Platelet adhesion signalling and the regulation of thrombus formation

Platelets perform a central role in haemostasis and thrombosis. They adhere to subendothelial collagens exposed at sites of blood vessel injury via the glycoprotein (GP) 1b-V-IX receptor complex, GPV1 and integrin alpha(2)beta(1)-These receptors perform distinct functions in the regulation of cell signalling involving non-receptor tyrosine kinases (e.g. Src, Fyn, Lyn, Syk and Btk), adaptor proteins, phospholipase C and lipid kinases such as phosphoinositide 3-kinase. They are also coupled to an increase in cytosolic calcium levels and protein kinase C activation, leading to the secretion of paracrine/autocrine platelet factors and an increase in integrin receptor affinities. Through the binding of plasma fibrinogen and von Willebrand Factor to integrin alphaIIbbeta(3), a platelet thrombus is formed. Although increasing evidence indicates that each of the adhesion receptors GPIb-V-IX and GPV1 and integrins alpha(2)beta(1) and alpha(IIb)beta(3) contribute to the signalling that regulates this process, the individual roles of each are only beginning to be dissected. By contrast, adhesion receptor signalling through platelet endothelial cell adhesion molecule 1 (PECAM-1) is implicated in the inhibition of platelet function and thrombus formation in the healthy circulation. Recent studies indicate that understanding of platelet adhesion signalling mechanisms might enable the development of new strategies to treat and prevent thrombosis.

Proteomic profiling of neuromas reveals alterations in protein composition and local protein synthesis in hyper-excitable nerves

Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis ( 2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a > 1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel alpha 2 delta-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S(35)methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability.

Alterations in receptor binding properties of recent human influenza H3N2 viruses are associated with reduced natural killer cell lysis of infected cells

Natural killer (NK) cell recognition of influenza virus-infected cells involves hemagglutinin (HA) binding to sialic acid (SA) on activating NK receptors. SA also acts as a receptor for the binding of influenza virus to its target host cells. The SA binding properties of H3N2 influenza viruses have been observed to change during circulation in humans: recent isolates are unable to agglutinate chicken red blood cells and show reduced affinity for synthetic glycopolymers representing SA-alpha-2,3-lactose (3'SL-PAA) and SA-alpha-2,6-N-acetyl lactosamine (6'SLN-PAA) carbohydrates. Here, NK lysis of cells infected with human H3N2 influenza viruses isolated between 1969 and 2003 was analyzed. Cells infected with recent isolates (1999 to 2003) were found to be lysed less effectively than cells infected with older isolates (1969 to 1996). This change occurred concurrently with the acquisition of two new potential glycosylation site motifs in RA. Deletion of the potential glycosylation site motif at 133 to 135 in HA1 from a recent isolate partially restored the agglutination phenotype to a recombinant virus, indicating that the HA-SA interaction is inhibited by the glycosylation modification. Deletion of either of the recently acquired potential glycosylation sites from HA led to increased NK lysis of cells infected with recombinant viruses carrying modified HA. These results indicate that alterations in RA glycosylation may affect NK cell recognition of influenza virus-infected cells in addition to virus binding to host cells.

Modulation of endothelial cell KCa3.1 Channels during endothelium-derived hyperpolarizing factor signaling in mesenteric resistance arteries

Arterial hyperpolarization to acetylcholine (ACh) reflects coactivation of KCa3.1 (IKCa) channels and KCa2.3 (SKCa) channels in the endothelium that transfers through myoendothelial gap junctions and diffusible factor(s) to affect smooth muscle relaxation (endothelium-derived hyperpolarizing factor [EDHF] response). However, ACh can differentially activate KCa3.1 and KCa2.3 channels, and we investigated the mechanisms responsible in rat mesenteric arteries. KCa3.1 channel input to EDHF hyperpolarization was enhanced by reducing external [Ca2+]o but blocked either with forskolin to activate protein kinase A or by limiting smooth muscle [Ca2+]i increases stimulated by phenylephrine depolarization. Imaging [Ca2+]i within the endothelial cell projections forming myoendothelial gap junctions revealed increases in cytoplasmic [Ca2+]i during endothelial stimulation with ACh that were unaffected by simultaneous increases in muscle [Ca2+]i evoked by phenylephrine. If gap junctions were uncoupled, KCa3.1 channels became the predominant input to EDHF hyperpolarization, and relaxation was inhibited with ouabain, implicating a crucial link through Na+/K+-ATPase. There was no evidence for an equivalent link through KCa2.3 channels nor between these channels and the putative EDHF pathway involving natriuretic peptide receptor-C. Reconstruction of confocal z-stack images from pressurized arteries revealed KCa2.3 immunostain at endothelial cell borders, including endothelial cell projections, whereas KCa3.1 channels and Na+/K+-ATPase {alpha}2/{alpha}3 subunits were highly concentrated in endothelial cell projections and adjacent to myoendothelial gap junctions. Thus, extracellular [Ca2+]o appears to modify KCa3.1 channel activity through a protein kinase A-dependent mechanism independent of changes in endothelial [Ca2+]i. The resulting hyperpolarization links to arterial relaxation largely through Na+/K+-ATPase, possibly reflecting K+ acting as an EDHF. In contrast, KCa2.3 hyperpolarization appears mainly to affect relaxation through myoendothelial gap junctions. Overall, these data suggest that K+ and myoendothelial coupling evoke EDHF-mediated relaxation through distinct, definable pathways.

Muscle Protein Alterations in LGMD21 Patients With Different Mutations in the Fukutin-related Protein Gene

Fukutin-related protein (FKRP) is a protein involved in the glycosylation of cell surface molecules. Pathogenic mutations in the FKRP gene cause both the more severe congenital muscular dystrophy Type 1C and the milder Limb-Girdle Type 21 form (LGMD21). Here we report muscle histological alterations and the analysis of 11 muscle proteins: dystrophin, four sarcoglycans, calpain 3, dysferlin, telethonin, collagen VI, alpha-DG, and alpha 2-laminin, in muscle biopsies from 13 unrelated LGMD21 patients with 10 different FKRP mutations. In all, a typical dystrophic pattern was observed. In eight patients, a high frequency of rimmed vacuoles was also found. A variable degree of alpha 2-laminin deficiency was detected in 12 patients through immunofluorescence analysis, and 10 patients presented a-DG deficiency on sarcolemmal membranes. Additionally, through Western blot analysis, deficiency of calpain 3 and dystrophin bands was found in four and two patients, respectively. All the remaining proteins showed a similar pattern to normal controls. These results suggest that, in our population of LGMD21 patients, different mutations in the FKRP gene are associated with several secondary muscle protein reductions, and the deficiencies of alpha 2-laminin and alpha-DG on sections are prevalent, independently of mutation type or clinical severity.

Animal models for genetic neuromuscular diseases

The neuromuscular disorders are a heterogeneous group of genetic diseases, caused by mutations in genes coding sarcolemmal, sarcomeric, and citosolic muscle proteins. Deficiencies or loss of function of these proteins leads to variable degree of progressive loss of motor ability. Several animal models, manifesting phenotypes observed in neuromuscular diseases, have been identified in nature or generated in laboratory. These models generally present physiological alterations observed in human patients and can be used as important tools for genetic, clinic, and histopathological studies. The mdx mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD). Although it is a good genetic and biochemical model, presenting total deficiency of the protein dystrophin in the muscle, this mouse is not useful for clinical trials because of its very mild phenotype. The canine golden retriever MD model represents a more clinically similar model of DMD due to its larger size and significant muscle weakness. Autosomal recessive limb-girdle MD forms models include the SJL/J mice, which develop a spontaneous myopathy resulting from a mutation in the Dysferlin gene, being a model for LGMD2B. For the human sarcoglycanopahties (SG), the BIO14.6 hamster is the spontaneous animal model for delta-SG deficiency, whereas some canine models with deficiency of SG proteins have also been identified. More recently, using the homologous recombination technique in embryonic stem cell, several mouse models have been developed with null mutations in each one of the four SG genes. All sarcoglycan-null animals display a progressive muscular dystrophy of variable severity and share the property of a significant secondary reduction in the expression of the other members of the sarcoglycan subcomplex and other components of the Dystrophin-glycoprotein complex. Mouse models for congenital MD include the dy/dy (dystrophia-muscularis) mouse and the allelic mutant dy(2J)/dy(2J) mouse, both presenting significant reduction of alpha 2-laminin in the muscle and a severe phenotype. The myodystrophy mouse (Large(myd)) harbors a mutation in the glycosyltransferase Large, which leads to altered glycosylation of alpha-DG, and also a severe phenotype. Other informative models for muscle proteins include the knockout mouse for myostatin, which demonstrated that this protein is a negative regulator of muscle growth. Additionally, the stress syndrome in pigs, caused by mutations in the porcine RYR1 gene, helped to localize the gene causing malignant hypertermia and Central Core myopathy in humans. The study of animal models for genetic diseases, in spite of the existence of differences in some phenotypes, can provide important clues to the understanding of the pathogenesis of these disorders and are also very valuable for testing strategies for therapeutic approaches.

Mechanism of acetylcholine-induced calcium signaling during neuronal differentiation of P19 embryonal carcinoma cells in vitro

Muscarinic (mAChRs) and nicotinic acetylcholine receptors (nAChRs) are involved in various physiological processes, including neuronal development. We provide evidence for expression of functional nicotinic and muscarinic receptors during differentiation of P19 carcinoma embryonic cells, as an in vitro model of early neurogenesis. We have detected expression and activity alpha(2)-alpha(7), beta(2), beta(4) nAChR and M1-M5 mAChR subtypes during neuronal differentiation. Nicotinic alpha(3) and beta(2) mRNA transcription was induced by addition of retinoic acid to P19 cells. Gene expression Of alpha(2), alpha(4)-alpha(7), beta(4) nAChR subunits decreased during initial differentiation and increased again when P19 cells underwent final maturation. Receptor response in terms of nicotinic agonist-evoked Ca2+, flux was observed in embryonic and neuronal-differentiated cells. Muscarinic receptor response, merely present in undifferentiated P19 cells, increased during neuronal differentiation. The nAChR-induced elevation of intracellular calcium ([Ca2+](i)) response in undifferentiated cells was due to Ca2+ influx. In differentiated P19 neurons the nAChR-induced [Ca2+](i) response was reduced following pretreatment with ryanodine, while the mAChR-induced response was unaffected indicating the contribution of Ca2+ release from ryanodine-sensitive stores to nAChR- but not mAChR-mediated Ca2+ responses. The presence of functional nAChRs in embryonic cells suggests that these receptors are involved in triggering Ca2+ waves during initial neuronal differentiation. (C) 2007 Elsevier Ltd. All rights reserved.

Age-related changes in cerebellar phosphatase-1 reduce Na,K-ATPase activity

We evaluated whether changes in protein content and activity of PP-1 and PP-2A were the mechanism underneath the basal age-related reduction in alpha(2/3)-Na,K-ATPase activity in rats cerebella and whether this occurred through the cyclic GMP-PKG pathway. PP1 activity, but not its expression, increased with age, whereas PP-2 was not changed. The activity Of ot2/3-Na,K-ATPase varied with age. and there was a negative association between the PP-1 and alpha(2/3)-Na,K-ATPase activities. In young rats, the inhibition of PP-1 and PP-2A by okadaic acid (OA) increased in a dose-dependent manner alpha(1)- and alpha(2/3)-Na,K-ATPase, but had no effect on Mg-ATPase activity. A direct stimulation of PKG with 8-Br-cyclic GMP did not surmount the effect of OA. This analogue of cyclic GMP inhibited PP-1 activity only, indicating that at least part of the increase in alpha(1)- and alpha(2/3)-Na,K-ATPase activity induced by OA was mediated by the cyclic GMP-PKG-PP-1 cascade. Taking into account that PP1 inhibition increased alpha(2/3)-Na,K-ATPase activity, we propose that an age-related increase in PP-1 activity due to a decrease in cyclic GMP-PKG modulation plays a role for the age-related reduction of alpha(2/3)-Na,K-ATPase activity in rat cerebellum. (C) 2007 Elsevier Inc. All rights reserved.

Domestic Cats Constitute a Natural Reservoir of Human Enteropathogenic Escherichia coli Types

Feces of 70 diarrhoeic and 230 non-diarrhoeic domestic cats from Sao Paulo, Brazil were investigated for enteropathogenic (EPEC), enterohaemorrhagic (EHEC) and enterotoxigenic (ETEC) Escherichia coli types. While ETEC and EHEC strains were not found, 15 EPEC strains were isolated from 14 cats, of which 13 were non-diarrhoeic, and one diarrhoeic. None of 15 EPEC strains carried the bfpA gene or the EPEC adherence factor plasmid, indicating atypical EPEC types. The EPEC strains were heterogeneous with regard to intimin types, such as eae-theta (three strains), eae-kappa (n = 3), eae-alpha 1 (n = 2), eae-iota (n = 2), one eae-alpha 2, eae-beta 1 and eae-eta each, and two were not typeable. The majority of the EPEC isolates adhered to HEp-2 cells in a localized adherence-like pattern and were positive for fluorescence actin staining. The EPEC strains belonged to 12 different serotypes, including O111:H25 and O125:H6, which are known to be pathogens in humans. Multi locus sequence typing revealed a close genetic similarity between the O111:H25 and O125:H6 strains from cats, dogs and humans. Our results show that domestic cats are colonized by EPEC, including serotypes previously described as human pathogens. As these EPEC strains are also isolated from humans, a cycle of mutual infection by EPEC between cats and its households cannot be ruled out, though the transmission dynamics among the reservoirs are not yet understood clearly.

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha 1 (residues Gly-9 to Arg-21), alpha 2 (residues Glu-27 to Asn-40), alpha 3 (residues Arg-44 to Thr-54), alpha 4 (residues Leu-57 to Tyr-64), and alpha 5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

Alpha7 Nicotinic Acetylcholine Receptor Expression and Activity During Neuronal Differentiation of PC12 Pheochromocytoma Cells

Nicotinic acetylcholine receptors (nAChR) exert pivotal roles in synaptic transmission, neuroprotection and differentiation. Particularly, homomeric alpha 7 receptors participate in neurite outgrowth, presynaptic control of neurotransmitter release and Ca(2+) influx. However, the study of recombinant alpha 7 nAChRs in transfected cell lines is difficult due to low expression of functional receptor channels. We show that PC12 pheochromocytoma cells induced to differentiation into neurons are an adequate model for studying differential nAChR gene expression and receptor activity. Whole-cell current recording indicated that receptor responses increased during the course of differentiation. Transcription of mRNAs coding for alpha 3, alpha 5, alpha 7, beta 2 and beta 4 subunits was present during the course of differentiation, while mRNAs coding for alpha 2, alpha 4 and beta 3 subunits were not expressed in PC12 cells. alpha 7 subunit expression was highest following 1 day of induction to differentiation. Activity of alpha 7 nAChRs, however, was most elevated on day 2 as revealed by inhibition experiments in the presence of 10 nM methyllycaconitine, rapid current decay and receptor responsiveness to the alpha 7 agonist choline. Increased alpha 7 receptor activity was noted when PC12 were induced to differentiation in the presence of choline, confirming that chronic agonist treatment augments nAChR activity. In summary, PC12 cells are an adequate model to study the role and pharmacological properties of this receptor during neuronal differentiation.

Prigogine-Defay Ratio for an Ionic Glass-Former: Molecular Dynamics Simulations

The pressure dependence of the glass-transition temperature, T(g)(P), of the ionic glass-former 2Ca(NO(3))(2) center dot 3KNO(3), CKN, has been obtained by molecular dynamics (MD) simulations The liquid-glass difference of thermal expansivity, Delta alpha, heat capacity, Delta C(p), and isothermal compressibility, Delta kappa, have been calculated as a function of pressure. It has been found that the Ehrenfest relation dT(g)/dP = TV Delta alpha/Delta C(p) predicts the pressure dependence of T, but the other Ehrenfest relation, dT(g)/dP = Delta kappa/Delta alpha, does not. Consequently, the Prigogine-Defay ratio, Pi = Delta C(p)Delta kappa/TV Delta alpha(2), is Pi similar to 1.2 at low pressures, but increases 1 order of magnitude at high pressures. The pressure dependence of the Prigogine-Defay ratio is interpreted in light of recent explanations for the finding Pi > 1.

Efeito da dexmedetomidina sobre a arritmia cardíaca induzida pela adrenalina em cães anestesiados pelo sevofluorano

Avaliou-se o efeito da dexmedetomidina sobre o ritmo cardíaco em 20 cães, sem raça definida, de ambos os sexos e considerados sadios, anestesiados pelo sevofluorano e submetidos a doses crescentes de adrenalina. Os animais foram, aleatoriamente, distribuídos em dois grupos (placebo e dexmedetomidina). No grupo placebo, os animais receberam, por via intravenosa, solução de NaCl a 0,9%, na dose de 0,3ml/kg. Foram considerados dois momentos, M0 e M1, imediatamente antes e após a aplicação, respectivamente. Após 10 minutos, realizou-se a indução anestésica com sevofluorano, por meio de máscara facial vedada, até a perda do reflexo laringotraqueal. em seguida, procedeu-se à intubação orotraqueal e a manutenção da anestesia foi realizada com a administração de sevofluorano na concentração de 1,5CAM, em circuito anestésico com reinalação parcial de gases. Decorridos 20 minutos da indução anestésica, iniciou-se a administração intravenosa contínua de solução de adrenalina a 2% em doses crescentes de 1, 2, 3, 4 e 5mg/kg/min, por meio de bomba de infusão, com aumento da dose em intervalos de 10 minutos. Imediatamente antes desse acréscimo eram feitas as mensurações (M2 a M6). No grupo dexmedetomidina empregou-se a mesma metodologia substituindo-se a solução de NaCl a 0,9% por hidrocloridrato de dexmedetomidina, na dose de 1µg/kg. Foram registradas as pressões arteriais, em M0 e em M2 a M6, e o traçado eletrocardiográfico, na derivação DII (M2 a M6), considerando-se para efeito estatístico o número total de bloqueios atrioventriculares (BAV) de primeiro e segundo graus e de complexos ventriculares prematuros (ESV), coincidentes com cada dose de adrenalina. Os dados foram submetidos à análise de variância seguida pelo teste de Tukey (P<0,05). Verificou-se que a dexmedetomidina interfere significativamente na condução atrioventricular levando a maior ocorrência de BAV e reduz o número de ESV nas doses infundidas de 2 e 3mg/kg/min de adrenalina. Logo após a aplicação de dexmedetomidina, observaram-se redução da freqüência cardíaca e da pressão arterial, cuja diminuição persistiu por até uma hora.

Fractal correlation of heart rate variability in obese children

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fractal correlation property of heart rate variability in chronic obstructive pulmonary disease

Background: It was reported that autonomic nervous system function is altered in subjects with chronic obstructive pulmonary disease (COPD). We evaluated short-and long-term fractal exponents of heart rate variability (HRV) in COPD subjects.Patients and methods: We analyzed data from 30 volunteers, who were divided into two groups according to spirometric values: COPD (n = 15) and control (n = 15). For analysis of HRV indices, HRV was recorded beat by beat with the volunteers in the supine position for 30 minutes. We analyzed the linear indices in the time (SDNN [standard deviation of normal to normal] and RMSSD [root-mean square of differences]) and frequency domains (low frequency [LF], high frequency [HF], and LF/HF), and the short-and long-term fractal exponents were obtained by detrended fluctuation analysis. We considered P < 0.05 to be a significant difference.Results: COPD patients presented reduced levels of all linear exponents and decreased short-term fractal exponent (alpha-1: 0.899 +/- 0.18 versus 1.025 +/- 0.09, P = 0.026). There was no significant difference between COPD and control groups in alpha-2 and alpha-1/alpha-2 ratio.Conclusion: COPD subjects present reduced short-term fractal correlation properties of HRV, which indicates that this index can be used for risk stratification, assessment of systemic disease manifestations, and therapeutic procedures to monitor those patients.