists of an equimolar mixture of all amino acids, the Z position is fixed with one of the proteinogenic amino acids (cysteine was excluded), Abz (ortho-aminobenzoic acid) or MCA ([7-amino-4-methyl]coumarin) is the fluorescence donor and Q-EDDnp (glutamine-[N-(2,4-dinitrophenyl)-ethylenediamine]) is the fluorescence acceptor. The peptide libraries MCA-GXXX↓XXQ-EDDnp and MCA-GXXZ↓XXQ-EDDnp were cleaved as indicated (↓) by trypsin, chymotrypsin, cathepsin L, pepsin A, and Eqolisin as confirmed by Edman degradation of the products derived from the digestion of these libraries. The best hydrolyzed Abz-GXXZXXQ-EDDnp sublibraries by these proteases, including Dengue 2 virus NS2B-NS3 protease, contained amino acids at the Z position that are reported to be well accepted by their S(1) subsite. The pH profiles of the hydrolytic activities of these canonical proteases on the libraries were similar to those reported for typical substrates. The FRET peptide libraries provide an efficient and simple approach for detecting nanomolar concentrations of endopeptidases and are useful for initial specificity characterization as performed for two proteases secreted by a Bacillus subtilis.
Assuntos
Endopeptidases/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Luminescentes , Biblioteca de Peptídeos , Sequência de Aminoácidos , Animais , Bovinos , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/química , Proteínas Luminescentes/genéticaRESUMO
This investigation was designed to evaluate the frequency of erythematous candidosis (EC) and Candida species, proteinase and phospholipase exoenzyme production, and to compare clinical features in patients with complete dentures and HIV+/Acquired Immunodeficiency Disease Syndrome (AIDS). Fifty-one patients were selected from a total of 285 with EC: denture wearers (n = 30) and HIV+/AIDS (n = 21). The yeast prevalence and the production of exoenzymes, such as proteinase and phospholipase by Candida species were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. The frequency of Candida albicans was significantly higher (P < 0.05) in both groups although other yeast species (Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondi and Candida tropicalis) were also found. Candida albicans showed greater levels of proteinase production in the denture wearers, when compared with the HIV+/AIDS group. There was no difference between groups with regard to phospholipase production. The protein bands presented similar molecular weights, showing the presence of proteinases in both groups. It could be concluded that the clinical manifestation of EC may be related to its proteinase production capacity. Combination therapies using proteinase inhibitors play an important role in inhibiting exoenzyme production by Candida species, mainly C. albicans.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/epidemiologia , Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Candida/isolamento & purificação , Candidíase Bucal/epidemiologia , Candidíase Bucal/microbiologia , Prótese Total/microbiologia , Infecções por HIV/epidemiologia , Síndrome da Imunodeficiência Adquirida/epidemiologia , Síndrome da Imunodeficiência Adquirida/microbiologia , Brasil/epidemiologia , Candida/classificação , Comorbidade , Prótese Total/estatística & dados numéricos , Eletroforese em Gel de Poliacrilamida , Endopeptidases/análise , Endopeptidases/isolamento & purificação , Feminino , Infecções por HIV/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Boca/microbiologia , Técnicas de Tipagem Micológica , Fosfolipases/análise , Fosfolipases/isolamento & purificação , PrevalênciaRESUMO
BACKGROUND: Actinic cheilitis (AC) is a pre-malignant lesion caused by ultraviolet (UV) radiation and characterized by epithelial and connective tissue alterations. Mast cells (MCs), key contributors to solar elastosis in murine UV-irradiated skin, were characterized in order to assess their potential contribution to connective tissue degeneration in AC. METHODS: Actinic cheilitis (n = 15) and normal lip (n = 8) biopsies were stained immunohistochemically for tryptase and enzymehistochemically for chymase to determine MC density and protease content. MC subpopulations (i.e. MC(T) containing only tryptase, and MC(TC) containing chymase and tryptase) and their distribution were also determined. RESULTS: Mast cells and their proteases were increased in AC as compared with normal lip (P < 0.0001), and appeared degranulated especially around elastotic areas. MC(T) predominated over MC(TC) in AC and normal lip (P < 0.05). However, in AC MC(T) were increased in the epithelium/connective junction and connective area (P < 0.05), while in normal lip MC(T) predominated in connective and submucosal areas (P < 0.05). CONCLUSION: The results suggest that increased MC density and protease content may contribute to elastosis formation in AC. In addition, changes in MC(T) distribution may favor AC malignization.
Assuntos
Queilite/patologia , Endopeptidases/análise , Mastócitos/patologia , Adulto , Idoso , Contagem de Células , Degranulação Celular , Queilite/enzimologia , Quimases , Tecido Conjuntivo/enzimologia , Tecido Conjuntivo/patologia , Epitélio/enzimologia , Epitélio/patologia , Feminino , Humanos , Mediadores da Inflamação/análise , Lábio/enzimologia , Lábio/patologia , Neoplasias Labiais/enzimologia , Neoplasias Labiais/patologia , Masculino , Mastócitos/enzimologia , Pessoa de Meia-Idade , Mucosa Bucal/enzimologia , Mucosa Bucal/patologia , Transtornos de Fotossensibilidade/enzimologia , Transtornos de Fotossensibilidade/patologia , Lesões Pré-Cancerosas/enzimologia , Lesões Pré-Cancerosas/patologia , Serina Endopeptidases/análise , TriptasesRESUMO
A comparative study of proteolytic enzymes and cell-surface protein composition in virulent and avirulent Leishmania (Leishmania) amazonensis promastigote forms was carried out using one- and two-dimensional dodecyl sulfate sodium-polyacrylamide gel electrophoresis (SDS-PAGE). The surface iodinated protein profiles showed two major polypeptides of 65-60 and 50-47 kDa that were expressed in both virulent and avirulent promastigote forms. However, minor quantitative differences were observed in the cell-surface profile between the avirulent and virulent promastigotes. These included polypeptides of 115, 52, 45, 32, and 25 kDa that were preferentially expressed in the virulent forms. Two-dimensional SDS-PAGE showed an accentuated expression of acidic polypeptides; some of them differentially expressed in the promastigote forms analyzed. Live parasites treated with glycosylphosphatidylinositol (GPI)-specific phospholipase C (PLC) from Trypanosoma brucei and immunoprecipitated with the cross-reacting determinant (CRD) antibody recognized three major polypeptides of 65-60, 52, and 50-47 kDa, hence suggesting that these peptides were anchored to the plasma membrane domains through GPI anchor. Moreover, the polypeptides of 65-60 and 52 kDa were also recognized by the gp63 antiserum. Several metalloproteinase activities were similar in both virulent and avirulent promastigote forms, whereas cysteine proteinase activities, sensitive to E-64, were preferentially expressed in virulent promastigotes. These results suggest that cell-surface polypeptides and intracellular cysteine proteinases might play an important role in the virulence of L. (L.) amazonensis.
Assuntos
Endopeptidases/biossíntese , Leishmania mexicana/patogenicidade , Proteínas de Membrana/biossíntese , Proteínas de Protozoários/biossíntese , Animais , Cricetinae , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Endopeptidases/análise , Humanos , Leishmania mexicana/enzimologia , Leishmania mexicana/metabolismo , Proteínas de Membrana/análise , Camundongos , Testes de Precipitina , Proteínas de Protozoários/análise , VirulênciaRESUMO
Trypanosoma equiperdum and Trypanosoma evansi were purified by three or four cycles of low-speed centrifugation and final filtration through DEAE cellulose. The purified trypanosomes were used in comparative biochemical and immunological studies. Comparative polypeptide pattern analysis revealed that T. equiperdum showed 21 polypeptide bands, whose Mr ranged from >200 to 14.8 kDa. T. evansi showed 25 polypeptide bands in the Mr range 97-14.8 kDa. The main differences were associated with the presence of secondary bands, relative intensity and the number of bands. Both species gave seven glycoprotein bands; those of 97 and 68 kDa were present in T. equiperdum but absent in T. evansi. Bands of 61 and 28 kDa were present in T. evansi but not in T. equiperdum. Anti-T. equiperdum sera recognized four homologous antigens and cross-reacted with three antigens of T. evansi. Anti-T. evansi sera recognized three homologous antigens and cross-reacted with four T. equiperdum antigens. Four identical proteolytic protease bands were present for both species, while only one surface protein was detected for each species: 66 kDa for T. equiperdum and 62 kDa for T. evansi.
Assuntos
Trypanosoma/química , Trypanosoma/imunologia , Animais , Antígenos de Protozoários/análise , Endopeptidases/análise , Glicoproteínas/análise , Peptídeos/análise , Especificidade da Espécie , Trypanosoma/enzimologiaRESUMO
The occurrence of yeasts on ripe fruits and frozen pulps of pitanga (Eugenia uniflora L), mangaba (Hancornia speciosa Gom.), umbu (Spondias tuberosa Avr. Cam.), and acerola (Malpighia glaba L) was verified. The incidence of proteolytic, pectinolytic, and mycocinogenic yeasts on these communities was also determined. A total of 480 colonies was isolated and grouped in 405 different strains. These corresponded to 42 ascomycetous and 28 basidiomycetous species. Candida sorbosivorans, Pseudozyma antarctica, C. spandovensis-like, C. spandovensis, Kloeckera apis, C. parapsilosis, Rhodotorula graminis, Kluyveromyces marxianus, Cryptococcus laurentii, Metchnikowia sp (isolated only from pitanga ripe fruits), Issatchenkia occidentalis and C. krusei (isolated only from mangaba frozen pulps), were the most frequent species. The yeast communities from pitanga ripe fruits exhibited the highest frequency of species, followed by communities from acerola ripe fruits and mangaba frozen pulps. Yeast communities from frozen pulp and ripe fruits of umbu had the lowest number of species. Except the yeasts from pitanga, yeast communities from frozen pulp exhibited higher number of yeasts than ripe fruit communities. Mycocinogenic yeasts were found in all of the substrates studied except in communities from umbu ripe fruits and pitanga frozen pulps. Most of the yeasts found to produce mycocins were basidiomycetes and included P. antarctica, Cryptococcus albidus, C. bhutanensis-like, R. graminis and R. mucilaginosa-like from pitanga ripe fruits as well as black yeasts from pitanga and acerola ripe fruits. The umbu frozen pulps community had the highest frequency of proteolytic species. Yeasts able to hydrolyse casein at pH 5.0 represented 38.5% of the species isolated. Thirty-seven percent of yeast isolates were able to hydrolyse casein at pH 7.0. Pectinolytic yeasts were found in all of the communities studied, excepted for those of umbu frozen pulps. The highest frequency of pectinolytic activity was found in mangaba frozen pulp communities. Around 30% of all isolates produced pectinases. The ability to split arbutin was observed in all communities ranging from 8% in yeasts from pitanga frozen pulps to 40.6% in acerola ripe fruit communities. Among 432 species tested, 125 were active for beta-glucosidase production, and Kloeckera apis, P. antarctica, C. sorbosivorans, and C. spandovensis-like were the most active species.
Assuntos
Frutas/microbiologia , Leveduras/isolamento & purificação , Arbutina/metabolismo , Brasil , Endopeptidases/análise , Alimentos Congelados/microbiologia , Poligalacturonase/análise , Clima Tropical , Leveduras/classificaçãoRESUMO
The adult female Culex quinquefasciatus midgut comprises a narrow anterior and a dilated posterior region, with epithelia composed of a monolayer of adjacent epithelial cells joined at the apical portion by septate junctions. Densely packed apical microvilli and an intricate basal labyrinth characterise each cell pole. Our morphological studies suggest that, during blood digestion, the anterior midgut region also participates in an initial absorptive stage which is probably related to the intake of water, salts and other small molecules. This activity peaked by 6h after bloodmeal feeding (ABF) and ended approximately 18 h ABF, when the peritrophic membrane was already formed. After this time, absorption only occurred in the posterior region, with morphologic and biochemical evidence of high synthetic activity related to the secretion of proteases. Chymotrypsin, elastase, aminopeptidase, and trypsin reached their maximum activity at around 36 h ABF. Digestion products were apparently absorbed and transported to the basal labyrinth, from where they should be released to the hemolymph. At 72 h ABF, proteolysis had already ended and protein levels had returned to those observed before blood meal. The epithelium of the posterior region, however, did not return to its initial morphology, appearing quite disorganised. Additionally, from 48 h ABF onwards some epithelial cells showed morphological signals of apoptosis.
Assuntos
Culex/fisiologia , Culex/ultraestrutura , Fenômenos Fisiológicos do Sistema Digestório , Sistema Digestório/ultraestrutura , Animais , Sangue/metabolismo , Endopeptidases/análise , Endopeptidases/metabolismo , Feminino , Absorção Intestinal/fisiologia , Período Pós-Prandial/fisiologiaRESUMO
Juvenile piracanjuba, Brycon orbignyanus, in the wild consume protein from both plant and animal sources. Digestion of protein in piracanjuba begins in the stomach with pepsin, at low pH, and is followed by hydrolysis at alkaline pH in the lumen of the intestine. The digestive system in piracanjuba was evaluated to characterize the enzymes responsible for the digestion of feed protein and their composition. The gastric tissue synthesizes pepsin and the intestine tissues trypsin and chymotrypsin. Operational variables were evaluated and defined for future studies of the digestive system physiology. The enzymatic activity in the intestine and the relative concentration of enzymes were heavily influenced by the composition of the feed and the feeding regime, as detected by substrate-SDS-PAGE. Piracanjuba possess a mechanism of enzyme adaptation responding to food quality and regime, by varying the amount and composition of digestive proteases. This is a requisite study to determine the enzymes digesting protein in food and their characteristics and to gain some clues about the possible regulation mechanisms of enzyme synthesis in piracanjuba.
Assuntos
Digestão , Sistema Digestório/enzimologia , Endopeptidases/metabolismo , Peixes/fisiologia , Adaptação Fisiológica , Ração Animal , Animais , Peso Corporal , Quimotripsina/metabolismo , Dieta , Endopeptidases/análise , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Intestinos/enzimologia , Masculino , Tamanho do Órgão , Pepsina A/metabolismo , Estômago/enzimologia , Especificidade por Substrato , Temperatura , Tripsina/metabolismoRESUMO
BACKGROUND: Several Trichoderma strains have been reported to be effective in controlling plant diseases, and the action of fungal hydrolytic enzymes has been considered as the main mechanism involved in the antagonistic process. However, although Trichoderma strains were found to impair development of Crinipellis perniciosa, the causal agent of cocoa plant witches' broom disease, no fungal strain is available for effective control of this disease. We have then undertaken a program of construction of hydrolytic enzyme-overproducing Trichoderma strains aiming improvement of the fungal antagonistic capacity. The protease of an indian Trichoderma isolate showing antagonistic activity against C. perniciosa was purified to homogeneity and characterized for its kinetic properties and action on the phytopathogen cell wall. RESULTS: A protease produced by the Trichoderma harzianum isolate 1051 was purified to homogeneity by precipitation with ammonium sulfate followed by hydrophobic chromatography. The molecular mass of this protease as determined by SDS-polyacrylamide gel electrophoresis was about 18.8 kDa. Its N-terminal amino acid sequence shares no homology with any other protease. The purified enzyme substantially affected the cell wall of the phytopathogen C. perniciosa. Western-blotting analysis showed that the enzyme was present in the culture supernatant 24 h after the Trichoderma started to grow in casein-containing liquid medium. CONCLUSIONS: The capacity of the Trichoderma harzianum protease to hydrolyze the cell wall of C. perniciosa indicates that this enzyme may be actually involved in the antagonistic process between the two fungi. This fact strongly suggest that hydrolytic enzyme over-producing transgenic fungi may show superior biocontrol capacity.
Assuntos
Agaricales/efeitos dos fármacos , Antifúngicos/análise , Endopeptidases/análise , Trichoderma/enzimologia , Agaricales/ultraestrutura , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Cacau/microbiologia , Parede Celular/efeitos dos fármacos , Endopeptidases/isolamento & purificação , Endopeptidases/farmacologia , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Varredura , Controle Biológico de Vetores , Doenças das Plantas/microbiologia , Temperatura , Trichoderma/isolamento & purificaçãoRESUMO
Loxoscelism, the term used to describe lesions and clinical manifestations induced by brown spider's venom (Loxosceles genus), has attracted much attention over the last years. Brown spider bites have been reported to cause a local and acute inflammatory reaction that may evolve to dermonecrosis (a hallmark of envenomation) and hemorrhage at the bite site, besides systemic manifestations such as thrombocytopenia, disseminated intravascular coagulation, hemolysis, and renal failure. The molecular mechanisms by which Loxosceles venoms induce injury are currently under investigation. In this review, we focused on the latest reports describing the biological and physiopathological aspects of loxoscelism, with reference mainly to the proteases recently described as metalloproteases and serine proteases, as well as on the proteolytic effects triggered by L. intermedia venom upon extracellular matrix constituents such as fibronectin, fibrinogen, entactin and heparan sulfate proteoglycan, besides the disruptive activity of the venom on Engelbreth-Holm-Swarm basement membranes. Degradation of these extracellular matrix molecules and the observed disruption of basement membranes could be related to deleterious activities of the venom such as loss of vessel and glomerular integrity and spreading of the venom toxins to underlying tissues
Assuntos
Humanos , Animais , Membrana Basal/efeitos dos fármacos , Endopeptidases/metabolismo , Matriz Extracelular/efeitos dos fármacos , Hemostasia/efeitos dos fármacos , Venenos de Aranha/enzimologia , Aranhas , Endopeptidases/análise , Venenos de Aranha/química , Venenos de Aranha/toxicidadeRESUMO
Acanthamoeba species can cause granulomatous encephalitis and keratitis in man. The mechanisms that underlie tissue damage and invasion by the amoebae are poorly understood, but involvement of as yet uncharacterized proteinases has been suggested. Here, we employed gelatin-containing gels and azocasein assays to examine proteinase activities in cell lysates and in medium conditioned by Acanthamoeba polyphaga trophozoites. Azocasein hydrolysis by cell lysates was optimally detected at pH 4.0-5.0 and was predominantly associated with the activity of cysteine proteinases. Compatible with enzyme activation during secretion, culture supernatants additionally contained a prominent azocasein hydrolyzing activity attributable to serine proteinases; these enzymes were better detected at pH 6.0 and above, and resolved at 47, 60, 75, 100, and >110 kDa in overlay gelatin gels. Although a similar banding profile was observed in gels of trophozoite lysates, intracellular serine proteinases were shown to be activated during electrophoresis and to split the substrate during migration in sodium dodecyl sulfate gels. Blockage of serine proteinases with phenylmethylsulfonylfluoride prior to electrophoresis permitted the detection of 43-, 59-, 70-, and 100-130-kDa acidic cysteine proteinases in cell lysates, and of 3 (43, 70, and 130 kDa) apparently equivalent enzymes in culture supernatants. Under the conditions employed, no band associated with a metalloproteinase activity could be depicted in substrate gels, although the discrete inhibition of supernatants' azocaseinolytic activity by 1,10-phenanthroline suggested secretion of some metalloproteinase.
Assuntos
Acanthamoeba/enzimologia , Endopeptidases/análise , Animais , Caseínas/metabolismo , Meios de Cultivo Condicionados , Cisteína Endopeptidases/análise , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Inibidores de Proteases/farmacologia , Serina Endopeptidases/análiseRESUMO
A neutral activity of Boophilus microplus in the intestine was identified by electrophoresis in polyacrilamide gel copolymerized with gelatin. The maximum of activity was attained at pH 6.0. The highest specific activity at that pH was obtained with casein substrate. The disappearance of this activity was observed in both substrates after the addition of phenylmethylsulphonyl fluoride in the reaction mixtures. It was very interesting to find out an endopeptidase activity with these characteristics in the intestine, in spite of the fact that the digestive activity in ticks is intracellular at very acid pH, which does not occur in other insects.
Assuntos
Endopeptidases/análise , Intestinos/enzimologia , Ixodidae/enzimologia , Animais , Eletroforese em Gel de PoliacrilamidaRESUMO
From the nutrition al point of view milk is one of the most complete food in the diet of mammals. It contains nearly all the nutrients necessary to sustain life, but milk can deteriorate very easily, either by microbiological contamination or by biochemical reactions during processing and/or storage. The objective of this research study was to design a modified UHT treatment to achieve commercial sterilization and maximize the stabilization of the heat-treated product during storage. To search for a modified UHT process, a mathematical model coupled with an optimization routine (complex method) was developed. The mathematical model considers Kinetics for the inactivation of Bacillus stearothermophilus and several quality factors. To attain the objective function, several commercial UHT milk were analyzed and for the computer search the minimization of hydroxy methyl furfural (HMF) formation was considered and also including constraints for protease and lipase inactivation. The complex optimization procedure was implemented to search for the optimum modified UHT treatment.. One of the optimum modified UHT treatments was the combination of two pre-treatment (3.16 minutes at 62.30 degrees C and 6 minutes at 75 degrees C) in addition with a UHT treatment (0.75 s at 148.8 degrees C). This treatment attains the maximum product stability with a negligible effect on composition and color formation in the treated milk (HMF formation less than 3 mg/mL).
Assuntos
Indústria Alimentícia/métodos , Leite , Esterilização/métodos , Animais , Endopeptidases/análise , Estudos de Avaliação como Assunto , Indústria Alimentícia/normas , Cinética , Lipase/análise , Leite/química , Leite/normas , Controle de Qualidade , Esterilização/normasRESUMO
Giardia duodenalis isolates from asymptomatic or symptomatic patients and from animals present similarities and differences in the protein composition, antigenic profile, pattern of proteases and isoenzymes, as well as in nucleic acids analysis. In the present overview, these differences and similarities are reviewed with emphasis in the host-parasite interplay and possible mechanisms of virulence of the protozoon.
Assuntos
Antígenos de Protozoários , Endopeptidases/metabolismo , Giardia , Ácidos Nucleicos , Proteínas de Protozoários/metabolismo , Animais , Variação Antigênica , Antígenos de Protozoários/análise , DNA Bacteriano/análise , Endopeptidases/análise , Giardia/enzimologia , Giardia/genética , Giardia/imunologia , Interações Hospedeiro-Parasita , Isoenzimas/análise , Ácidos Nucleicos/análise , Proteínas de Protozoários/análiseRESUMO
El moho zigomiceto Mucor miehei produce una proteasa de tipo ácido (EC: 3.4.23.10) semejante a la renina o cuajo de ternero. Se ha encontrado que la síntesis de la enzima está parcialmente asociada al crecimiento, y que altas velocidades de consumo de glucosa dan como resultado una mayor producción de la renina microbiana (Escobar and Barnett, 1993, 1995). Durante el proceso de produc-ción de la proteasa se observó que cuando la velocidad de producción de la misma aún es alta, los niveles de glucosa alcanzan a ser mínimos, niveles que se consideraron como una de las posibles causas de la finalización de la produc-ción de la enzima (Escobar and Barnett, 1993,1995). Frente a esta limitación fisiológica, se planteó un proceso de dos etapas para mejorar la producción de la proteasa y superar el fenómeno mencionado.La primera consistió en estudiar la relación entre la pro-ducción de la renina y el consumo de azúcares (especial-mente la glucosa) en el transcurso de la fermentación, para determinar aquellos momentos en los que la rata de pro-ducción de enzima es alta y la concentración de glucosa se encuentra cercana a cero. En la segunda etapa se aplicó un proceso de alimentación por lote de glucosa durante esos momentos, para observar si la producción de la enzi-ma aumentaba.Se obtuvo un valor máximo de actividad enzimática (AE) de 165 unidades coagulantes (UC)/ml para el proceso en cochada y una velocidad de consumo de azúcares prome-dio de 0,1813 g de glucosa/1/h. Con base en los resultadosanteriores se determinaron condiciones para el proceso de alimentación por lote de glucosa, tales como veloci-dad, tiempo y concentración. Las condiciones para el pro-ceso de alimentación por lote fueron un flujo de 0,06 ml/ min y una concentración de glucosa de 50 g/1 sin obtener-se aumento considerable en el valor de la AE (95 UC/ml). Se obtuvo una concentración celular promedio de 11 g/1 y un rendimiento del nutriente en masa celular (YX/SA) pro-medio de 0,3 g de células/g...
Assuntos
Queijo , Quimosina , Endopeptidases/análiseRESUMO
Los anticuerpos anticitoplasma de neutrófilos, ANCA, se encuentran involucrados en la patogénesis de las diferentes formas de las vasculitis inmunes. Su descubrimiento y posterior estudio ha permitido su utilización como elemento de ayuda diagnóstica y de seguimiento en enfermedades tales como la granulomatosis de Wegener, panarteritis microscópica y glomerulonefritis cresenticas necrotizantes. El objetivo de esta revisión es tratar de comprender las diferentes formas de vasculitis y así orientarse con respecto al diagnóstico de las mismas. La técnica de inmunofluorescencia indirecta (IFI) que hasta el momento es la única normatizada, sigue siendo una herramienta fundamental para la detección de ANCA dada su elevada especificidad; pero no debe olvidarse que posee como interferentes a los anticuerpos antinucleares (ANA). Se desarrolló un absorvente a partir de un extracto nucleoproteico de timo vacuno que permite eliminar la interferencia que producen los ANA cuando están presente en una muestra donde se quiere determinar ANCA. De esta manera se logró optimizar la IFI-ANCA, lo cual quedó demostrado que los resultados obtenidos cuando se utilizó esta técnica en conjunto con ELISA antígeno específico, LIA blot antígeno específico y Dot blot gránulos Ó desarrollados. Resultados preexistentes en relación con los ANCA y colangenopatías como el lupus eritematoso sistémico (LES) pediátrico y la artritis reumatoidea (AR) mostraban resultados dispares entre diferentes autores, lo que generó la inquietud de estudiar la seroprevalencia en estas enfermedades y su importancia clínica. Se demostró relación entre ANCA e insuficiencia renal en LES pediátrico, no así en AR. Se generó la hipótesis que la presencia de ANCA sea un epifenómeno derivado de la respuesta inflamatoria. También se pudo demostrar la presenciade ANCA en pacientes con glomerulopatías sin evidencias clínicas ni anatomopatológicas de vasculitis como son las glomerulopatías: de la diabetes, IgA, membranoproliferativas, mesangial, membranosa, nefroesclerosis, esclerosis focal y segmentaria, todas ellas asociadas a microhematuria y/o proteinuria en diferentes grados. Finalmente la revisión de este tema y en particular el desarrollo y optimización de los métodos de detección ha permitido ampliar y profundizar el campo de estudio en relación con los ANCA arribando a las conclusiones aquí expresadas
Assuntos
Humanos , Anticorpos Anticitoplasma de Neutrófilos , Glomerulonefrite/imunologia , Granulomatose com Poliangiite/imunologia , Grânulos Citoplasmáticos , Vasculite/diagnóstico , Anticorpos Antinucleares , Anticorpos Antinucleares/sangue , Anticorpos Anticitoplasma de Neutrófilos/sangue , Antígenos , Artrite Reumatoide/imunologia , Catepsinas , Técnicas de Laboratório Clínico/normas , Glomerulonefrite/fisiopatologia , Granulomatose com Poliangiite/diagnóstico , Hepatite Autoimune/imunologia , Lactoferrina , Lúpus Eritematoso Sistêmico/imunologia , Neoplasias/imunologia , Neutrófilos/química , Endopeptidases/análise , Endopeptidases , Poliarterite Nodosa/diagnóstico , Valor Preditivo dos Testes , Vasculite por IgA/diagnóstico , Insuficiência Renal/imunologia , Insuficiência Respiratória/imunologia , Sensibilidade e Especificidade , Doença Antimembrana Basal Glomerular/diagnóstico , Vasculite/classificação , Vasculite/imunologiaRESUMO
Los anticuerpos anticitoplasma de neutrófilos, ANCA, se encuentran involucrados en la patogénesis de las diferentes formas de las vasculitis inmunes. Su descubrimiento y posterior estudio ha permitido su utilización como elemento de ayuda diagnóstica y de seguimiento en enfermedades tales como la granulomatosis de Wegener, panarteritis microscópica y glomerulonefritis cresenticas necrotizantes. El objetivo de esta revisión es tratar de comprender las diferentes formas de vasculitis y así orientarse con respecto al diagnóstico de las mismas. La técnica de inmunofluorescencia indirecta (IFI) que hasta el momento es la única normatizada, sigue siendo una herramienta fundamental para la detección de ANCA dada su elevada especificidad; pero no debe olvidarse que posee como interferentes a los anticuerpos antinucleares (ANA). Se desarrolló un absorvente a partir de un extracto nucleoproteico de timo vacuno que permite eliminar la interferencia que producen los ANA cuando están presente en una muestra donde se quiere determinar ANCA. De esta manera se logró optimizar la IFI-ANCA, lo cual quedó demostrado que los resultados obtenidos cuando se utilizó esta técnica en conjunto con ELISA antígeno específico, LIA blot antígeno específico y Dot blot gránulos O desarrollados. Resultados preexistentes en relación con los ANCA y colangenopatías como el lupus eritematoso sistémico (LES) pediátrico y la artritis reumatoidea (AR) mostraban resultados dispares entre diferentes autores, lo que generó la inquietud de estudiar la seroprevalencia en estas enfermedades y su importancia clínica. Se demostró relación entre ANCA e insuficiencia renal en LES pediátrico, no así en AR. Se generó la hipótesis que la presencia de ANCA sea un epifenómeno derivado de la respuesta inflamatoria. También se pudo demostrar la presenciade ANCA en pacientes con glomerulopatías sin evidencias clínicas ni anatomopatológicas de vasculitis como son las glomerulopatías: de la diabetes, IgA, membranoproliferativas, mesangial, membranosa, nefroesclerosis, esclerosis focal y segmentaria, todas ellas asociadas a microhematuria y/o proteinuria en diferentes grados. Finalmente la revisión de este tema y en particular el desarrollo y optimización de los métodos de detección ha permitido ampliar y profundizar el campo de estudio en relación con los ANCA arribando a las conclusiones aquí expresadas (AU)
Assuntos
Humanos , Anticorpos Anticitoplasma de Neutrófilos/diagnóstico , Vasculite/diagnóstico , Glomerulonefrite/imunologia , Granulomatose com Poliangiite/imunologia , Grânulos Citoplasmáticos/metabolismo , Anticorpos Anticitoplasma de Neutrófilos/sangue , Glomerulonefrite/fisiopatologia , Sensibilidade e Especificidade , Anticorpos Antinucleares/sangue , Anticorpos Antinucleares/diagnóstico , Granulomatose com Poliangiite/diagnóstico , Valor Preditivo dos Testes , Poliarterite Nodosa/diagnóstico , Lúpus Eritematoso Sistêmico/imunologia , Vasculite por IgA/diagnóstico , Doença Antimembrana Basal Glomerular/diagnóstico , Neoplasias/imunologia , Insuficiência Respiratória/imunologia , Hepatite Autoimune/imunologia , Insuficiência Renal/imunologia , Artrite Reumatoide/imunologia , Lactoferrina , Antígenos/diagnóstico , Catepsinas , Vasculite/imunologia , Vasculite/classificação , Endopeptidases/diagnóstico , Endopeptidases/análise , Técnicas de Laboratório Clínico/normas , Neutrófilos/químicaRESUMO
The aim of this work was to evaluate the microbiological quality of raw type B milk, kept under refrigeration at 3 degrees C, for a period of 15 days. The milk was enumerated and isolated a total of 180 microorganisms, of which 80 were psychrotrophics. The ability of these microorganisms to produce lipolysis and/or proteolysis in the milk was evaluated. Concerning the microbiological analysis, the samples analyzed presented high values for total counting. The results from the initial counting were 2.7 x 10(4) UFC/ml for psychrotrophic, with a predominance of Gram negative bacilli, which were highly lipolytic and had proteolytic activities associated.
Assuntos
Proteínas de Bactérias/análise , Criopreservação , Endopeptidases/análise , Microbiologia de Alimentos , Conservação de Alimentos , Bactérias Gram-Negativas/isolamento & purificação , Lipase/análise , Leite/microbiologia , Animais , Brasil , Bovinos , Temperatura Baixa , Feminino , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Negativas/fisiologia , Lipólise , Leite/química , RefrigeraçãoRESUMO
Three actinomycete strains isolated from soil treated with 2,4-D were able to degrade the herbicide Diuron in vitro. Strain CCT 4916 was the most efficient, degrading up to 37% of applied Diuron (100 mg Kg-1 soil) in 7 days, as measured by HPLC and UV/VIS spectroscopy. All strains showed protease and urease activity; intracellular activity of metapyrocatechase and pyrocatechase were not found. Actinomycete strain CCT 4916 produced manganese peroxidase, which could be potentially related to degradation of Diuron.