Platelet glycoprotein Ib beta/IX mediates glycoprotein Ib alpha localization to membrane lipid domain critical for von Willebrand factor interaction at high shear

The localization of the platelet glycoprotein GP Ib-IX complex (GP Ibα, GP Ibβ, and GP IX) to membrane lipid domain, also known as glycosphingolipid-enriched membranes (GEMs or raft) lipid domain, is essential for the GP Ib-IX complex mediated platelet adhesion to von Willebrand factor (vWf) and subsequent platelet activation. To date, the mechanism for the complex association with the GEMs remains unclear. Although the palmitate modifications of GP Ibβ and GP IX were thought to be critical for the complex presence in the GEMs, we found that the removal of the putative palmitoylation sites of GP Ibβ and GP IX had no effects on the localization of the GP Ib-IX complex to the GEMs. Instead, the disruption of GP Ibα disulfide linkage with GP Ibβ markedly decreased the amount of the GEM-associated GP Ibα without altering the GEM association of GP Ibβ and GP IX. Furthermore, partial dissociation with the GEMs greatly inhibited GP Ibα interaction with vWf at high shear instead of in static condition or under low shear stress. Thus, for the first time, we demonstrated that GP Ibβ/GP IX mediates the disulfide-linked GP Ibα localization to the GEMs, which is critical for vWf interaction at high shear.     

Activation state of alpha4beta1 integrin on sickle red blood cells is linked to the duffy antigen receptor for chemokines (DARC) expression

In sickle cell anemia, reticulocytes express enhanced levels of α4β1 integrin that interact mainly with vascular cell adhesion molecule-1 and fibronectin, promoting vaso-occlusion. These interactions are known to be highly sensitive to the inflammatory chemokine IL-8. The Duffy antigen receptor for chemokines (DARC) modulates the function of inflammatory processes. However, the link between α4β1 activation by chemokines and DARC erythroid expression is not or poorly explored. Therefore, the capacity of α4β1 to mediate Duffy-negative and Duffy-positive sickle reticulocyte (SRe) adhesion to immobilized vascular cell adhesion molecule-1 and fibronectin was evaluated. Using static adhesion assays, we found that, under basal conditions, Duffy-positive SRe adhesion was 2-fold higher than that of Duffy-negative SRes. Incubating the cells with IL-8 or RANTES (regulated on activation normal T cell expressed and secreted) increased Duffy-positive SRe adhesion only, whereas Mn(2+) increased cell adhesion independently of the Duffy phenotype. Flow cytometry analyses performed with anti-β1 and anti-α4 antibodies, including a conformation-sensitive one, in the presence or absence of IL-8, revealed that Duffy-positive and Duffy-negative SRes displayed similar erythroid α4β1 expression levels, but with distinct activation states. IL-8 did not affect α4β1 affinity in Duffy-positive SRes but induced its clustering as corroborated by immunofluorescence microscopy. Our results indicate that in Duffy-negative SRes α4β1 integrin is constitutively expressed in a low affinity state, whereas in Duffy-positive SRes α4β1 is expressed in a higher chemokine-sensitive affinity state. This activation state associated with DARC RBC expression may influence the intensity of the inflammatory responses encountered in sickle cell anemia and participate in its interindividual clinical expression variability.     

High molecular weight kininogen activates B2 receptor signaling pathway in human vascular endothelial cells

The nonenzymatic cofactor high molecular weight kininogen (HK) is a precursor of bradykinin (BK). The production of BK from HK by plasma kallikrein has been implicated in the pathogenesis of inflammation and vascular injury. However, the functional role of HK in the absence of prekallikrein (PK), the proenzyme of plasma kallikrein, on vascular endothelial cells is not fully defined. In addition, no clinical abnormality is seen in PK-deficient patients. Therefore, an investigation into the effect of HK, in the absence of PK, on human pulmonary artery endothelial cell (HPAEC) function was performed. HK caused a marked and dose-dependent increase in the intracellular calcium [Ca(2+)](i) level in HPAEC. Gd(3+) and verapamil potentiated the HK-induced increase in [Ca(2+)](i). HK-induced Ca(2+) increase stimulated endothelial nitric oxide (NO) and prostacyclin (PGI(2)) production. The inhibitors of B(2) receptor-dependent signaling pathway impaired HK-mediated signal transduction in HPAEC. HK had no effect on endothelial permeability at physiological concentration. This study demonstrated that HK regulates endothelial cell function. HK could play an important role in maintaining normal endothelial function and blood flow and serve as a cardioprotective peptide.     

Detection of etiological agents of malaria in howler monkeys from Atlantic Forests, rescued in regions of São Paulo city, Brazil

Background In some states of the Brazilian extra‐Amazonian region, such as the Atlantic Forest area, autochthonous human cases of malaria were related to simian malarias and vice versa. Methods To verify the presence of Plasmodium, 50 blood samples of howler monkeys (Alouatta guariba clamitans) rescued from the Metropolitan Region of Saõ Paulo city, where the Atlantic Forest is present, were analyzed. The samples were submitted to microscopy (thin and thick blood smears), enzyme‐linked immunosorbent assays (ELISA), indirect immunofluorescent assay (IFA), and polymerase chain reaction (PCR). Results Only one smear showed forms reminiscent of Plasmodium vivax. In ELISA, the frequencies of antibodies against synthetic peptides corresponding to circumsporozoite protein of P. vivax VK210 ‘classic’ (Pvc), P. vivax VK247, human P. vivax‐like (Pvk and Pvl), P. malariae/P. brasilianum (Pm), and P. falciparum (Pf) were 24.0% (12/50) for Pvc, 8.0% (04/50) for Pvk, 6.0% (03/50) for Pvl, 24.0% (12/50) for Pm, and 28.0% (14/50) for Pf, while the frequency of antibodies against PvMSP119 recombinant proteins was 42.0% (21/50). No serum reacted against PfMSP1‐19. In IFA,the seropositivity of antibodies against asexual forms of P. malariae was 31.3% (15/48). We utilized three PCR protocols to develop a molecular consensus (positive results in, at least, two protocols). The frequency of Plasmodium infections detected by PCR was 18.0% (09/50) for P. vivax, 4.0% (02/50) for P. malariae, and 76.0% (38/50) of samples were negative. The molecular consensus was not seen in 4.0% (02/50) of samples. Conclusions These results suggest that a possible interaction between human and simian malaria coming from a zoonotic cycle cannot be discarded because simians that live in the areas of the Atlantic Forest could play a role as a reservoir for Plasmodium.     

A new type of orally active anti-diabetic Zn(II)-dithiocarbamate complex

In order to find orally active Zn(II) complexes that can treat diabetes mellitus (DM) at low doses, four new Zn(II)-dithiocarbamate complexes with Zn(II)-sulfur coordination bonds were prepared and their in vitro insulinomimetic activity and in vivo anti-diabetic ability were evaluated. Among the Zn(II)-dithiocarbamate complexes, the bis(pyrrolidine-N-dithiocarbamate)zinc(II) (Zn(pdc)(2)) complex was found to be the most effective in terms of inhibiting free fatty acid-release and enhancing glucose-uptake in adipocytes. After oral administration of the Zn(pdc)(2) complex to KK-A(y) mice with obesity and type 2 DM, we observed that the high blood glucose levels in the mice were lowered from approximately 500 mg/dL to 350 mg/dL within 6 days, and the effect was maintained during the administration period. Also, indicators of insulin resistance such as serum insulin, leptin, and triglyceride levels were also reduced compared with those in untreated mice. Moreover, the Zn(pdc)(2) complex improved not only the hypertension in the mice, but also the adiponectin level in the serum. On the basis of the results, the Zn(pdc)(2) complex is proposed to improve hyperglycemia and insulin resistance in type 2 DM animals on daily oral administrations.     

Fluoxetine and nortriptyline affect NTPDase and 5'-nucleotidase activities in rat blood serum

Depression is a serious condition associated with considerable morbidity and mortality. Selective serotonin reuptake inhibitors and tricyclic antidepressants, such as fluoxetine and nortriptyline, respectively, were commonly used in treatment for depression. Selective serotonin reuptake inhibitors have been associated with increased risk of bleeding complications, possibly as a result of inhibition of platelet aggregation. ATP, ADP and adenosine are signaling molecules in the vascular system and nucleotidases activities are considered an important thromboregulatory system which functions in the maintenance of blood fluidity. Therefore, here we investigate the effect of in vivo (acute and chronic) and in vitro treatments with the antidepressant drugs on nucleotidases activities in rat blood serum. In acute treatment, nortriptyline decreased ATP hydrolysis (41%), but not altered ADP and AMP hydrolysis. In contrast, fluoxetine did not alter NTPDase and ecto-5'-nucleotidase activities. A significant inhibition of ATP, ADP, and AMP hydrolysis were observed in chronic treatment with fluoxetine (60%, 32%, and 42% for ATP, ADP, and AMP hydrolysis, respectively). Similar effects were shown in chronic treatment with nortriptyline (37%, 41%, and 30% for ATP, ADP, and AMP hydrolysis, respectively). In addition, there were no significant changes in NTPDase and ecto-5'-nucleotidase activities when fluoxetine and nortriptyline (100, 250, and 500 microM) were tested in vitro. Our results have shown that fluoxetine and nortriptyline changed the nucleotide catabolism, suggesting that homeostasis of vascular system can be altered by antidepressant treatments.     

Contribution to the Data on Copper Concentration in Blood and Urine in Patients with Wilson’s Disease and in Normal Subjects

Determination of copper in human tissues and body fluids may be crucial in the diagnosis of Wilson’s disease. In this study we evaluated urinary copper excretion and urine and blood concentration in 14 patients in whom Wilson’s disease was confirmed (group A) and in 21 subjects in whom the disease was only suspected (group B). The following values (mean ± SD) were found: 24-h urine (μg Cu/24 h), 152 ± 135 (A) and 31.8 ± 10.9 (B); urine (μg Cu/ml), 0.091 ± 0.087 (A) and 0.028 ± 0.011 (B); and blood (μg Cu/ml), 0.62 ± 0.25 (A) and 0.72 ± 0.09 (B). By comparison, urine copper concentration in the group of apparently healthy subjects was 0.035 ± 0.010 (n = 50), and blood copper concentration in autopsy cases of nonpoisoned people was 0.85 ± 0.19 (n = 73).     

Blood cleansing in heart atrium of <Emphasis Type="Italic">Trichogaster leerii</Emphasis> (Bleeker, 1852), Anabantidae: Teleostei

The capability and capacity of endocardial tissue in heart of Trichogaster leerii (Pearl gourami) to take up intraperitoneally injected horse-spleen ferritin and the regular content of indigestible wastes in this tissue are described. The entire heart wall was spongy composed of muscle trabeculae enveloped by flat endocardial cells. In atrium these cells were filled by Prussian blue precipitations in sections treated with acid ferrocyanide, when the period of time between ferritin-injection and sacrifice was 6 h or more. The endocardial cell layers enveloping the muscle trabeculae in ventricle or covering the wall and valves of the ventricular apertures nearly lacked such precipitations. Those endocardial cell layers capable to endocytose much foreign ferritin, also reacted strongly with Schmorl’s solution in control hearts, i.e. these atrial cells are normally rich in indigestible wastes, probably lipofuscin. We suggest that the endocytic and lipofuscin-rich endocardial cells in heart atrium may play a role in the blood clearance in T. leerii and that this cleansing in heart is restricted to atrium only in this species.     

Redistribution of blood within the body is important for thermoregulation in an ectothermic vertebrate (<Emphasis Type="Italic">Crocodylus porosus</Emphasis>)

Changes in blood flow are a principal mechanism of thermoregulation in vertebrates. Changes in heart rate will alter blood flow, although multiple demands for limited cardiac output may compromise effective thermoregulation. We tested the hypothesis that regional differences in blood flow during heating and cooling can occur independently from changes in heart rate. We measured heart rate and blood pressure concurrently with blood flow in the crocodile, Crocodylus porosus. We measured changes in blood flow by laser Doppler flowmetry, and by injecting coloured microspheres. All measurements were made under different heat loads, with and without blocking cholinergic and β-adrenergic receptors (autonomic blockade). Heart rates were significantly faster during heating than cooling in the control animals, but not when autonomic receptors were blocked. There were no significant differences in blood flow distribution between the control and autonomic blockade treatments. In both treatments, blood flow was directed to the dorsal skin and muscle and away from the tail and duodenum during heating. When the heat source was switched off, there was a redistribution of blood from the dorsal surface to the duodenum. Blood flow to the leg skin and muscle, and to the liver did not change significantly with thermal state. Blood pressure was significantly higher during the autonomic blockade than during the control. Thermal time constants of heating and cooling were unaffected by the blockade of autonomic receptors. We concluded that animals partially compensated for a lack of differential heart rates during heating and cooling by redistributing blood within the body, and by increasing blood pressure to increase flow. Hence, measures of heart rate alone are insufficient to assess physiological thermoregulation in reptiles.     

Acid proteinase,phospholipase, and biofilm production of <Emphasis Type="Italic">Candida</Emphasis> species isolated from blood cultures

Three virulence factors comprising proteinase, phospholipase, and biofilm among 68 Candida albicans and 31 non-albicans Candida strains (11 C. tropicalis, 8 C. parapsilosis, 6 C. glabrata, 4 C. guillermondii, 2 C. krusei) isolated from blood cultures were analyzed. In total, 61 (89.7%) C. albicans strains were detected as proteinase positive whereas eight (25.8%) non-albicans Candida strains were proteinase positive (P < 0.05). Phospholipase production was detected in 41 (60.3%) C. albicans strains. All non-albicans Candida strains were phospholipase negative. Biofilm production was determined by both visual and spectrophotometric methods. Eight (11.8%) of C. albicans strains and 13 (41.93%) of 31 non-albicans Candida strains were biofilm positive with two of the methods (P < 0.05). According to our results, we may suggest that detection of hydrolytic enzyme and biofilm production abilities of the Candida isolates in clinical mycology laboratories may warn the clinican for a possible hematogenous infection.