RESUMO
BACKGROUND/AIMS: A twofold increased risk for breast cancer has been reported recently for women with late onset diabetes. Most studies showed that there were differences in serum concentrations of insulin-like growth factors and related proteins between women with and without diabetes who have breast cancer. This study investigated the expression of these markers at the cellular level in a cohort of women with and without type 2 diabetes who underwent biopsy because of a breast lump. METHODS: Relative quantitative analysis of specific mRNA sequences was performed after extraction and reverse transcription polymerase chain reaction amplification from formalin fixed and paraffin wax embedded tissues. Sixty seven breast surgical specimens from women with and without diabetes who did not have cancer and from women with and without diabetes who did have cancer were studied for insulin-like growth factor I (IGF-I), the IGF-I receptor (IGF-IR), insulin-like growth factor binding protein 3 (IGFBP-3), and oestrogen receptor 1 gene expression. RESULTS: The expression of IGF-I and IGF-IR was significantly lower in the cancer groups, whereas there was no significant difference for IGFBP-3 between women with and without cancer. Moreover, there was a good correlation between the expression of IGF-I and IGF-IR in women without cancer: this link was still present in breast tissue from patients with diabetes and cancer, whereas it was lost in patients without diabetes but with cancer. CONCLUSIONS: These differences in IGF-I/IGF-IR expression could contribute to the increased risk for breast cancer in women with type 2 diabetes.
Assuntos
Neoplasias da Mama/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Fator de Crescimento Insulin-Like I/genética , RNA Mensageiro/análise , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Estudos de Casos e Controles , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica/métodos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Pessoa de Meia-Idade , Receptor IGF Tipo 1/genética , Receptores de Estrogênio/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de RiscoRESUMO
Human immunodeficiency virus protease activity can be regulated by reversible oxidation of a sulfur-containing amino acid at the dimer interface. We show here that oxidation of this amino acid in human immunodeficiency virus type 1 protease prevents dimer formation. Moreover, we show that human T-cell leukemia virus type 1 protease can be similarly regulated through reversible glutathionylation of its two conserved cysteine residues. Based on the known three-dimensional structures and multiple sequence alignments of retroviral proteases, it is predicted that the majority of retroviral proteases have sulfur-containing amino acids at the dimer interface. The regulation of protease activity by the modification of a sulfur-containing amino acid at the dimer interface may be a conserved mechanism among the majority of retroviruses.
Assuntos
Endopeptidases/metabolismo , Regulação Viral da Expressão Gênica , Retroviridae/enzimologia , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Gatos , Bovinos , Dimerização , Endopeptidases/química , Endopeptidases/genética , Ativação Enzimática , Protease de HIV/química , Protease de HIV/genética , Protease de HIV/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Oxirredução , Alinhamento de SequênciaAssuntos
Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , HIV-2/enzimologia , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/genética , Cromatografia Líquida de Alta Pressão , Protease de HIV , HIV-2/genética , Humanos , Peróxido de Hidrogênio , Técnicas In Vitro , Espectrometria de Massas , Metionina/química , Metionina Sulfóxido Redutases , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Oxirredutases/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Previous studies have indicated that human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) are less active at blocking viral replication in HIV-1 infected peripheral blood monocytes/macrophages (M/M) than in HIV-1-infected T cells. We explored the hypothesis that oxidative modification and/or metabolism of the PIs in M/M might account for this reduced potency. We first tested the susceptibility of several PIs (kynostatin-272 [KNI-272], saquinavir, indinavir, ritonavir, or JE-2147) to oxidation after exposure to hydrogen peroxide (H(2)O(2)): only KNI-272 was highly susceptible to oxidation. Treatment of KNI-272 with low millimolar concentrations of H(2)O(2) resulted in mono-oxidation of the sulfur in the S-methyl cysteine (methioalanine) moiety, as determined by reversed-phase high-performance liquid chromatography and mass spectrometry (RP-HPLC/MS). Higher concentrations of H(2)O(2) led to an additional oxidation of the sulfur in the thioproline moiety of KNI-272. None of the PIs were metabolized or oxidized when added to T cells and cultured for up to 12 days. However, when KNI-272 was added to M/M, the concentration of the original KNI-272 steadily decreased with a corresponding increase in the production of three KNI-272 metabolites as identified by RP-HPLC/MS. The structures of these metabolites were different from those produced by H(2)O(2) treatment. The two major products of M/M metabolism of KNI-272 were identified as isomeric forms of KNI-272 oxidized solely on the thioproline ring. Both metabolites had reduced capacities to inhibit HIV-1 protease activity when tested in a standard HIV-1 protease assay. These studies demonstrate that antiviral compounds can be susceptible to oxidative modification in M/M and that this can affect their antiviral potency.
Assuntos
Inibidores da Protease de HIV/metabolismo , Protease de HIV/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Oligopeptídeos/metabolismo , Relação Dose-Resposta a Droga , Protease de HIV/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , Humanos , Peróxido de Hidrogênio/química , Técnicas In Vitro , Oligopeptídeos/farmacologia , Oxirredução , Linfócitos T/metabolismo , Fatores de TempoAssuntos
Hiperlipidemias/tratamento farmacológico , Hipolipemiantes/uso terapêutico , Anticolesterolemiantes/uso terapêutico , Colesterol/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Humanos , Hipercolesterolemia/tratamento farmacológico , Hiperlipidemias/sangue , Hipertrigliceridemia/tratamento farmacológico , Triglicerídeos/sangueRESUMO
BACKGROUND AND AIM: Impaired triglyceride-rich lipoprotein metabolism is most probably related to an enhanced cardiovascular risk, and may be associated with a pro-coagulant state. A double-blind, randomized study was undertaken to evaluate two widely utilized hypolipidemic drugs in the post-prandial phase and their impact on lipid, coagulation and fibrinolytic parameters. METHODS AND RESULTS: Thirty middle-aged men selected according to their low density lipoprotein-cholesterol (LDL-C) > or = 160 and < or = 240 mg/dl and borderline hypertriglyceridemia (110-220 mg/dl) after at least one month of a lipid-lowering diet received gemfibrozil (600 mg bid) or simvastatin (20 mg qd) and the corresponding placebo. On enrollment and after 2 months of drug treatment, they were tested with a standard oral fat load (OFL) (35 g fat/m2 body surface). On both occasions plasma total-cholesterol, LDL-C, HDL-C, triglycerides, lipoprotein[a] (Lp[a]), tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), antithrombin-III (AT-III), plasminogen and fibrinogen were determined just before the meal (t0) and at times 2 hours, 4 h, 6 h, 8 h after it (t2-t8). A two-factor (time and visit) multivariate analysis for repeated measurements was performed to evaluate the data. Total cholesterol, and LDL-C were significantly diminished 2 months after both gemfibrozil and simvastatin, the latter being more active. Plasma triglycerides showed a marked reduction with gemfibrozil at all times, while simvastatin regimen yielded only minor modifications. HDL-C was only slightly increased by simvastatin; Lp[a] plasma levels were almost unaffected. Small fibrinogen (t0, t2, t6, t8), PAI-1 (t6) and AT III (t0-t8) increases were observed after gemfibrozil, while simvastatin did not significantly modify these parameters. CONCLUSIONS: In the post-prandial phase, gemfibrozil and simvastatin induce different metabolic effects that beneficially influence the lipid pattern, whereas fibrinolytic and coagulative parameters display minor variations of undetermined significance.
Assuntos
Genfibrozila/farmacologia , Hipercolesterolemia/prevenção & controle , Hipertrigliceridemia/prevenção & controle , Hipolipemiantes/farmacologia , Sinvastatina/farmacologia , Adulto , Idoso , Área Sob a Curva , Coagulação Sanguínea/efeitos dos fármacos , Colesterol/sangue , Método Duplo-Cego , Feminino , Genfibrozila/uso terapêutico , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/complicações , Hipertrigliceridemia/sangue , Hipertrigliceridemia/complicações , Hipolipemiantes/uso terapêutico , Itália , Lipoproteínas/sangue , Lipoproteínas/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Período Pós-Prandial , Sinvastatina/uso terapêutico , Triglicerídeos/sangueRESUMO
The poor solubility of tyrosine (Tyr) limits the amount of this amino acid in total parenteral nutrition (TPN). In rats maintained on a standard pediatric TPN mixture, plasma and brain concentrations of Tyr are reduced to about 25% of the levels in chow-fed controls. To determine whether these low concentrations of Tyr affect the synthesis of catecholamines in neural tissue, the rate-limiting step (conversion of Tyr to dihydroxyphenylalanine [DOPA]) is studied by administering NSD-1015 to block the pyridoxal phosphate (PLP)-dependent decarboxylation of DOPA. However, in TPN rats, plasma concentrations of Tyr are increased by drug treatment. Because brain Tyr is also increased, these and other experiments using NSD-1015 clearly overestimate the rate of DOPA synthesis for drug-free rats on TPN. Nevertheless, in TPN rats, there is less DOPA in the brain in one experiment and less DOPA in the olfactory bulbs in another, versus control rats. Further examination of the metabolic effects of NSD-1015 reveals that the drug also elevates the concentration of branched-chain amino acids (BCAAs) in the plasma of TPN rats. These findings result from inhibition by NSD-1015 of the PLP-dependent aminotransferases that initiate catabolism of Tyr in the liver and BCAAs in the muscle. Despite the pronounced reduction in plasma Tyr, TPN rats showed a marked increase in the activity of hepatic Tyr aminotransferase compared with chow-fed controls. Conversely, although TPN elevates BCAA concentrations in plasma, the activity of branched-chain aminotransferase (BCAT) in the heart muscle of TPN rats is not different from control values. Different values but the same relationships are seen in drug-free rats.
Assuntos
Encéfalo/metabolismo , Di-Hidroxifenilalanina/biossíntese , Nutrição Parenteral Total , Transaminases/metabolismo , Tirosina Transaminase/metabolismo , Tirosina/deficiência , Aminoácidos de Cadeia Ramificada/sangue , Animais , Hidrazinas/farmacologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To examine the association of cognitive impairment with educational, demographic, and nutritional variables in older hospitalized people. DESIGN: Survey of older patients admitted consecutively to a hospital during two 2-month periods in 1993. SETTING: Patients admitted for general medical care at 35 hospitals participating in the GIFA study throughout Italy. PARTICIPANTS: A total of 3628 patients aged 65 or older were studied. MEASUREMENTS: The Hodkinson Abbreviated Mental Test (HAMT) was used as a screening method to assess the patients' basic cognitive function. Multiple logistic regression was used to examine the association between cognitive impairment and demographic, educational or nutritional variables. RESULTS: Twenty-nine percent of older inpatients were classified as having cognitive impairment, with similar distribution of HAMT score found in both genders. Educational attainment has a highly significant inverse relationship with cognitive impairment (highest education: OR 0.32; 95% CI 0.20-0.52). Moreover, cognitive impairment decreased with increasing body mass index (3rd tertile: OR 0.69; 95% CI: 0.51-0.93), cholesterol serum level (highest values: OR 0.59; 95% CI 0.37-0.93), circulating lymphocytes (highest values: OR 0.55; 95% CI 0.45-0.69), and serum albumin (highest values: OR 0.60; 95% CI 0.47-0.76), with a gradient of influence for each variable. CONCLUSIONS: Educational attainment affects cognitive function in older inpatients. The strong association between cognitive impairment and nutritional variables suggests that every effort to improve nutritional status is needed in approaching cognitive impairment in older patients.
Assuntos
Transtornos Cognitivos/epidemiologia , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Consumo de Bebidas Alcoólicas/epidemiologia , Índice de Massa Corporal , Colesterol/sangue , Transtornos Cognitivos/diagnóstico , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/fisiopatologia , Escolaridade , Feminino , Humanos , Itália/epidemiologia , Tempo de Internação/estatística & dados numéricos , Modelos Logísticos , Contagem de Linfócitos , Masculino , Estado Nutricional , Razão de Chances , Prevalência , Fatores de Risco , Albumina Sérica/metabolismo , Distribuição por SexoRESUMO
The mutual interaction between monocytes and low density lipoprotein (LDL) in atherogenesis prompted a test of the hypothesis that LDL-apheresis could reduce the adhesive properties of monocytes to endothelium; and therefore interfere with a key mechanism in atheroma formation. Five patients affected by heterozygous familial hypercholesterolemia were studied. All patients received LDL-apheresis treatment with selective adsorption of LDL-cholesterol on dextran-sulphate columns. Low density lipoprotein particles were isolated by sequential preparative ultracentrifugation and subfractionated by ion exchange high performance liquid chromatography. Thiobarbituric acid reacting products of lipid peroxidation were measured fluorometrically. Vitamin E was estimated by high performance liquid chromatographic technique. Monocytes were isolated from patients blood before and 1 day after LDL-apheresis by Percoll gradient. The blood samples for monocyte adhesion were drawn from control subjects for 2 consecutive days. The adhesion of monocytes to an endothelial monolayer was evaluated by assaying the peroxidase content of the adherent monocytes. Low density lipoprotein-apheresis reduced total cholesterol (-65%; p < 0.01), LDL-cholesterol (-75%; p < 0.01), triglycerides (-51%; p < 0.05), and fibrinogen (-28%; p < 0.01). With LDL-apheresis treatment, a reduction of 54% in oxidized LDLs was observed; vitamin E concentration significantly increased in LDLs (+ 14.2%; p < 0.05). The monocyte adhesion decreased by approximately 61% after apheresis; the variation became statistically significant (-65%; p < 0.01) when endothelial cells were stimulated by lipopolysaccaride.
Assuntos
Remoção de Componentes Sanguíneos , Hiperlipoproteinemia Tipo II/terapia , Lipoproteínas LDL/isolamento & purificação , Idoso , Arteriosclerose/sangue , Arteriosclerose/etiologia , Arteriosclerose/prevenção & controle , Adesão Celular , Células Cultivadas , Endotélio Vascular/fisiologia , Humanos , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/complicações , Técnicas In Vitro , Lipoproteínas LDL/sangue , Lipoproteínas LDL/fisiologia , Masculino , Pessoa de Meia-Idade , Monócitos/fisiologia , OxirreduçãoRESUMO
Clinical and autoptical studies have suggested a predisposing role of the allele E4 of apolipoprotein E (apoE) in the development of atherosclerosis and cardiovascular disease. To investigate the possible contribution of apoE allele polymorphism to the carotid intima-media thickness (IMT) as assessed by ultrasound, we studied 260 asymptomatic nondiabetic subjects (121 men, 139 women; mean +/- SD age, 53 +/- 7 years), randomly selected from the population register of the inhabitants of Trieste, Italy. B-mode ultrasound was used to quantify the maximum IMT at 12 sites on the near and far wall of the common, bifurcation, and internal carotid arteries. ApoE genotypes were determined from amplified apoE sequences by restriction isotyping. The frequencies of E2, E3, and E4 alleles were 0.073, 0.827, and 0.100, respectively. As expected, subjects with E4 allele had the highest levels of total serum cholesterol and LDL cholesterol, subjects with E2 allele had the lowest levels, and those with E3 genotype had intermediate levels. The echographic measurements of carotid IMT showed increasing values from E2 to E4 carriers. After adjustment for total and LDL cholesterol serum levels, triglycerides, ratio of LDL to HDL cholesterol, age, sex, and body mass index, ANCOVA showed that the common carotid IMT was significantly greater (P = .029) in subjects with E4 allele compared with E3 carriers. Our data confirm the influence of apoE4 on cholesterol levels and clearly show that apoE genotype affects carotid atherosclerosis in its early stages in middle-aged asymptomatic subjects.
Assuntos
Apolipoproteínas E/genética , Arteriosclerose/genética , Artérias Carótidas/patologia , Adulto , Idoso , Arteriosclerose/diagnóstico por imagem , Artérias Carótidas/diagnóstico por imagem , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , UltrassonografiaRESUMO
LDL-apheresis often induces an almost constant and progressive increase of the differential pressure of plasma flowing through the dextran sulphate cellulose column, reducing the efficacy of the treatment. On two occasions we were able to identify a fibrin plug by immunofluorescence. Our aim was to verify the modification of some coagulation indicators in patients undergoing LDL-apheresis and whether an activation of coagulation occurs in the LDL-apheresis device. Blood samples were obtained from six patients with familial hypercholesterolaemia who were undergoing LDL-apheresis. During the same session further blood/ plasma samples were taken from the LDL-apheresis device at different sites and at different volumes of filtered blood. In patients after LDL-apheresis the following modifications were found: a 25% decrease of fibrinogen and a slight increase in F1 + 2 plasma levels. No relevant changes in thrombin-antithrombin complexes and fibrinopeptide A plasma levels were noted. In the LDL-apheresis device the main results were: (a) fibrinogen was trapped in the dextran sulphate cellulose column in the early phases; (b) activation of coagulation was recognisable in the plasma separator during the procedure and progressively increased with duration of LDL-apheresis; (c) thrombin-antithrombin complexes, formed in the plasma separator, were retained by the dextran sulphate cellulose column. In conclusion, LDL-apheresis activates coagulation in the device. Shortening cycle time or using nafamostat mesilate as an anticoagulant, could be interesting alternatives for improving the procedure.
Assuntos
Coagulação Sanguínea , Remoção de Componentes Sanguíneos , Lipoproteínas LDL/sangue , Idoso , Antitrombina III/metabolismo , Remoção de Componentes Sanguíneos/instrumentação , Remoção de Componentes Sanguíneos/métodos , Celulose , Dextranos , Feminino , Fibrinogênio/metabolismo , Humanos , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/terapia , Masculino , Pessoa de Meia-Idade , Tempo de Tromboplastina Parcial , Peptídeo Hidrolases/metabolismo , SulfatosRESUMO
To assess whether the initial status of lipid metabolism in patients with chronic viral hepatitis might correlate with outcome of therapy, 52 patients (32 males and 20 female) with chronic hepatitis C were studied: 44 were treated with human recombinant interferon-alpha 2b (3 MU three times per week for up to 12 months), and 8 served as controls. At baseline, sera were tested for total and HDL cholesterol, HDL2, HDL3, apolipoprotein A-I, apolipoprotein B, interferon-alpha, tumor necrosis factor, and interleukin-6. Changes in blood lipids were evaluated after 3, 30, and 90 days of treatment. HDL cholesterol, apolipoprotein A-I, and HDL3 decreased by 9.4-11.4% within 4 weeks of starting interferon treatment, but this effect was sustained only in patients with a primary response to interferon. On multivariate analysis, a primary response to interferon correlated with higher apolipoprotein A-I and lower (< 2.23 pg/ml) interleukin-6 levels (p < 0.005 for both). In contrast, a sustained response was significantly more common in patients with low (< or = 13.3 pg/ml) serum interferon-alpha and lower interleukin-6 at baseline but did not correlate with any of the blood lipids. Thus, in chronic hepatitis C, interferon treatment induces specific changes in blood lipids. The concentration of apolipoprotein A-I at baseline is a strong predictor of primary response to treatment, and the likelihood of sustained response seems to be reflected by lower cytokine activation.
Assuntos
Hepatite C/tratamento farmacológico , Interferon-alfa/uso terapêutico , Lipídeos/sangue , Adolescente , Adulto , Idoso , Sequência de Bases , Estudos de Casos e Controles , Doença Crônica , Feminino , Hepatite C/sangue , Humanos , Interferon alfa-2 , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Análise Multivariada , Proteínas Recombinantes , Resultado do TratamentoRESUMO
Human erythrocyte pyridoxal phosphatase has an essential requirement for divalent cations. Its activation by Mg2+, Co2+, Ni2+, or Mn2+ followed Michaelis-Menten kinetics. Other divalent cations inhibited the enzyme. The kinetic properties of the enzyme were investigated with pyridoxal phosphate and Mg2+ alone and in the presence of the product, Pi, or dead-end inhibitors at pH 7.4 and 37 degrees C. The enzyme bound both the substrate and Mg2+ before products were released. Pi gave competitive inhibition vs substrate and noncompetitive inhibition vs Mg2+. Molybdate also was a competitive inhibitor vs substrate and noncompetitive inhibitor vs Mg2+. Ca2+ gave competitive inhibition vs Mg2+ and noncompetitive inhibition vs substrate. The effects of Mg2+ and substrate on the inactivation of pyridoxal phosphatase by a variety of group-specific reagents were studied. The inactivation of the enzyme by iodoacetate was potentiated by MgCl2. The Kd of the enzyme-Mg complex determined in the inactivation analysis was similar to the Km of the free enzyme for Mg2+, indicating that Mg2+ binds to the free enzyme. Low concentrations of a substrate, pyridoxine phosphate, or Pi protected pyridoxal phosphatase from inactivation by N-ethylmaleimide in the absence or presence of Mg2+. Thus, the substrate binds to the free enzyme and the enzyme-Mg complex. The steady-state kinetics and the kinetics of inactivation are consistent with random binding of pyridoxal phosphate and Mg2+ and with the formation of a dead-end complex of Pi with the enzyme-Mg complex.
Assuntos
Eritrócitos/enzimologia , Metais/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Ativação Enzimática , Humanos , CinéticaRESUMO
The study was carried out on 25 whole carotid arteries explanted from a corpse and perfused at constant pressure to reproduce the conditions of an in vivo examination as much as possible. Out of 5 samples with intimal thickening detected by echo, fibrosis of the tunica media was observed by the pathologist in 4 and microcalcification in 1. In 4 vessels with soft plaques at echo scanning, a wide necrotic area (2 cases), slack connective tissue (1 case) and cystic lesions (1 case) were observed. Hard lesions with (5 cases) or without (2 cases) a cone of shadow at echo evaluation corresponded to fibrous (2 cases) or fibrocalcific (3 cases) plaques. The histological study of the two echo-diagnosed thrombi showed an intermediate echographic pattern and the main feature of the non-occluding thrombus was the absence of a lumen-lesion interface. Mixed plaques were diagnosed at echo in 9 arteries and the correspondent histological aspect was a typical atheromatous lesion in all cases. Thus, the comparison of the ultrasound image with the histological findings proved the reliability of echography in the detection of atheromatous lesions with an excellent agreement between the results at the 2 examinations. Since the type of carotid lesions has an impact upon clinical events these results might support the use of vascular ultrasound images in clinical applications.
Assuntos
Arteriosclerose/diagnóstico por imagem , Arteriosclerose/patologia , Doenças das Artérias Carótidas/diagnóstico por imagem , Doenças das Artérias Carótidas/patologia , Artéria Carótida Primitiva/diagnóstico por imagem , Artéria Carótida Primitiva/patologia , Artéria Carótida Externa/diagnóstico por imagem , Artéria Carótida Externa/patologia , Artéria Carótida Interna/diagnóstico por imagem , Artéria Carótida Interna/patologia , Humanos , Reprodutibilidade dos Testes , UltrassonografiaRESUMO
Pyridoxal phosphatase purified from human erythrocytes catalyzes the dephosphorylation of pyridoxal phosphate (PLP) and pyridoxine phosphate. The enzyme had phosphotransferase activity and transferred 20-25% of the phosphoryl group from either substrate to ethanol. Incubation of the enzyme with [32P]PLP, followed by quenching in acid, resulted in trapping 0.14-0.24 mol of 32P per mol of subunit. The incorporation of 32P was not due to Schiff base formation. Phosphorylation of the enzyme by [32P]PLP required catalysis by the enzyme and did not occur in the presence of excess pyridoxine phosphate or with denatured enzyme. The phosphoenzyme intermediate was relatively acid stable and very labile at high pH or in the presence of hydroxylamine. Woodward's reagent K, which specifically modifies acidic amino acid residues, inactivated the phosphatase in a concentration- and time-dependent manner which followed pseudo-first-order kinetics. Substrates or Pi protected the enzyme from inactivation. It is concluded that PLP phosphatase catalyzes the hydrolysis of PLP by forming a covalent phosphoenzyme intermediate and the intermediate may be an acylphosphate. The 32P-labeled phosphatase was digested with pepsin, and two radioactive peaks were isolated by reversed-phase chromatography. However, definitive sequences were not obtained.
Assuntos
Eritrócitos/enzimologia , Monoéster Fosfórico Hidrolases/sangue , Catálise , Humanos , Fosfatos/metabolismo , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Fosfato de Piridoxal/metabolismoRESUMO
It has been shown that severe hypercholesterolemia is associated with carotid atherosclerosis but it is unclear whether this is true for moderate hypercholesterolemia. The aim of the study was to determine the prevalence of ultrasound detectable extent and severity of carotid intima-media thicknesses in 143 asymptomatic (79 males, 64 females, age range 45-64 years) primary moderate hypercholesterolemic patients (serum LDL cholesterol range 160-190 mg/dl). This group was compared with 143 asymptomatic normolipidemic subjects (serum LDL cholesterol < or = 130 mg/dl and serum triglycerides < 200 mg/dl) matched for age, sex and other cardiovascular risk factors. The maximum intima-media thickness (IMT) was measured using B-mode ultrasonography at 12 sites on the near and the far wall of the common, bifurcation and internal carotid arteries. The mean-maximum IMT at the 12 sites was compared in cases and controls. Moreover, the prevalence of intima-media thickening (i.e. at least one of the 12 sites with an IMT equal to or greater than 1.0 mm but less than 1.3) and plaques (i.e. at least one of the 12 sites with an IMT equal to or greater than 1.3 mm) was considered in the two groups. The mean-maximum intima-media thickness was 0.97 +/- 0.12 mm in hypercholesterolemic patients and 0.93 +/- 0.05 mm in controls (P < 0.0001). Intima-media thickening and plaques were detected in 76% of hypercholesterolemics vs. 57% of controls (P < 0.0002). Gender did not influence these differences.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Arteriosclerose/complicações , Doenças das Artérias Carótidas/complicações , Hipercolesterolemia/complicações , Arteriosclerose/diagnóstico por imagem , Artérias Carótidas/diagnóstico por imagem , Doenças das Artérias Carótidas/diagnóstico por imagem , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , UltrassonografiaRESUMO
Over a 1-year period, three patients were seen in our trauma service with delayed bleeding (> or = 7 days) from an initially inapparent splenic injury. This entity was defined as a late occurrence of signs and symptoms attributed to splenic injury not detected by diagnostic computed tomographic (CT) scanning during the initial examination. We believe that this represents an "injury in evolution" minor enough to go undetected on initial CT scans of the abdomen. A high index of suspicion and liberal utilization of imaging techniques are essential for the identification of delayed splenic rupture. Further multicenter studies are required to delineate the true incidence of its occurrence and its clinical significance. We conclude that "delayed rupture" of the spleen is a true clinical entity. The occurrence of a delayed rupture may prove hazardous to patients discharged early from the hospital after blunt abdominal injury. A classification system to assess this type of injury is suggested.
Assuntos
Ruptura Esplênica , Acidentes de Trânsito , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esplenectomia , Ruptura Esplênica/classificação , Ruptura Esplênica/diagnóstico por imagem , Ruptura Esplênica/cirurgia , Fatores de Tempo , Tomografia Computadorizada por Raios XRESUMO
Pyridoxal-specific phosphatase purified from human erythrocytes was inactivated by a variety of thiol-specific reagents in a time- and concentration-dependent manner. The presence of pyridoxal phosphate, a substrate, or inorganic phosphate, a competitive inhibitor, protected the enzyme from inactivation. Phosphatase inactivated by disulfide reagents was reactivated by the addition of excess dithiothreitol, indicating that the inactivation was due to formation of a mixed disulfide between the reagent and a free cysteinyl residue at or near the active site of the enzyme. Incorporation of either 1 mol of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 0.6 mol of iodo[3H]acetate, or 0.6 mol of N-[3H]ethylmaleimide per mol of subunit led to complete inactivation of the enzyme. High concentration of phosphate prevented the incorporation of DTNB and iodo[3H]acetate. Amino acid analysis of carboxymethylated enzyme and DTNB titration of the denatured phosphatase indicated that there may be only 1 cysteinyl residue per subunit. Modification by iodoacetate did not affect the quaternary structure of the enzyme. The phosphatase modified by iodo[3H]acetate was subjected to trypsin digestion, and the resulting peptides were separated on a reverse phase C18 column. Two radioactive peaks were obtained and contained a peptide with the N-terminal sequence of Ala-Gln-Gly-Val-Leu-Phe-Asp-Cys(Cm)-Asp-Gly-Val-Leu-X-Asn-Gly. Most of the radioactivity was released with Cys(Cm). These results indicate that the cysteinyl residue in this sequence is at or near the active site and is essential for activity. Residues 5-12 and 15 of this peptide are identical with a sequence of a yeast alkaline p-nitrophenylphosphatase, and the peptide has little homology with other mammalian phosphatases.
Assuntos
Cisteína , Eritrócitos/enzimologia , Monoéster Fosfórico Hidrolases/sangue , Reagentes de Sulfidrila/farmacologia , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Ácido Ditionitrobenzoico/metabolismo , Etilmaleimida/metabolismo , Humanos , Iodoacetatos/metabolismo , Ácido Iodoacético , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/isolamento & purificação , Ligação ProteicaRESUMO
The specificity and active site properties of vitamin B6 phosphatase purified from human erythrocytes were studied by kinetic analyses with vitamin B6 compounds and derivatives and chemical modification with group-specific reagents. The kinetic constants for pyridoxal phosphate (PLP), 4-pyridoxic acid phosphate, pyridoxine phosphate, and pyridoxamine phosphate were determined from pH 5 to 9. The values of Vmax/Km and pKm were highest for PLP and 4-pyridoxic acid phosphate and lowest for pyridoxamine phosphate. Vmax/Km and pKm for the four substrates were maximum between pH 6 and 8. Ionizable groups with pKa values about 6 and 8 affected substrate binding to the enzyme. Vmax values for all the substrates gradually decreased with increasing pH. The enzyme also catalyzed the dephosphorylation of 4'-secondary amine derivatives of vitamin B6-phosphate. The phosphatase had greatest catalytic efficiency with substrates that contained a negatively charged group on the 4'-position of the pyridine ring. It is concluded that there are one or two positively charged groups at the active site of the enzyme that interact with the substrate's phosphate ester and 4'-substituent. The phosphatase was inactivated by phenylglyoxal, and PLP protected the enzyme against this inactivation. Phenylglyoxal did not modify Lys or Cys residues or an alpha-amino group since the enzyme's NH2 terminus is blocked, and it did not affect the quaternary structure of the phosphatase. The enzyme was inactivated by the incorporation of 1 mol of phenylglyoxal/subunit. Diethylpyrocarbonate inactivated the enzyme by reacting with a group with a pKa of 6.7, and pyridoxine phosphate protected the enzyme against this inactivation. These data suggest that Arg and His residues are at or near the active site and may play roles in substrate binding and/or catalysis.