cDNA cloning and 1.75 Å crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 ± 15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed β(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-β-d-glucopyranose units in chitin. The full-length amino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 Å resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (βα) 8 barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182. © 2006 The Authors.

In vivo clastogenicity assessment of the Austroplenckia populnea (Celastraceae) leaves extract using micronucleus and chromosome aberration assay

The clastogenic effect of the A. populnea leaves extract was tested in vivo on bone marrow cells of Wistar rats by evaluating the induction of chromosome aberrations and micronuclei induction on polychromatic erythrocytes. The extract was administered by gavage at doses of 300, 600 and 900mg/kg body weight. Experimental and control animals were submitted to euthanasia 24 h after the treatment. Under the conditions used, A. populnea leaves extract did not induce decrease in mitotic index and did not induce a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes or chromosome aberrations in the bone marrow cells of Wistar rats. © 2007 The Japan Mendel Society.

Aspectos estruturais da membrana eritrocitária

This article describes the structures and functions of the erythrocyte membrane and its importance in transfusional medicine. The erythrocyte membrane is one of the best known membranes in terms of structure, function and genetic disorders. As any other plasma membrane, it mediates transport functions. It also provides the erythrocytes with their resilience and deformability. According to the International Society of Blood Transfusion (ISBT), more than 500 antigens are expressed in the erythrocyte membrane, and around 270 are involved in transfusion reaction cases and hemolytic diseases of the fetus and newborn. In the ISBT classification, the high frequency series is represented by antigens in more than 99% of population (high prevalence antigen). In transfusion, the absence of these antigens determines severe problems as for example, one woman without the P antigen suffered 6 repetitive miscarriages due to placental insufficiency, which was caused by an antibody formed against the absent P antigen. Some important erythrocyte membrane proteins are described here including Band 3, Glycophorins and spectrin. The most abundant integral membrane protein is Band 3 and its main function is to mediate exchange of chloride and bicarbonate anions across the plasma membrane. The second most abundant integral membrane protein in the human erythrocyte is sialoglycoprotein glycophorin A (GPA). With its high sialic acid content, GPA is the main contributor to the net negative cell-surface charge and is thus critical for minimizing cell-cell interactions and preventing red cell aggregation. Glycophorin C (GPC) is the receptor for PfEBP-2 (baebl, EBA-140), the newly identified erythrocyte binding ligand of Plasmodium falciparum. The ternary complex of spectrin, actin and 4.1R defines the nodes of the erythrocyte membrane skeletal network, and is inseparable from membrane stability when under mechanical stress. This erythrocyte membrane review is important for a better understanding of transfusion reactions, where the antibody formation against high prevalence antigens makes compatible transfusions difficult. The study of antigen diversity and biochemical characterization of different proteins will contribute to healthcare, as well as diagnosis, development of technology such as monoclonal antibody production and the therapeutic conduct of many diseases.

Assessment of the potential clastogenic/aneugenic risk of Casearia sylvestris extract using in vivo assays

Casearia sylvestris (Flacourtiaceae) is a plant which grows in wild and has been widely used in folk medicine. In this study, clastogenic/aneugenic properties of Casearia sylvestris crude ethanolic extract were evaluated using in vivo chromosomal aberrations (CAs) and micronucleus (MN) assays in rodents. The animals were treated by gavage with 3 concentrations of the extract: 150, 300 and 500 mg/kg body weight. Bone marrow cells from Wistar rats were collected 24 h after having been submitted to the MN and CAs test. Peripheral blood cells from Swiss mice were collected 48 and 72 h after having been submitted to the MN test. The results show that C. sylvestris extract does not induce a significant increase in mean values for micronucleated polychromatic erythrocytes (MNPCE) in Swiss mice and Wistar rats, or CAs in rat bone marrow cells, at the 3 tested doses, indicating that the extract showed no clastogenic/aneugenic effects on chromosomes of the rodent cells tested. © 2007 The Japan Mendel Society.

Citologia do lavado broncoalveolar de eqüinos da polícia militar do distrito federal

The objective of this study was evaluate the airways' cells types from horses of the military police of Federal District. It was used 24 horses, divided into 2 groups of 12: group I - animals in policy activity and group II - animals out of policy activity. At the inspection, physical exam and blood exam, the results agreed with no present clinical pathologies. It was infused 240mL of saline solution in the bronchoalveolar wash. In the bronchoalveolar wash, the group I registered 25.25±7.3 squamous cells, 20.2±5.5 epithelial cells, 22.2±6.23 macrophages and 2.02±5.77 erythrocytes, and in the group II, it was observed 31.0±9.47 squamous cells, 19.0±5.20 epithelial cells, 43.0±13.88 macrophages, 4.0±1.52 eosinophile and 3.0±1.0 erythrocytes. Verifying the occurrence of side effects after the experiment, there was no problem from the used technique.

Significado da presença de esquizócitos no sangue periférico de gestantes com pré-eclâmpsia

PURPOSE: to evaluate the significance of schizocytes presence in peripheral blood smear of pregnant women with pre-eclampsia, identifying and correlating them with other markers of hemolysis and of the disease severity. METHODS: Seventh six glass slides of peripheral blood smear of pregnant women with pre-eclampsia have been evaluated. After the smear, the slides have been stained with Leishman's dye and stored till they were examined with a Leica, model DLMB microscope, provided with the Qwin Lite 2.5 software that made it possible to record the images of selected fields in CD-ROM. Ten fields with approximately 100 erythrocytes were counted in each glass slide. Schizocytes (irregular fragment or helmet-shaped, bite-shaped or triangular) were considered as present, when their percentage was equal or higher than 0.2%, their presence being correlated with other hemolysis markers (hemoglobin, total bilirubin, lactic desidrogenasis and reticulocytes), pre-eclampsia markers (proteinuria and platelet number). The Statistical Package in Social Science for Windows (SPSS), 10.0 version has been used for statistical analysis, at p<0.05. RESULTS: schizocytes have been present in 31.6% of the pregnant women with pre-eclampsia. In most (75%) of the blood smears there have been three or four schizocytes. There has been no correlation between schizocyte presence and any other hemolysis marker, any pre-eclampsia marker or disease severity. CONCLUSIONS: schizocytes have been identified in a small number and in less than a third of the pregnant women with pre-eclampsia. There has been no correlation with other hemolysis marker parameters or with the disease severity. This way, the presence of schizocytes is not a marker of the clinical evolution of pre-eclampsia.

Metodologia para quantificação de leucóciotos totais em peixe Oreochromis Niloticus

Haematological investigation is an important part of disease diagnosis. The techniques used for mammals are generally applicable for fishes with slight modification. The presence of nucleated erythrocytes and thrombocytes in fish may cause some confusion in the identification of blood cells, mainly in total leukocytes count. This work evaluated two different methods (direct and indirect) of leukocytes counting. Specimens of tilapia were divided randomly into four groups of 48 fish each. Each group was further randomized into three replicate experiments of 16 fish per replicate in 40 L aquarium and maintained for 10 days. Counting of leukocytes in peripheral blood was determined in six fishes per group at the times: 0, 3, 7 and 10 days. The direct method in Neubauer chamber showed a large dispersion of data in regard to the average, the opposite was observed in indirect method determined in blood smears that showed more homogeneity among groups. Due to this factor the indirect method can be considered more accurate than the direct method for counting leukocytes in fishes.

Hematological and incubation parameters of chicks from young breeders eggs: Variation with sex and incubation temperature

This experiment analyzed the effect of sex and incubation temperature on daily mass loss and eggshell conductance, embryo mortality rates, incubation duration, hematological parameters and body, liver, heart and bursa weights of neonatal chicks from young breeders. The daily mass loss was higher at incubation temperature of 39°C. The eggshell conductance rate increased with the temperature. The total and partial duration of incubation were lower for eggs incubated at 39°C. The time taken by the chick to leave the eggshell did not differ below and above the thermoneutral temperature. The total and intermediate embryo mortality rates increased with the incubation temperature, whereas the early and late embryo mortality rates were higher at incubation temperature of 39°C. Sex did not influence the analyzed parameters, while the incubation temperature did not affect the body and bursa weight and the erythrocytes characteristics. The liver weight of chicks incubated at 36°C was higher than the incubated at 39°C, however there were no differences among the liver weight from chicks incubated at 36 and 39°C and those incubated at 37.5°C. The number of heterophils and the heterophil/lymphocyte ratio (H/L ratio) increased following the temperature, whereas the number of lymphocytes decreased at high temperatures. The other leukocyte parameters did not suffer influence of temperature. Males and females presented similar response to variation of incubation temperatures (36, 37.5 and 39°C) and demonstrated higher sensibility to temperatures above the thermoneutral. Moreover, temperatures below the thermoneutral demonstrated to be better for improvement of hatchability and development of chicks from light eggs. © Asian Network for Scientific Information, 2010.

O papel das argininas alfa-92 e alfa-141 na regulação das propriedades funcionais de hemoglobinas por íons cloreto

The oxygenation of human Hb (HbA) demands a three state model: two deoxy states To and Tx, free and complexed with anions respectively, and an oxy R state. The regulation between these states is modulated by the presence of anions, such as chloride, that binds to T state. The b inding if chloride, however, remains controversial. The aim of this work is the study of arginines 92a (a1ß2 interface) and 141a (C-terminal) as chloride binding sites. To investigate that, we have studied 92 and 141 site directed mutant species: natural mutants Hb J-Cape-Town (R92Q), desArg (R141Δ), Chesapeake (R92L), and the constructed Chesapeake desArg (R92L,141Δ). We expressed Hbs in Escherichia coli and purified. Through oxygen binding curves we measured affinity and cooperativity, in function of water effect and Bohr effect in presence and absence of chloride. Structural features were obtained through 1H NMR spectroscopy Oxygen binding properties and Bohr effect measured indicated a higher affinity and lower cooperativity in absence and presence of chloride for all mutants. Structural changes represent functional aspects of mutant Hbs, such as a significant rise in affinity or a change in cooperativity. Water activity studies conducted as a function of chloride concentration showed that the only Hb desArg follows the thre state model. The other mutant Hbs do not exhibit the Tx state, a fact confirmed by the number of water molecules bound to each Hb during the deoxy-oxy transition. This behavior suggests that the Arginine 92 site could be responsible for chloride binding to Hb, since oxygenation of 92 mutant Hbs cannot be adjusted by the three state model. However, Bohr effect showed that all mutant Hbs released~1 proton in chloride presence, different from HbA that releases ~2, suggesting a role for 141 arginine in the tertiary and quaternary Bohr effect.

Evaluations hematological and biochemical by the use of sodium diclofenac, meloxicam and firocoxib in rats

This work has evaluated the hematological and biochemical profile by the use of sodium diclofenac, meloxicam and firocoxib in Wistar rats. The rats were distributed in groups: G1 (control), G2 (diclofenac sodium: 15 mg/kg), G3 (meloxicam: 2.0 mg/ kg), G4 (meloxicam: 10.0 mg/ kg), G5 (firocoxib: 5.0 mg/ kg) e G6 (firocoxib: 25.0 mg/ kg). The drugs were administered intragastrically (gavage) once a day, during five days and evaluated in three moments: M1 (48 hours after the beginning of the treatment), M2 (96 hours after the beginning of the treatment) and M3 (72 hours after the ending of the treatment). In each moment of each group, five to seven animals were evaluated and laboratory exams were performed. There were no significant changes observed in the biochemical and hematological parameters by the use of meloxicam and firocoxib. One of the effects of the sodium diclofenac was eritrogram variation as hematocrit, erythrocytes, hemoglobin decrease during the treatment. In addition, the platelets and total white blood cells counts did not change except for basophil. There was no changes in AST, ALP, GGT, urea, creatinine, sodium, potassium values. However, the values of protein, globulin and albumin decreased. It was concluded that diclofenac sodium does not provide large variations in the hemogram and biochemical profile than the meloxicam and firocoxib do not provide delletery effects in laboratories tests.

Advances in sickle cell disease treatment: From drug discovery until the patient monitoring

Sickle Cell Disease (SCD) is one of the most prevalent hematological diseases in the world. Despite the immense progress in molecular knowledge about SCD in last years few therapeutical sources are currently available. Nowadays the treatment is performed mainly with drugs such as hydroxyurea or other fetal hemoglobin inducers and chelating agents. This review summarizes current knowledge about the treatment and the advancements in drug design in order to discover more effective and safe drugs. Patient monitoring methods in SCD are also discussed. © 2011 Bentham Science Publishers Ltd.

Eritrograma e medição dos eritrócitos de avestruzes (Struthio camelus) em Sãojosé do Rio Preto-SP, Brasil

Ostriches' erythrocytic parameters help on the diagnosis of specific pathologies and serve as basic knowledge for studies in comparative avian pathology. To obtain reference values of erythrocyte indices for ostriches (Struthio camelus) raised in a commercial system in Brazil and verify if there are differences between gender and age groups, 240 healthy from both sexes animals were bled. Heparinized blood samples were analyzed using standard techniques to determine the red blood cell (RBC) count, hemoglobin concentration, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) values, besides the red cell morphometry analysis using a computer software that calculates the greater and the smaller diameters of the erythrocytes. Prior to data analysis, ostriches were divided into three different age groups: from four to 13 months, from 13 to 23 months and from 23 to 30 months the. Younger ostriches presented lower erythrocyte indices than the older ones. The age group effect was only significant in females for the erythrocyte, hemoglobin, MCV, MCH, and MCHC variables. The female ostriches presented PCV, MCV, MCH, and MCHC values significantly higher than males in some age groups. It was observed that the erythrocytes of the female ostriches are more elongated and larger than males. It was concluded that erythrocytic parameters of ostriches in São José do Rio Preto-SP, Brazil are influenced by gender and age, highlighting the importance of consider besides these factors also the geoclimatic conditions to an adequate interpretation of the erythrogram.

Antioxidant and cytotoxic studies for kaempferol, quercetin and isoquercitrin

The aim of the present study was to investigate a cytotoxic oxidative cell stress related and the antioxidant profile of kaempferol, quercetin, and isoquercitrin. The flavonol compounds were able to act as scavengers of superoxide anion (but not hydrogen peroxide), hypochlorous acid, chloramine and nitric oxide. Although flavonoids are widely described as antioxidants and this activity is generally related to beneficial effects on human health, here we show important cytotoxic actions of three well known flavonoids. They were able to promote hemolysis which one was exacerbated on the presence of hypochlorous acid but not by AAPH radical. Therefore, despite they expected scavenger action over free radicals an oxidants, these compounds could be very lesive to living organisms by acting over erythrocytes and maybe other cellular types.

Absence of genotoxic effects of the coumarin derivative 4-methylesculetin in vivo and its potential chemoprevention against doxorubicin-induced DNA damage

4-Methylesculetin (4-ME) is a synthetic derivative of coumarin that displays a potent reactive oxygen species (ROS) scavenger and metal chelating agent and therefore has been produced to help reduce the risk of human disease. The main objective of this study was to investigate the in vivo genotoxicity of 4-ME and initially to verify its potential antigenotoxicity on doxorubicin (DXR)-induced DNA damage. Different doses of 4-ME (500, 1000 and 2000mgkg -1 body weight) were administered by gavage only or with a simultaneous intraperitoneal (i.p.) injection of DXR (80mgkg -1). The following endpoints were analyzed: DNA damage in peripheral blood, liver, bone marrow, brain and testicle cells according to an alkaline (pH>13) comet assay and micronucleus induction in bone marrow cells. Cytotoxicity was assessed by scoring polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). No differences were observed between the negative control and the groups treated with a 4-ME dose for any of the endpoints analyzed, indicating that it lacks genotoxic and cytotoxic effects. Moreover, 4-ME demonstrated protective effects against DXR-induced DNA damage at all tested doses and in all analyzed cell types, which ranged from 34.1% to 93.3% in the comet assay and 54.4% to 65.9% in the micronucleus test.

Hemoparasite and hematological parameters in Nile tilapia

The accelerated growth of finfish aquaculture has resulted in a series of health problems, including blood disorders by hemoparasites; there are scarce studies about these agents and their impact in actual intensive farming. The aim of this study was to identify and quantify the hemoparasites present in monocytes and erythrocytes in the blood of tilapia and their correlation with the hematological profile. Blood samples were collected from caudal vessels of 15 cage-reared Nile tilapia (Oreochromis niloticus), with 70 ± 10 g weight on average, from Itambaracá Municipality, Parana State, Brazil. The total red blood cells, mean corpuscular volume, hematocrit, total and differential white blood cells counts, and the number of thrombocytes were determined in blood smears stained with May-Grünwald-Giemsa-Wright and Quick Panoptic. The results showed the presence of pleomorphic cytoplasmic inclusions with corrugated appearance and basophilic staining, mainly in monocytes, suggesting Anaplasmataceae parasitemia and inclusions with the same morphological characteristics in erythrocytes of one Nile tilapia. The hematological analysis showed no significant difference (P < 0.05) between infected and not infected fish, and therefore, there was no correlation between parasitemia and hematological profile. These observations allow us to infer that the intracytoplasmic inclusions in monocytes and erythrocytes are compatible with the family Anaplasmataceae. There was no correlation between the blood profile and low level of parasitemia. © 2012 Springer-Verlag London.