Proteinase activity regulation by glycosaminoglycans

There are few reports concerning the biological role and the mechanisms of interaction between proteinases and carbohydrates other than those involved in clotting. It has been shown that the interplay of enzymes and glycosaminoglycans is able to modulate the activity of different proteases and also to affect their structures. From the large number of proteases belonging to the well-known protease families and also the variety of carbohydrates described as widely distributed, only few events have been analyzed more deeply. The term "family" is used to describe a group of proteases in which every member shows an evolutionary relationship to at least one other protease. This relationship may be evident throughout the entire sequence, or at least in that part of the sequence responsible for catalytic activity. The majority of proteases belong to the serine, cysteine, aspartic or metalloprotease families. By considering the existing limited proteolysis process, in addition to the initial idea that the proteinases participate only in digestive processes, it is possible to conclude that the function of the enzymes is strictly limited to the cleavage of intended substrates since the destruction of functional proteins would result in normal tissue damage. In addition, the location as well as the eventual regulation of protease activity promoted by glycosaminoglycans can play an essential role in the development of several physiopathological conditions.

Slime production and proteinase activity of Candida species isolated from blood samples and the comparison of these activities with minimum inhibitory concentration values of antifungal agents

Slime and proteinase activity of 54 strains consisting of 19 Candida parapsilosis and 35 C. albicans strains isolated from blood samples were investigated in this study. Ketoconazole, amphothericin B, and fluconazole susceptibility of Candida species were compared with slime production and proteinase activity of these species. For both Candida species, no correlation was detected between the slime activity and minimum inhibitory concentration (MIC) values of the three antifungal agents. For both Candida species no correlation was detected between the proteinase activity and the MIC values of amphothericin B, and fluconazole however, statistically significant difference, was determined between the proteinase activity and MIC values of ketoconazole (p = 0.007). Slime production was determined by using modified Christensen macrotube method and proteinase activity was measured by the method of Staib. Antifungal susceptibility was determined through the guidelines of National Committee for Laboratory Standards (NCCLS M27-A).

Influence of air temperature on proteinase activity and beverage quality in Coffea arabica

Fruits were collected from trees of Coffea arabica cv. Obatã grown at Mococa and Adamantina in São Paulo State, Brazil, which are regions with marked differences in air temperature that produce coffee with distinct qualities. Mococa is a cooler location that produces high-quality coffee, whereas coffee from Adamantina is of lower quality. The amino acid and protein contents, amino acid profile, and proteinase activity and type in endosperm protein extracts were analysed. Proteinase genes were identified, and their expression was assayed. All results indicate that temperature plays a role in controlling proteinase activity in coffee endosperm. Proteinase activity was higher in the endosperm of immature fruits from Adamantina, which was correlated with higher amino acid content, changes in the amino acid profile, and increased gene expression. Cysteine proteinases were the main class of proteinases in the protein extracts. These data suggest that temperature plays an important role in coffee quality by altering nitrogen compound composition.

Correlations between differences in amino-terminal sequences and different hemolytic activity of sticholysins

Sticholysins I and II (St I and St II) are cytolysins produced by the sea anemone Stichodactyla helianthus. In spite of their 93% sequence homology, St II is more hemolytic against human erythrocytes than St 1. In order to establish the possible causes of this difference, we studied the hemolytic activity of synthetic peptides containing sequences from the N-termini of both proteins. The results demonstrated that the differences in hemolytic activity of the toxins could be ascribed at least partly to differences in their N-termini. (c) 2007 Elsevier Ltd. All rights reserved.

Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition.

Tobacco plants were transformed with a cDNA clone of chymotrypsin/trypsin-specific potato proteinase inhibitor II (PI2) under the control of a constitutive promoter. Although considerable levels of transgene expression could be demonstrated, the growth of Spodoptera exigua larvae fed with detached leaves of PI2-expressing plants was not affected. Analysis of the composition of tryptic gut activity demonstrated that only 18% of the proteinase activity of insects reared on these transgenic plants was sensitive to inhibition by PI2, whereas 78% was sensitive in insects reared on control plants. Larvae had compensated for this loss of tryptic activity by a 2.5-fold induction of new activity that was insensitive to inhibition by PI2. PI2-insensitive proteolytic activity was also induced in response to endogenous proteinase inhibitors of tobacco; therefore, induction of such proteinase activity may represent the mechanism by which insects that feed on plants overcome plant proteinase inhibitor defense.

A sensitive HPLC method for measuring bacterial proteolysis and proteinase activity in UHT milk

A sensitive quantitative reversed-phase HPLC method is described for measuring bacterial proteolysis and proteinase activity in UHT milk. The analysis is performed on a TCA filtrate of the milk. The optimum concentration of TCA was found to be 4%; at lower concentrations, non-precipitated protein blocked the HPLC while higher concentrations yielded lower amounts of peptides. The method showed greater sensitivity and reproducibility than a fluorescamine-based method. Quantification of the HPLC method was achieved by use of an external dipeptide standard or a standard proteinase. (c) 2006 Elsevier Ltd. All rights reserved.

Proteinase activity in rheumatoid arthritis

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Isolates of Candida albicans that differ in virulence for mice elicit strain-specific antibody-mediated protective responses

Three distinct isolates of Candida albicans were used to establish systemic and oral infections in inbred mice that are genetically resistant or susceptible to tissue damage. Patterns of infection differed significantly between both yeasts and mouse strains. Systemic infection conferred significant protection against re-challenge with the homologous, but not the heterologous yeast; however, the protective effect was more evident in the tissue-susceptible CBA/CaH mice than in the resistant BALB/c strain. In contrast, oral infection induced protection against both homologous and heterologous oral challenge, although this was significant only in the CBA/CaH mice. CBA/CaH mice produced antibodies of both IgG1 and IgG2a subclasses, whereas BALB/c mice produced predominantly IgG1. Western blotting demonstrated considerable differences between epitopes recognised by serum antibodies from mice of both strains after immunisation with each of the three yeasts. Thus, different strains of yeast show considerable specificity in antibody responses elicited by either systemic or oral infection. (c) 2005 Elsevier SAS. All rights reserved.

In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida

INTRODUCTION: Candida yeasts are commensals; however, if the balance of normal flora is disrupted or the immune defenses are compromised, Candida species can cause disease manifestations. Several attributes contribute to the virulence and pathogenicity of Candida, including the production of extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate the in vitro activity of phospholipases and acid proteinases in clinical isolates of Candida spp. METHODS: Eighty-two isolates from hospitalized patients collected from various sites of origin were analyzed. Phospholipase production was performed in egg yolk medium and the production of proteinase was verified in a medium containing bovine serum albumin. The study was performed in triplicate. RESULTS: Fifty-six (68.3%) of isolates tested were phospholipase positive and 16 (44.4%) were positive for proteinase activity. C. tropicalis was the species with the highest number of positive isolates for phospholipase (91.7%). Statistically significant differences were observed in relation to production of phospholipases among species (p<0,0001) and among the strains from different sites of origin (p=0.014). Regarding the production of acid protease, the isolates of C. parapsilosis tested presented a larger number of producers (69.2%). Among the species analyzed, the percentage of protease producing isolates did not differ statistically (χ2=1.9 p=0.5901 (χ2=1.9 p=0.5901). CONCLUSIONS: The majority of C. non-albicans and all C. albicans isolates were great producers of hydrolytic enzymes and, consequently, might be able to cause infection under favorable conditions.

Lipodystrophy in HIV/AIDS patients with different levels of physical activity while on antiretroviral therapy

INTRODUCTION: Lipodystrophy is related to the use of highly active antiretroviral therapy (HAART) and can cause aesthetic stigma and increase the risk of developing cardiovascular diseases. Physical activity may be a valid alternative for the treatment and prevention of lipodystrophy. However, few studies address this issue. The objective of this study was to assess lipodystrophy related to highly active antiretroviral therapy in HIV/AIDS patients with different physical activity habits. METHODS: The sample was composed of 42 HIV/AIDS patients taking HAART medication who were visiting the Counseling and Testing Center (CTC) in Presidente Prudente. The level of physical activity was obtained using the International Physical Activity Questionnaire (IPAQ); lipodystrophy was diagnosed using a self-report questionnaire that was administered to the patient and then followed up by medical confirmation. The percentage of trunk fat was estimated by dual X-Ray absorptiometry (DEXA). Information about sex, age, length of HAART treatment, CD4+ T lymphocyte count (CD4) and viral load was also collected. RESULTS: A higher prevalence of lipodystrophy was observed in the sedentary group when compared to the physically active group, which indicates that physical activity may be a protective factor in relation to the occurrence of lipodystrophy. The group that had a higher CD4 had a higher proportion of lipodystrophy and a higher proportion of younger and physically active individuals. The patients with lipodystrophy had a higher percentage of trunk fat and were more sedentary than active individuals. CONCLUSIONS: A physically active lifestyle has a protective effect against the occurrence of lipodystrophy related to HAART.

Lipodystrophy in HIV/AIDS patients with different levels of physical activity while on antiretroviral therapy

INTRODUÇÃO: A lipodistrofia relacionada ao uso de terapia antirretroviral (TARV) pode causar estigma estético e elevar o risco de doenças cardiovasculares. A atividade física pode ser uma alternativa válida para o tratamento e prevenção da lipodistrofia. Entretanto, poucos estudos tratam dessa temática. O objetivo deste estudo foi verificar a ocorrência de lipodistrofia relacionada ao uso de TARV em portadores de HIV/AIDS, com diferentes hábitos de atividades físicas. MÉTODOS: A casuística foi formada por 42 portadores de HIV em uso de TARV, do Centro de Testagem e Aconselhamento de Presidente Prudente. Para obtenção do nível de atividade física aplicou-se o Questionário Internacional de Atividade Física (IPAQ); a lipodistrofia foi diagnosticada pelo autorrelato do paciente e a confirmação médica. O percentual de gordura de tronco foi estimado pela absortometria por raio-X de dupla energia (DEXA). Foram coletados também dados referentes a sexo, idade, tempo de uso de TARV, valores de CD4 e carga viral. RESULTADOS: Verificou-se maior ocorrência de lipodistrofia no grupo sedentário quando comparado ao ativo, além de fator protetor da prática da atividade física em relação à ocorrência da lipodistrofia. O grupo com valores mais elevados de CD4 também apresentou maior proporção de sujeitos com lipodistrofia, além de maior proporção de ativos e de indivíduos com menor faixa etária. Os acometidos pela lipodistrofia apresentaram maiores valores de percentual de gordura de tronco, bem como, os sedentários em relação aos ativos. CONCLUSÕES: O estilo de vida fisicamente ativa resultou em efeito protetor para ocorrência da lipodistrofia relacionada ao uso da TARV.

Candida albicans and Candida tropicalis in Oral Candidosis: Quantitative Analysis, Exoenzyme Activity, and Antifungal Drug Sensitivity

Candida albicans and C. tropicalis obtained from whole saliva of patients presenting signs of oral candidosis were assayed for quantification of colony forming units, exoenzyme activity (phospholipase and proteinase) and antifungal drug sensitivity (amphotericin B, fluconazole and itraconazole) by the reference method of the Clinical and Laboratory Standards Institute. The number of colony forming units per milliliter varied according to the Candida species involved and whether a single or mixed infection was present. Proteinase activity was observed in both C. albicans and C. tropicalis, but phospholipase activity was noted only in C. albicans. In vitro resistance to antifungals was verified in both species, but C. tropicalis appears to be more resistant to the tested antifungals than C. albicans.

Production of proteinases by psychrotrophic bacteria in raw milk stored at low temperature

Raw milk samples from two different sources were stored at 2degreesC, 4degreesC and 7degreesC for 10 days and the growth of psychrotrophic bacteria, production of proteinase and proteolysis in the milks were measured during storage. Peptide analyses by the fluorescamine method and RP-HPLC were used in determination of proteolysis and proteinase activity. The average times taken for the psychrotroph counts to reach 10(7) cfu/mL at 2degreesC, 4degreesC and 7degreesC were approximately 9, 7 and 4 days, although there was considerable variation in growth rates in the different milks. There was little correlation between psychrotroph counts and either proteolysis or proteinase activity levels. At 2degreesC, no milk stored showed significant proteolysis by the fluorescamine method after 10 days' storage, but significant proteinase activity could be measured in some of these milks at 8 and 10 days. RP-HPLC analysis was a more sensitive means of detecting peptides than the fluorescamine method.

Insights into human carboxylesterase 2 stability and activity in vivo and in vitro

Dissertation to obtain a Master Degree in Biotechnology