RESUMO
Quinoa proteins (QP) have promise as a potential source of novel food ingredients, and it is of great interest to know how high-intensity ultrasound (HIUS) treatments affect the properties of QP. This work aimed to study the impact of on-off time-pulses of HIUS treatments on the structural and physicochemical properties of QP; samples were treated at 5, 10, 20, and 30â¯min with on-off pulses of 10â¯s/10â¯s, 5â¯s/1â¯s, and 1â¯s/5â¯s). Structural changes were evaluated using PAGE-SDS, circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. Meanwhile, physicochemical properties were also examined, including solubility, Z-average, polydispersity index PDI, and Z-potential. PAGE-SDS showed the appearance of polypeptides over 190â¯kDa in HIUS samples-treated. All samples presented 15.6% α-helices, 31.3% ß-sheets, 21.8% ß-rotations, and 31.4% random coils independent of the HIUS treatment. ß-Turn structures and "random coils" were not affected by HIUS. When US 10â¯s/10â¯s and 1â¯s/5â¯s were applied, an increase in the % α-helix and a decrease in ß-fold were observed, which could indicate a small conversion of ß-folds to α-helices. Fluorescence spectra for all HIUS showed a significant increase (23%) of average fluorescence intensity and a decrease of λmax in relation to that of the control (346â¯dnm and 340â¯nm average HIUS treatment). DSC showed one endotherm in all cases (81.6-99.8⯰C), and an increase in Td was observed due to the effect of the HIUS treatment. HIUS caused a 48% increase in solubility. The Z-average of the HIUS samples compared to that of the controls showed an increase from 37.8 to 47.3â¯nm. PDI and Z-potential values from the QP controls and the HIUS samples did not show significance differences and presented average values of 0.466⯱â¯0.021 (PDI) and -16.63⯱â¯0.89 (Z-potential). It is possible to conclude that HIUS treatments affect the secondary and tertiary structure of quinoa proteins, and these changes resulted in an increase of solubility and particle size. HIUS treatment as a new and promising technology that can improve the QP solubility properties and in that way allow its use as an ingredient with a good source of protein to develop different types of beverages/protein sauces.
Assuntos
Fenômenos Químicos , Chenopodium quinoa/química , Proteínas de Plantas/química , Ondas Ultrassônicas , Conformação Proteica , Solubilidade , Fatores de TempoRESUMO
Noble metals deposited on TiO2 act as electron traps facilitating electron-hole separation and promoting the interfacial electron transfer process. In particular, silver nanoparticles have the ability to absorb visible light due to localized surface plasmon resonance. Here we report a photochemical and photocatalytic method for depositing Ag nanoparticles (2-20 nm) on TiO2 by using UV light at room temperature. UV-Vis diffuse reflectance spectroscopy, transmission electron microscopy, X-ray photoelectron spectroscopy and time-resolved microwave conductivity were used as characterization techniques. The photocatalytic activity was investigated by measuring the decomposition of rhodamine B under UV and visible light irradiation. The fastest bleaching of RhB under visible-light irradiation has been obtained by Ag/TiO2 plasmonic photocatalyst prepared by the photocatalytic route. These results were explained in terms of the more efficient photon absorption due to the presence of the surface plasmon resonance.
Assuntos
Nanopartículas Metálicas/química , Prata/química , Titânio/química , Adsorção , Luz , Teste de Materiais , Nanopartículas Metálicas/efeitos da radiação , Prata/efeitos da radiação , Titânio/efeitos da radiaçãoRESUMO
HTLV-1 Tax expression exerts an inhibitory effect on the Foxp3 transcription factor in CD4+CD25+ T-regulatory cells (Treg). For a better understanding of the role of Tax mRNA in the gene expression of cellular markers we measured Tax, Foxp3, CTLA-4, GITR, TGF-β, and IL-10 mRNA in Treg cells of 50 patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP; 27 women and 23 men; mean age: 56.7 years). The control group consisted of 23 non-infected subjects (12 women and 11 men) with a mean age of 51.3 years. Real-time PCR was used to measure mRNA of Tax proteins and several cellular markers of Treg function. Determinations revealed a high level of Tax mRNA in HAM/TSP (124.35 copies/100 CD4+CD25+ T cells). Foxp3, GITR, and CTLA-4 mRNA levels were lower in HAM/TSP patients (mean ± SD, 22.07 ± 0.78, 9.63 ± 0.36, and 4.54 ± 0.39, respectively) than in non-infected controls (47.15 ± 12.94, 22.14 ± 1.91, and 21.07 ± 2.31). Both groups had similar levels of TGF-β and IL-10. An inverse relationship was found between Tax levels and Foxp3, CTLA-4, and GITR levels. Conversely, there was a direct correlation between levels of Foxp3, GITR, and CTLA-4. Disease severity and evolution time did not correlate with Tax or Foxp3 levels. The present results suggest that Tax and Foxp3 mRNA vary with the same degree of disease severity in HAM/TSP patients. Tax fluctuations may affect CTLA-4 and GITR expression via the Foxp3 pathway, causing virus-induced dysfunction of CD4+CD25+ T cells in HAM/TSP patients.
Assuntos
Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , /metabolismo , Fatores de Transcrição Forkhead/metabolismo , Produtos do Gene tax/metabolismo , Proteína Relacionada a TNFR Induzida por Glucocorticoide/metabolismo , Vírus Linfotrópico T Tipo 1 Humano , Paraparesia Espástica Tropical/sangue , Biomarcadores/sangue , Biomarcadores/metabolismo , Estudos de Casos e Controles , /sangue , Fatores de Transcrição Forkhead/sangue , Produtos do Gene tax/sangue , Proteína Relacionada a TNFR Induzida por Glucocorticoide/sangue , Paraparesia Espástica Tropical/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , RNA Mensageiro/sangue , Índice de Gravidade de Doença , Fator de Crescimento Transformador beta/sangue , Fator de Crescimento Transformador beta/metabolismoRESUMO
HTLV-1 Tax expression exerts an inhibitory effect on the Foxp3 transcription factor in CD4(+)CD25(+) T-regulatory cells (Treg). For a better understanding of the role of Tax mRNA in the gene expression of cellular markers we measured Tax, Foxp3, CTLA-4, GITR, TGF-ß, and IL-10 mRNA in Treg cells of 50 patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP; 27 women and 23 men; mean age: 56.7 years). The control group consisted of 23 non-infected subjects (12 women and 11 men) with a mean age of 51.3 years. Real-time PCR was used to measure mRNA of Tax proteins and several cellular markers of Treg function. Determinations revealed a high level of Tax mRNA in HAM/TSP (124.35 copies/100 CD4(+)CD25(+) T cells). Foxp3, GITR, and CTLA-4 mRNA levels were lower in HAM/TSP patients (mean ± SD, 22.07 ± 0.78, 9.63 ± 0.36, and 4.54 ± 0.39, respectively) than in non-infected controls (47.15 ± 12.94, 22.14 ± 1.91, and 21.07 ± 2.31). Both groups had similar levels of TGF-ß and IL-10. An inverse relationship was found between Tax levels and Foxp3, CTLA-4, and GITR levels. Conversely, there was a direct correlation between levels of Foxp3, GITR, and CTLA-4. Disease severity and evolution time did not correlate with Tax or Foxp3 levels. The present results suggest that Tax and Foxp3 mRNA vary with the same degree of disease severity in HAM/TSP patients. Tax fluctuations may affect CTLA-4 and GITR expression via the Foxp3 pathway, causing virus-induced dysfunction of CD4(+)CD25(+) T cells in HAM/TSP patients.
Assuntos
Antígeno CTLA-4/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Produtos do Gene tax/metabolismo , Proteína Relacionada a TNFR Induzida por Glucocorticoide/metabolismo , Vírus Linfotrópico T Tipo 1 Humano , Paraparesia Espástica Tropical/sangue , Adulto , Idoso , Biomarcadores/sangue , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos , Antígeno CTLA-4/sangue , Estudos de Casos e Controles , Feminino , Fatores de Transcrição Forkhead/sangue , Produtos do Gene tax/sangue , Proteína Relacionada a TNFR Induzida por Glucocorticoide/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Paraparesia Espástica Tropical/metabolismo , RNA Mensageiro/sangue , Reação em Cadeia da Polimerase em Tempo Real , Índice de Gravidade de Doença , Fator de Crescimento Transformador beta/sangue , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVE: Neutrophils play a crucial role in the defense of invading bacteria by releasing biologically active molecules. The response of peripheral blood neutrophils was studied in periodontitis-affected patients and in healthy controls towards stimulation to Porphyromonas gingivalis (Pg) and Actinobacillus actinomycetemcomitans (Aa) extracts. MATERIALS AND METHODS: Peripheral venous blood was drawn from 23 adult patients with moderate to advanced chronic periodontitis (probing depth >or=5 mm, attachment loss >or=3 mm), and 30 healthy volunteers. Neutrophil response followed by metalloproteinase-9 (MMP-9) and interleukin-8 (IL-8) secretion was assayed by zymography and enzyme-linked immunosorbent assay, respectively, on both whole blood and purified neutrophils. In addition to periodontal pathogen extracts, known stimulating agents were tested, such as Escherichia coli-lipopolysaccharide (LPS), phytohemagglutinin, and zymosan A. RESULTS: Neutrophil response, expressed as a secretion ratio under stimulated and non-stimulated conditions, measured in whole blood, showed no differences between periodontitis and healthy controls. Instead, in purified neutrophils from patients, MMP-9 exhibited a significantly higher secretion ratio with LPS and Pg (1.5- to 2-fold), whereas IL-8 showed a larger increase in secretion ratio (3- to 7-fold) in the presence of Pg, Aa, LPS, and zymosan A. CONCLUSION: Peripheral neutrophils of periodontitis-affected patients are more reactive as suggested by their significantly higher response toward periodontal pathogen extracts and other stimulating agents.
Assuntos
Interleucina-8/análise , Metaloproteinase 9 da Matriz/análise , Neutrófilos/metabolismo , Periodontite/microbiologia , Adulto , Aggregatibacter actinomycetemcomitans , Estudos de Casos e Controles , Índice de Placa Dentária , Feminino , Humanos , Masculino , Índice Periodontal , Periodontite/sangue , Porphyromonas gingivalisRESUMO
Apyrase/ATP-diphosphohydrolase hydrolyzes di- and triphosphorylated nucleosides in the presence of a bivalent ion with sequential release of orthophosphate. We performed studies of substrate specificity on homogeneous isoapyrases from two potato tuber clonal varieties: Desiree (low ATPase/ADPase ratio) and Pimpernel (high ATPase/ADPase ratio) by measuring the kinetic parameters K(m) and k(cat) on deoxyribonucleotides and fluorescent analogues of ATP and ADP. Both isoapyrases showed a broad specificity towards dATP, dGTP, dTTP, dCTP, thio-dATP, fluorescent nucleotides (MANT-; TNP-; ethene-derivatives of ATP and ADP). The hydrolytic activity on the triphosphorylated compounds was always higher for the Pimpernel apyrase. Modifications either on the base or the ribose moieties did not increase K(m) values, suggesting that the introduction of large groups (MANT- and TNP-) in the ribose does not produce steric hindrance on substrate binding. However, the presence of these bulky groups caused, in general, a reduction in k(cat), indicating an important effect on the catalytic step. Substantial differences were observed between potato apyrases and enzymes from various animal tissues, concerning affinity labeling with azido-nucleotides and FSBA (5'-p-fluorosulfonylbenzoyl adenosine). PLP-nucleotide derivatives were unable to produce inactivation of potato apyrase. The lack of sensitivity of both potato enzymes towards these nucleotide analogues rules out the proximity or adequate orientation of sulfhydryl, hydroxyl or amino-groups to the modifying groups. Both apyrases were different in the proteolytic susceptibility towards trypsin, chymotrypsin and Glu-C.
Assuntos
Apirase/química , Apirase/metabolismo , Tubérculos/enzimologia , Solanum tuberosum/enzimologia , Marcadores de Afinidade , Sítios de Ligação , Isoenzimas , Cinética , Proteínas de Plantas , Desnaturação Proteica , Especificidade por SubstratoRESUMO
BACKGROUND: The aim of this work was to improve the assessment of the periodontal disease status through measurements of extracellular matrix metalloproteinases (MMPs) and their tissular inhibitors (TIMPs) in the gingival crevicular fluid from patients diagnosed with chronic periodontitis. METHODS: Gingival crevicular fluid samples from patients (n = 13) were taken from 60 sites initially, and from 51 and 41 sites, respectively, 3 and 6 months after scaling and root planing. Gingival crevicular fluid samples were also taken from healthy subjects (n = 11, 24 sites). The presence of MMP-9 and MMP-8 was assessed by zymography and immunowestern blotting, respectively. The actual MMP activity (gelatinase and collagenase) was measured using the fluorogenic substrate assay. TIMP-1 and -2 levels were measured by immunodot blot. RESULTS: The fluorogenic substrate assay determinations showed higher MMP activity in sites with probing depth > or = 4 mm, with significant reduction post-treatment. Gelatinase activity followed by zymography consisted mainly of MMP-9. A different pattern of MMP-8 in control and patient sites was found. Controls only showed species of a partially active form (69 kDa), whereas patient sites showed a high frequency of the active form (56 kDa), and in some cases the latent form (85 kDa) was also observed. The active form reduced its frequency in sites with probing depth > or = 4 mm. TIMP-1 and -2 levels in patients were significantly lower than in controls, and after treatment the recovery of TIMP-1 level similar to control was observed. CONCLUSION: Significant correlations between the severity of the periodontal disease and the actual MMP activity, the active form of MMP-8 and the low level of both TIMP-1 and TIMP-2 were found.
Assuntos
Líquido do Sulco Gengival/metabolismo , Metaloproteinases da Matriz/metabolismo , Periodontite/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , Adulto , Análise de Variância , Western Blotting/métodos , Feminino , Fluorometria/métodos , Líquido do Sulco Gengival/enzimologia , Humanos , Estudos Longitudinais , Masculino , Metaloproteinases da Matriz/análise , Neutrófilos/enzimologia , Periodontite/enzimologia , Periodontite/terapia , Estatísticas não Paramétricas , Inibidores Teciduais de Metaloproteinases/análiseRESUMO
The cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the CNS, thus biochemical processes in the CNS could potentially be reflected in the CSF. Changes in extracellular matrix (ECM) proteins can be studied through their analysis in the CSF. ECM plays an essential role in CNS homeostasis and several proteins such as laminin (LN), fibronectin (FN), thrombospondin (TS) and heparan sulphate proteoglycan (HS, perlecan) form part of its structure. Possible changes in the levels of these proteins were investigated in two different pathologies--tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) (n=25) and Creutzfeldt-Jakob disease (CJD) (n=19)--and compared with those in a control group with or without neurological disease (n=25). CSF analyses were carried out using monoclonal or monospecific polyclonal antibodies. In comparison with the control group, it was found that TSP/HAM patients presented significantly higher levels of LN, TS and HS, while in CJD patients the levels of FN, TS and HS were increased. In CJD patients the HS level was almost double that of the TSP/HAM patients. These results suggest a distinct pattern of ECM proteins in CSF in relation to the type of neurological disease. TSP/HAM is a chronic motor disease that affects the white matter of the spinal cord, while CJD is a subacute dementia that affects cerebral neurons and their synapsis.
Assuntos
Síndrome de Creutzfeldt-Jakob/líquido cefalorraquidiano , Síndrome de Creutzfeldt-Jakob/complicações , Proteínas da Matriz Extracelular/análise , Infecções por HTLV-I/líquido cefalorraquidiano , Infecções por HTLV-I/complicações , Paraparesia Espástica Tropical/líquido cefalorraquidiano , Paraparesia Espástica Tropical/etiologia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Paraparesia Espástica Tropical/complicaçõesRESUMO
Preliminary findings suggest that abnormalities in matrix metalloproteinase (MMP) activity may be found in the cerebrospinal fluid (CSF) of patients with Creutzfeldt-Jakob disease (CJD). In this study of 16 subjects with CJD and 16 age-, and sex-matched controls, we determined the presence of MMP-2 and MMP-9 in their active and proenzyme forms, the relative levels of MMP-3 and four inhibitors of MMP activity (TIMP-1, TIMP-2, TIMP-3 and TIMP-4), and the concentration of 4-3-3 protein. The methodology used involved zymography and immunological techniques. The results indicate that, compared with controls, CJD patients have a significantly higher positive frequency of pro-MMP-9 and of the active form of MMP-2, along with significantly higher levels of TIMP-1 and TIMP-2, classical inhibitors of MMP-9 and MMP-2, respectively. We also found a positive correlation between 14-3-3 protein concentration and that of TIMP-1 and TIMP-2 levels (correlation coefficients of 0.793 and 0.798, respectively). These results suggest that abnormalities in MMP and TIMP profiles may be helpful in the biochemical characterisation of CJD.
Assuntos
Síndrome de Creutzfeldt-Jakob/enzimologia , Metaloproteinases da Matriz/líquido cefalorraquidiano , Inibidores Teciduais de Metaloproteinases/líquido cefalorraquidiano , Tirosina 3-Mono-Oxigenase/líquido cefalorraquidiano , Proteínas 14-3-3 , Adulto , Idoso , Estudos de Casos e Controles , Síndrome de Creutzfeldt-Jakob/líquido cefalorraquidiano , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Comparative studies of intrinsic and extrinsic fluorescence of apyrases purified from two potato tuber varieties (Pimpernel and Desirée) were performed to determine differences in the microenvironment of the nucleotide binding site. The dissociation constants (K(d)) of Pimpernel apyrase for the binding of different fluorescent substrate analogs: methylanthranoyl (MANT-), trinitrophenyl (TNP-), and epsilon -derivatives of ATP and ADP were determined from the quenching of Trp fluorescence, and compared with K(d) values previously reported for Desirée enzyme. Binding of non-fluorescent substrate analogues decreased the Trp emission of both isoapyrases, indicating conformational changes in the vicinity of these residues. Similar effect was observed with fluorescent derivatives where, in the quenching effect, the transfer of energy from tryptophan residues to the fluorophore moiety could be additionally involved. The existence of energy transfer between Trp residues in the Pimpernel enzyme was demonstrated with epsilon -analogues, similar to our previous observations with the Desirée. From these results we deduced that tryptophan residues are close to or in the nucleotide binding site in both enzymes. Experiments with quenchers like acrylamide, Cs(+) and I(-), both in the presence and absence of nucleotide analogues, suggest the existence of differences in the nucleotide binding site of the two enzymes. From the results obtained in this work, we can conclude that the differences found in the microenvironment of the nucleotide binding site can explain, at least in part, the kinetic behaviour of both isoenzymes.
Assuntos
Apirase/metabolismo , Nucleotídeos/metabolismo , Solanum tuberosum/enzimologia , Triptofano/química , Acrilamida/química , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Apirase/química , Sítios de Ligação , Césio/química , Iodetos/química , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Nucleotídeos/química , Fotodegradação , Solanum tuberosum/química , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
Primary hyperparathyroidism is the main cause of hypercalcemia in ambulatory patients. Classic surgical resolution of this disease includes bilateral cervical exploration with exhibition of the four parathyroid glands and evaluation of the suspicious gland with a rapid biopsy. We report a 79 years old female with a primary hyperparathyroidism in whom the adenoma causing the disease was located with the help of an intraoperative gamma-probe that detected the zone of higher radiation counting of Tc99m Sestamibi, that was injected preoperatively. The patient was discharged with normal serum calcium and phosphate levels.
Assuntos
Adenoma/cirurgia , Hiperparatireoidismo/cirurgia , Neoplasias das Paratireoides/cirurgia , Compostos Radiofarmacêuticos , Tecnécio Tc 99m Sestamibi , Adenoma/diagnóstico por imagem , Idoso , Feminino , Humanos , Procedimentos Cirúrgicos Minimamente Invasivos , Neoplasias das Paratireoides/diagnóstico por imagem , Período Pós-Operatório , CintilografiaRESUMO
The fetal llama (Lama glama; a species adapted to live in chronic hypoxia in the highlands of the Andes) did not increase cerebral blood flow and reduce the brain oxygen uptake during hypoxemia. Although nitric oxide (NO) is a normal mediator in the regulation of vascular tone and synaptic transmission, NO overproduction by hypoxemia could produce neuronal damage. We hypothesized that nitric oxide synthase (NOS) activity is either maintained or reduced in the central nervous system of the llama fetuses submitted to chronic hypoxemia. Approximately 85% of the Ca(2+)-dependent NOS activity was soluble, at least 12% was associated with the mitochondrial fraction, and less than 5% remains associated with microsomes. To understand the role of NO in chronic hypoxemia, we determined the effect of 24-h hypoxemia on NOS activity in the central nervous system. No changes in activity or the subcellular distribution of NOS activity in brain tissues after hypoxemia were found. We proposed that the lack of changes in NOS activity in the llama under hypoxemia could be a cytoprotective mechanism inherent to the llama, against possible toxic effects of NO.
Assuntos
Encéfalo/embriologia , Camelídeos Americanos/embriologia , Hipóxia/veterinária , Óxido Nítrico Sintase/metabolismo , Animais , Western Blotting , Feminino , Doenças Fetais/enzimologia , Doenças Fetais/veterinária , Hipóxia/enzimologia , Gravidez , Valores de ReferênciaRESUMO
BACKGROUND: Among hypertensive patients, other risk factors for mortality and morbidity, besides blood pressure, must be considered when therapeutic decisions are done. AIM: To assess the incidence and relevance of cardiovascular risk factors in a cohort of patients with essential hypertension. MATERIAL AND METHODS: A cohort of 1,072 treated patients with essential hypertension was followed for a period up to 25 years. Four hundred eighty six were men and 586 were women, age ranged from 31 to 70 years. At entry, 56% of subjects did not have any organic complications associated to hypertension (stage I WHO criteria), 30% had mild alterations (Stage II) and 14% had major complications (myocardial infarction, stroke, heart failure or renal failure). Likewise, 43.8% had mild, 14.5%, moderate and 41.7%, severe hypertension. Patients were treated with monotherapy or combined therapy based on diuretics, beta blockers, calcium antagonists and angiotensin converting enzyme inhibitors. Goal of therapy was 140/90 mm Hg. Risk factors associated diseases and complications were registered carefully. Causes of death were obtained from hospital records and death certificates. Mortality was analyzed using life tables (intention to treat method included). RESULTS: Blood pressure dropped significantly during follow up from a mean of 182/110 to 154/92 mm Hg. During follow up, 143 patients died and 429 complications (lethal or non lethal) were recorded. Twenty four percent of patients smoked, 24% reported alcohol intake, 56% had hypercholesterolemia, 11% were obese, 13% had diabetes and 3% had gout. The proportional hazard model showed that the existence of previous complications, the presence of more than 3 risk factors, and age over 60 and mean systolic and diastolic pressure during therapy, were independent and significant risk factors for mortality. CONCLUSIONS: The incidence of risk factors among our hypertensive patients is very similar to that of other national or international cohorts.
Assuntos
Hipertensão/mortalidade , Adulto , Idoso , Análise de Variância , Distribuição de Qui-Quadrado , Chile/epidemiologia , Estudos de Coortes , Feminino , Seguimentos , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Prevalência , Fatores de RiscoRESUMO
BACKGROUND: Proteolytic modifications of neuronal surfaces and the surrounding extracellular matrix are very important in neuronal development and regeneration. Increased activity of matrix metalloproteinases (MMPs) and their tissue inhibitors, due to secretion by macrophages and lymphocytes, occur in inflammatory processes that disrupt the blood brain barrier. However, neurons and microglia can also secrete these enzymes. AIM: To identify the type of MMP present in the cerebrospinal fluid (CSF) and changes in the expression of tissue inhibitors of metalloproteinases (TIMPs) in patients with HTLV-1 associated tropical spastic paraparesis. PATIENTS AND METHODS: CSF samples from 12 patients with HTLV-1 associated tropical spastic paraparesis and 12 healthy controls were obtained by an atraumatic lumbar puncture. The presence of MMPs was measured by zymography and the relative amounts of TIMPs were measured by immunowestern blot. RESULTS: In the CSF of both controls and patients, a similar gelatinolytic band corresponding to proMMP-2 (latent form) was observed. In 83.3% of patients with HTLV 1 associated tropical spastic paraparesis, the MMP-9 was also present. TIMP-1, TIMP-2 and TIMP-3 were elevated 2.24 +/- 0.72, 3.85 +/- 1.38 and 5.89 +/- 3.4 fold, respectively, in the CSF of patients as compared to controls. CONCLUSIONS: Patients with HTLV-1 associated tropical spastic paraparesis have elevated activity of MMP-9 and levels of TIMPs in the CSF, when compared to healthy controls.
Assuntos
Metaloproteinases da Matriz/líquido cefalorraquidiano , Paraparesia Espástica Tropical/líquido cefalorraquidiano , Inibidores Teciduais de Metaloproteinases/líquido cefalorraquidiano , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade , Paraparesia Espástica Tropical/enzimologia , Paraparesia Espástica Tropical/etiologiaRESUMO
Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.
Assuntos
Difosfato de Adenosina/metabolismo , Apirase/metabolismo , Nucleotídeos/metabolismo , Proteínas de Plantas/metabolismo , Solanum tuberosum/enzimologia , Apirase/química , Apirase/isolamento & purificação , Isoenzimas/química , Isoenzimas/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Solanum tuberosum/química , Espectrometria de FluorescênciaRESUMO
Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.
Assuntos
Difosfato de Adenosina/metabolismo , Apirase/metabolismo , Nucleotídeos/metabolismo , Solanum tuberosum/enzimologia , Apirase/química , Apirase/isolamento & purificação , Césio/química , Césio/metabolismo , Iodo/química , Iodo/metabolismo , Isoenzimas/química , Solanum tuberosum/química , Espectrometria de FluorescênciaRESUMO
We have tested several chemical modifiers to investigate which amino acid residues, present in the primary structure of the ecto-apyrase, could be involved in catalysis. Synaptosomes from cerebral cortex of rats were prepared and the ATP diphosphohydrolase activity was assayed in absence or the presence of the modifiers. Percentages of residual activity for ATPase and ADPase obtained when the following reagents were tested, are respectively: phenylglyoxal (an arginine group modifier) 17 and 30%; Woodward's reagent (a carboxylic group modifier) 33 and 23%; Koshland's reagent (a tryptophan group modifier) 10 and 12%; maleic anhidride (an amino group modifier) 11 and 25% and carbodiimide reagent (a carboxylic group modifier) 56 and 72%. Otherwise, PMSF, a seryl protein modifier and DTNB, a SH-group modifier did not affect either ATPase or ADPase activity. Inhibitions observed after treatment with phenylglyoxal and Woodward's reagent were significantly prevented when the synaptosomal fraction was preincubated with ATP and ADP, indicating that the arginine and the side chain of glutamate or aspartate (carboxyl groups) participate in the structure of the active site. This interpretation was confirmed by using GTP and GDP, two other apyrase substrates. Phenylglyoxal and Woodward's reagent also inhibited the GTPase and GDPase activities and this inhibition was prevented by preincubation with these substrates.
Assuntos
Adenosina Trifosfatases/metabolismo , Encéfalo/enzimologia , 2-Hidroxi-5-nitrobenzil Brometo/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Animais , Antígenos CD , Apirase/metabolismo , Carbodi-Imidas/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Isoxazóis/farmacologia , Anidridos Maleicos/farmacologia , Fenilglioxal/farmacologia , Ratos , Sinaptossomos/enzimologiaRESUMO
This paper reports findings from an ethnographic study of self-care behaviors and illness concepts among Mexican-American non-insulin dependent diabetes mellitus (NIDDM) patients. Open-ended interviews were conducted with 49 NIDDM patients from two public hospital outpatient clinics in South Texas. They are self-identified Mexican-Americans who have had NIDDM for at least 1 yr, and have no major impairment due to NIDDM. Interviews focused on their concepts and experiences in managing their illness and their self-care behaviors. Clinical assessment of their glucose control was also extracted from their medical records. The texts of patient interviews were content analyzed through building and refining thematic matrixes focusing on their causal explanations and treatment behaviors. We found patients' causal explanations of their illness often are driven by an effort to connect the illness in a direct and specific way to their personal history and their past experience with treatments. While most cite biomedically accepted causes such as heredity and diet, they elaborate these concepts into personally relevant constructs by citing Provoking Factors, such as behaviors or events. Their causal models are thus both specific to their personal history and consistent with their experiences with treatment success or failure. Based on these findings, we raise a critique of the Locus of Control Model of treatment behavior prevalent in the diabetes education literature. Our analysis suggests that a sense that one's own behavior is important to the disease onset may reflect patients' evaluation of their experience with treatment outcomes, rather than determining their level of activity in treatment.
Assuntos
Adaptação Psicológica , Diabetes Mellitus Tipo 2/psicologia , Americanos Mexicanos/psicologia , Autocuidado/psicologia , Papel do Doente , Adulto , Idoso , Dieta para Diabéticos/psicologia , Feminino , Comportamentos Relacionados com a Saúde , Humanos , Controle Interno-Externo , Masculino , Pessoa de Meia-Idade , Cooperação do Paciente/psicologia , Educação de Pacientes como Assunto , TexasRESUMO
Periplasmic 5'-nucleotidase from Escherichia coli, in addition to the monophosphoesterase activity has a diphosphohydrolase activity, acting on nucleoside di- and triphosphates. We proposed that the monophosphoesterase and diphosphohydrolase activities have their own active site. This proposal is based on the different types of bonds being broken. Chemical modification with selective group reagents did not show differences in the essentiality of some residues, like histidyl, carboxyl and arginyl groups, of these two hydrolytic activities. While kinetic approaches employing the competition plot and unidirectional substrate inhibition point to that diphosphohydrolase activity (ATPase-ADPase) do not share the same active site with monophosphoesterase activity. Western blotting developed with polyclonal anti-placental apyrase antibody revealed a single protein in the periplasmic fraction of 66.5 kDa similar to the Mr of the purified enzyme by isoelectrofocusing.
Assuntos
5'-Nucleotidase/metabolismo , Adenosina Trifosfatases/metabolismo , Apirase/metabolismo , Escherichia coli/enzimologia , Complexos Multienzimáticos/metabolismo , Sítios de Ligação , Western Blotting , Membrana Celular/enzimologia , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Hidrólise , Focalização Isoelétrica , Cinética , Diester Fosfórico Hidrolases/metabolismoRESUMO
The human placental microvillar membrane contains several ectoenzymes, including 5'-nucleotidase, alkaline phosphatase and ATP-diphosphohydrolase (ATP-DPH), which might be involved in the extracellular metabolism of nucleotides. The type of anchorage to the plasma membrane of the two first enzymes has been shown to be via a glycosyl-phosphatidylinositol. In the present study, using an enzymatic approach, we show that the ATP-DPH should be attached to the plasma membrane through a different type of anchorage. We were also interested in the search of compounds which could interact differentially with this enzyme to be used as a tool for studying the other two hydrolytic enzymes in the presence of ATP-DPH. Here we report several inhibitors of ecto-ATPases which seem to be a useful tool for studying these three enzymes.