The role of platelet and plasma markers of antioxidant status and oxidative stress in thrombocytopenia among patients with vivax malaria

Malaria remains an important health problem in tropical countries like Brazil. Thrombocytopenia is the most common hematological disturbance seen in malarial infection. Oxidative stress (OS) has been implicated as a possible mediator of thrombocytopenia in patients with malaria. This study aimed to investigate the role of OS in the thrombocytopenia of Plasmodium vivax malaria through the measurement of oxidant and antioxidant biochemical markers in plasma and in isolated platelets. Eighty-six patients with P. vivax malaria were enrolled. Blood samples were analyzed for total antioxidant and oxidant status, albumin, total protein, uric acid, zinc, magnesium, bilirubin, total thiols, glutathione peroxidase (GPx), malondialdehyde (MDA), antibodies against mildly oxidized low-density lipoproteins (LDL-/nLDL ratio) and nitrite/nitrate levels in blood plasma and GPx and MDA in isolated platelets. Plasma MDA levels were higher in thrombocytopenic (TCP) (median 3.47; range 1.55-12.90 µmol/L) compared with the non-thrombocytopenic (NTCP) patients (median 2.57; range 1.95-8.60 µmol/L). Moreover, the LDL-/nLDL autoantibody ratio was lower in TCP (median 3.0; range 1.5-14.8) than in NTCP patients (median 4.0; range 1.9-35.5). Finally, GPx and MDA were higher in the platelets of TPC patients. These results suggest that oxidative damage of platelets might be important in the pathogenesis of thrombocytopenia found in P. vivax malaria as indicated by alterations of GPx and MDA.

Cells and mediators of inflammation (C-reactive protein, nitric oxide, platelets and neutrophils) in the acute and convalescent phases of uncomplicated Plasmodium vivax and Plasmodium falciparum infection

The haematological changes and release of soluble mediators, particularly C-reactive protein (CRP) and nitric oxide (NO), during uncomplicated malaria have not been well studied, especially in Brazilian areas in which the disease is endemic. Therefore, the present study examined these factors in acute (day 0) and convalescent phase (day 15) patients infected with Plasmodium falciparum and Plasmodium vivax malaria in the Brazilian Amazon. Haematologic parameters were measured using automated cell counting, CRP levels were measured with ELISA and NO plasma levels were measured by the Griess reaction. Our data indicate that individuals with uncomplicated P. vivax and P. falciparum infection presented similar inflammatory profiles with respect to white blood cells, with high band cell production and a considerable degree of thrombocytopaenia during the acute phase of infection. Higher CRP levels were detected in acute P. vivax infection than in acute P. falciparum infection, while higher NO was detected in patients with acute and convalescent P. falciparum infections. Although changes in these mediators cannot predict malaria infection, the haematological aspects associated with malaria infection, especially the roles of platelets and band cells, need to be investigated further.

Mineral and vitamin content of beef, chicken, and turkey hydrolysates mineral and vitamin content of protein hydrolysates

The purpose of this study was to assess the concentration of vitamins and minerals in meat protein hydrolysates. Calcium, phosphorus and iron were analyzed by inductively coupled-plasma atomic emission spectrophotometry; vitamin C was analyzed by the reduction of cupric ions and vitamins B1 and B2 by fluorescence. Regarding minerals, the beef hydrolysate (BH) had more iron than the turkey hydrolysate (TH) and the chicken hydrolysate (CH); TH had a little more phosphorus. BH had the largest amount of vitamin C, and similar amounts of vitamins B1 and B2. The amount of these nutrients found in the hydrolysates suggests that it is possible to use them to enrich special dietary formulations.

Fracionamento de cobre em proteínas do plasma, músculo e fígado de tilápia do Nilo

Copper fractionation in plasma, muscle and liver of Nile tilapia was performed after protein separation by 2D-PAGE. SR XRF analysis indicated the presence of copper in three protein spots of plasma, and in two protein spots of muscle and liver, respectively. Copper ions were found to be distributed mostly in proteins that had a molar mass of less than 54 kDa and greater than 13 kDa and a pI in the 5.3-9.3 range. The copper concentration bound to these proteins was determined by GFAAS which showed concentrations in the 1.20-4.82 mg g-1 range.

Determinação de bussulfano em plasma através de cromatografia líquida de alta eficiência com detector de arranjo de diodos e derivatização com dietilditiocarbamato de sódio

A high performance liquid chromatographic-diode array detection method for the determination of busulfan in plasma was developed and validated. Sample preparation consisted of protein precipitation followed by derivatization with sodium diethyldithiocarbamate and liquid-liquid extraction with methyl-tert-butyl ether. Chromatograms were monitored at 277 nm. Separation was carried out on a Lichrospher RP 18 column (5 µm, 250 x 4 mm). The mobile phase consisted of water and acetonitrile (20:80, v/v). The method presented adequate specificity, linearity, precision and accuracy and allowed reliable determination of busulfan in clinical plasma samples, being applied to three patients submitted to bone marrow transplantation.

Determination of levodopa in human plasma by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS): application to a bioequivalence study

A sensitive, accurate and simple method using HPLC-MS/MS was developed and validated for levodopa quantitation in human plasma. Analysis was achieved on a pursuit® C18 analytical column (5 µm; 150 x 4.6 mm i.d.) using a mobile phase (methanol and water , 90:10, v/v) containing formic acid 0.5% v/v, after extracting the samples using a simple protein plasma precipitation with perchloric acid. The developed method was validated in accordance with ANVISA guidelines and was successfully applied to a bioequivalence study in 60 healthy volunteers demonstrating the feasibility and reliability of the proposed method.

Regulation of gap junctions by protein phosphorylation

Gap junctions are constituted by intercellular channels and provide a pathway for transfer of ions and small molecules between adjacent cells of most tissues. The degree of intercellular coupling mediated by gap junctions depends on the number of gap junction channels and their activity may be a function of the state of phosphorylation of connexins, the structural subunit of gap junction channels. Protein phosphorylation has been proposed to control intercellular gap junctional communication at several steps from gene expression to protein degradation, including translational and post-translational modification of connexins (i.e., phosphorylation of the assembled channel acting as a gating mechanism) and assembly into and removal from the plasma membrane. Several connexins contain sites for phosphorylation for more than one protein kinase. These consensus sites vary between connexins and have been preferentially identified in the C-terminus. Changes in intercellular communication mediated by protein phosphorylation are believed to control various physiological tissue and cell functions as well as to be altered under pathological conditions.

Increase in skeletal muscle protein content by the ß-2 selective adrenergic agonist clenbuterol exacerbates hypoalbuminemia in rats fed a low-protein diet

This investigation examined how the nutritional status of rats fed a low-protein diet was affected when the animals were treated with the ß-2 selective agonist clenbuterol (CL). Males (4 weeks old) from an inbred, specific-pathogen-free strain of hooded rats maintained at the Dunn Nutritional Laboratory were used in the experiments (N = 6 rats per group). CL treatment (Ventipulmin, Boehringer-Ingelheim Ltd., 3.2 mg/kg diet for 2 weeks) caused an exacerbation of the symptoms associated with protein deficiency in rats. Plasma albumin concentrations, already low in rats fed a low-protein diet (group A), were further reduced in CL rats (A = 25.05 ± 0.31 vs CL = 23.64 ± 0.30 g/l, P<0.05). Total liver protein decreased below the level seen in either pair-fed animals (group P) or animals with free access to the low-protein diet (A = 736.56 ± 26 vs CL = 535.41 ± 54 mg, P<0.05), whereas gastrocnemius muscle protein was higher than the values normally described for control (C) animals (C = 210.88 ± 3.2 vs CL = 227.14 ± 1.7 mg/g, P<0.05). Clenbuterol-treated rats also showed a reduction in growth when compared to P rats (P = 3.2 ± 1.1 vs CL = -10.2 ± 1.9 g, P<0.05). This was associated with a marked decrease in fat stores (P = 5.35 ± 0.81 vs CL = 2.02 ± 0.16 g, P<0.05). Brown adipose tissue (BAT) cytochrome oxidase activity, although slightly lower than in P rats (P = 469.96 ± 16.20 vs CL = 414.48 ± 11.32 U/BAT x kg body weight, P<0.05), was still much higher than in control rats (C = 159.55 ± 11.54 vs CL = 414.48 ± 11.32 U/BAT x kg body weight, P<0.05). The present findings support the hypothesis that an increased muscle protein content due to clenbuterol stimulation worsened amino acid availability to the liver and further reduced albumin synthesis causing exacerbation of hypoalbuminemia in rats fed a low-protein diet.

Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice

The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins.

Association of apolipoprotein E polymorphism with plasma lipids and Alzheimer's disease in a Southern Brazilian population

Apolipoprotein E (protein: apo E; gene: APOE) plays an important role in the multifactorial etiology of both Alzheimer's disease (AD) and lipid level concentrations. The polymerase chain reaction (PCR) was used to investigate the APOE gene polymorphism in 446 unrelated Caucasians, among them 23 AD patients, and 100 Afro-Brazilians living in Porto Alegre, Brazil. The frequencies of the APOE*2, APOE*3 and APOE*4 alleles were 0.075, 0.810 and 0.115 in Caucasians and 0.075, 0.700 and 0.225 in Afro-Brazilians, respectively (c2 = 8.72, P = 0.013). A highly significant association was observed between the APOE*4 allele and AD in this population-based sample. The APOE*4 frequency in AD patients (39%) was about four times higher than in the general Caucasian population (11.5%). The influence of each of the three common APOE alleles on lipid traits was evaluated by the use of the average excess statistic. The E*2 allele is associated with lower levels of triglycerides and of total and non-HDL cholesterol in both men and women. Conversely, the E*4 allele is associated with higher levels of these traits in women only. The effect of APOE alleles was of greater magnitude in women.

Isolation of bovine plasma albumin by liquid chromatography and its polymerization for use in immunohematology

The aim of the method described here is to remove hemoglobin, the major contaminant in the bovine plasma obtained from slaughterhouses, by adding a mixture of 19% cold ethanol and 0.6% chloroform, followed by fibrinogen and globulin precipitation by the Cohn method and nonspecific hemagglutinin by thermocoagulation. The experimental volume of bovine plasma was 2,000 ml per batch. Final purification was performed by liquid chromatography using the ion-exchange gel DEAE-Sepharose FF. The bovine albumin thus obtained presented > or = 99% purity, a yield of 25.0 ± 1.2 g/l plasma and >71.5% recovery. N-acetyl-DL-tryptophan (0.04 mmol/g protein) and sodium caprylate (0.04 mmol/g protein) were used as stabilizers and the final concentration of albumin was adjusted to 22.0% (w/v), pH 7.2 to 7.3. Viral inactivation was performed by pasteurization for 10 h at 60°C. The bovine albumin for the hemagglutination tests used in immunohematology was submitted to chemical treatment with 0.06% (w/v) glutaraldehyde and 0.1% (w/v) formaldehyde at 37°C for 12 h to obtain polymerization. A change in molecular distribution was observed after this treatment, with average contents of 56.0% monomers, 23.6% dimers, 12.2% trimers and 8.2% polymers. The tests performed demonstrated that this polymerized albumin enhances the agglutination of Rho(D)-positive red cells by anti-Rho(D) serum, permitting and improving visualization of the results.

von Willebrand factor antigen levels in plasma of patients with malignant breast disease

von Willebrand factor (vWF) is a protein that mediates platelet adherence to the subendothelium during primary hemostasis. High plasma vWF concentrations have been reported in patients with various types of cancer, such as head and neck, laryngeal and prostatic cancer, probably representing an acute phase reactant. In the present study we determined the plasma levels of vWF antigen (vWF:Ag) by quantitative immunoelectrophoresis in 128 female patients with breast cancer as well as in 47 women with benign breast disease and in 27 healthy female controls. The levels of vWF:Ag were 170.7 ± 78 U/dl in patients with cancer, 148.4 ± 59 U/dl in patients with benign disease and 130.6 ± 45 U/dl in controls (P<0.005). We also detected a significant increase in the levels of vWF:Ag (P<0.0001) in patients with advanced stages of the disease (stage IV = 263.3 ± 113 U/dl, stage IIIB = 194.0 ± 44 U/dl) as compared to those with earlier stages of the disease (stage I = 155.3 ± 65 U/dl, stage IIA = 146.9 ± 75 U/dl). In conclusion, vWF levels were increased in plasma of patients with malignant breast disease, and these levels correlated with tumor progression.

GLUT4 protein expression in obese and lean 12-month-old rats: insights from different types of data analysis

GLUT4 protein expression in white adipose tissue (WAT) and skeletal muscle (SM) was investigated in 2-month-old, 12-month-old spontaneously obese or 12-month-old calorie-restricted lean Wistar rats, by considering different parameters of analysis, such as tissue and body weight, and total protein yield of the tissue. In WAT, a ~70% decrease was observed in plasma membrane and microsomal GLUT4 protein, expressed as µg protein or g tissue, in both 12-month-old obese and 12-month-old lean rats compared to 2-month-old rats. However, when plasma membrane and microsomal GLUT4 tissue contents were expressed as g body weight, they were the same. In SM, GLUT4 protein content, expressed as µg protein, was similar in 2-month-old and 12-month-old obese rats, whereas it was reduced in 12-month-old obese rats, when expressed as g tissue or g body weight, which may play an important role in insulin resistance. Weight loss did not change the SM GLUT4 content. These results show that altered insulin sensitivity is accompanied by modulation of GLUT4 protein expression. However, the true role of WAT and SM GLUT4 contents in whole-body or tissue insulin sensitivity should be determined considering not only GLUT4 protein expression, but also the strong morphostructural changes in these tissues, which require different types of data analysis.

Hormone replacement therapy increases levels of antibodies against heat shock protein 65 and certain species of oxidized low density lipoprotein

Hormone replacement therapy (HRT) reduces cardiovascular risks, although the initiation of therapy may be associated with transient adverse ischemic and thrombotic events. Antibodies against heat shock protein (Hsp) and oxidized low density lipoprotein (LDL) have been found in atherosclerotic lesions and plasma of patients with coronary artery disease and may play an important role in the pathogenesis of atherosclerosis. The aim of the present study was to assess the effects of HRT on the immune response by measuring plasma levels of antibodies against Hsp 65 and LDL with a low and high degree of copper-mediated oxidative modification of 20 postmenopausal women before and 90 days after receiving orally 0.625 mg equine conjugate estrogen plus 2.5 mg medroxyprogesterone acetate per day. HRT significantly increased antibodies against Hsp 65 (0.316 ± 0.03 vs 0.558 ± 0.11) and against LDL with a low degree of oxidative modification (0.100 ± 0.01 vs 0.217 ± 0.02) (P<0.05 and P<0.001, respectively, ANOVA). The hormone-mediated immune response may trigger an inflammatory response within the vessel wall and potentially increase plaque burden. Whether or not this immune response is temporary or sustained and deleterious requires further investigation.

Abnormal subcellular distribution of GLUT4 protein in obese and insulin-treated diabetic female dogs

The GLUT4 transporter plays a key role in insulin-induced glucose uptake, which is impaired in insulin resistance. The objective of the present study was to investigate the tissue content and the subcellular distribution of GLUT4 protein in 4- to 12-year-old control, obese and insulin-treated diabetic mongrel female dogs (4 animals per group). The parametrial white adipose tissue was sampled and processed to obtain both plasma membrane and microsome subcellular fractions for GLUT4 analysis by Western blotting. There was no significant difference in glycemia and insulinemia between control and obese animals. Diabetic dogs showed hyperglycemia (369.9 ± 89.9 mg/dl). Compared to control, the plasma membrane GLUT4, reported per g tissue, was reduced by 55% (P < 0.01) in obese dogs, and increased by 30% (P < 0.05) in diabetic dogs, and the microsomal GLUT4 was increased by ~45% (P < 0.001) in both obese and diabetic animals. Considering the sum of GLUT4 measured in plasma membrane and microsome as total cellular GLUT4, percent GLUT4 present in plasma membrane was reduced by ~65% (P < 0.001) in obese compared to control and diabetic animals. Since insulin stimulates GLUT4 translocation to the plasma membrane, percent GLUT4 in plasma membrane was divided by the insulinemia at the time of tissue removal and was found to be reduced by 75% (P < 0.01) in obese compared to control dogs. We conclude that the insulin-stimulated translocation of GLUT4 to the cell surface is reduced in obese female dogs. This probably contributes to insulin resistance, which plays an important role in glucose homeostasis in dogs.