Anti-HLA class I antibody-mediated activation of the PI3K/Akt signaling pathway and induction of Bcl-2 and Bcl-xL expression in endothelial cells

Anti-human leukocyte antigen (HLA) antibodies (Ab) have long been implicated in the process of acute and chronic allograft rejection, yet their mechanism(s) of action is not well understood. The aim of this study was to determine whether ligation of HLA class I molecules by anti-HLA Ab on the surface of human endothelial cells (EC) activates the PI3 Kinase (PI3K)/Akt signaling pathway and downstream target proteins of the cell death apparatus. We report that Ab ligation of major histocompatibility complex (MHC) class I molecules on the surface of EC triggers phosphorylation of Akt, PI3K, and recruitment of PI3K and Akt into a signaling unit with focal adhesion kinase. Signaling through class I also stimulated phosphorylation of Bad and upregulated expression of Bcl-2 and Bcl-xL. Pretreatment of EC with the PI3K inhibitor wortmannin blocked class I-mediated expression of Bcl-2, but not Bcl-xL, suggesting a role for the PI3K/Akt signaling pathway in regulation of class I-induced Bcl-2 expression. The intracellular events initiated by class I ligation were influenced by the concentration of the anti-HLA Ab with the lowest tested concentrations of Ab stimulating the highest level of Akt phosphorylation, Bcl-xL and Bcl-2 expression. Consistent with the in vitro experiments, analysis of biopsy samples from heart transplant recipients with evidence of Ab-mediated rejection exhibited increased Bcl-2 expression on the vascular endothelium. These results suggest that exposure of the graft endothelium to low concentrations of anti-HLA Ab may promote cell survival by transducing signals resulting in upregulation of cell survival genes.     

Association of the dopamine receptor D4 (DRD4) gene 7-repeat allele with children with attention-deficit/hyperactivity disorder (ADHD): an update.

Polymorphisms of the dopamine receptor D4 gene DRD4, 11p15.5, have previously been associated with attention-deficit/hyperactivity disorder (ADHD) [Bobb et al., 2005; Am J Med Genet B Neuropsychiatr Genet 132:109-125; Faraone et al., 2005; Biol Psychiatry 57:1313-1323; Thapar et al., 2005; Hum Mol Genet 14 Spec No. 2:R275-R282]. As a follow up to a pilot study [see Castellanos et al., 1998; Mol Psychiatry 3:431-434] consisting of 41 probands and 56 controls which found no significant association between the DRD4 7-repeat allele in exon 3 and ADHD, a greatly expanded study sample (cases n = 166 and controls n = 282) and long term follow-up (n = 107, baseline mean age n = 9, follow-up mean age of n = 15) prompted reexamination of this gene. The DRD4 7-repeat allele was significantly more frequent in ADHD cases than controls (OR = 1.2; P = 0.028). Further, within the ADHD group, the 7-repeat allele was associated with better cognitive performance (measured by the WISC-III) (P = 0.013-0.07) as well as a trend for association with better long-term outcome. This provides further evidence of the role of the DRD4 7-repeat allele in the etiology of ADHD and suggests that this allele may be associated with a more benign form of the disorder.     

Plasma homocysteine concentration and its relationship with the development of preeclampsia. Effect of prenatal administration of folic acid

The increase of plasmatic homocysteine (Hc) in pregnant women, who later develop preeclampsia/eclampsia, the cause of this increment and its pathogenic role in toxemia of pregnancy, are still controversial. The objectives of the present research were to determine the plasmatic He concentrations during the first and second trimesters of pregnancy and the effect of folic acid administration on these values, and in the prevention of preeclampsia. Ninety six pregnant women of low economic background were studied on the first prenatal consultation: 27 women in the first trimester of pregnancy and 59 in the second. After 8 hours of fasting, venous blood was extracted and each patient was provided with 1 mg folic acid tablets and instructed to ingest one tablet daily, and to come back to the laboratory after three months. Plasma homocysteine and serum folic acid were determined for each patient before and after the folic acid treatment, by using the IMX system (Abbott Lab) and radioimmunoassay, respectively. Basal homocysteine concentrations were 4.0 +/- 2.1 micromol/L and 4.8 +/- 2.1 micromol/L in the first and second trimesters respectively, with no significant modifications after three months of folic acid. Although the degree of desertion from the study was high, it was possible to determine the evolution of 65 pregnancies. Ten of them developed preeclampsia (15.4%). No significant differences were found in Hc concentrations, or the frequency of hyperhomocysteinemia in the different stages of pregnancy, between women with normal gestation and those who developed preeclampsia. The small sample size of these groups, preclude any valid conclusion, however the results do not suggest that Hc concentration or folic acid administration influence the development of toxemia of pregnancy.     

Granulocyte turnover in the feline intestine

The objective of this study was to determine the turnover rate of the extravascular pool of granulocytes in different regions of the feline gastrointestinal tract. Leukocyte emigration from the vasculature was prevented over a 48-h period by repeated intravenous injections of a monoclonal antibody (MAb IB₄) directed against the leukocyte adhesion glycoprotein complex CD11/CD18. Tissue-associated myeloperoxidase (MPO) activity was used to monitor the total tissue granulocyte pool at 0.5, 12, 24, and 48 h after MAb IB₄ administration. The mucosal layer of the duodenum, jejunum, ileum, and colon exhibited different kinetics of granulocyte clearance, with average life-spans (t1/2) ranging between 6.9 (colon) and 10.4 h (duodenum). Granulocyte clearance rates of 0.5 × 10⁶ and 2.4 x 10⁶ cells/h/g tissue were estimated (from measured values oft1/2 and tissue granulocyte pool) for the small bowel and colonie mucosae, respectively. The submucosal layer of the intestine exhibited a biphasic reduction in tissue MPO activity following immunoneutralization of CD11/CD18, with an initialt1/2 0.5 h followed by at1/2 of 36–60 h. The initial rapid decline in tissue MPO suggests that a significant fraction of granulocytes in the submucosa is localized in a readily exchangeable pool (e.g., marginated cells within the vasculature). The results of this study indicate that the average life-span of resident granulocytes varies significantly between different regions of the gastrointestinal tract, with the intestinal mucosa exhibiting at1/2 comparable to that previously reported for circulating feline neutrophils (R 8 h).     

Percepciones estudiantiles sobre las propiedades del examen clínico objetivo estructurado en Uruguay. Disponiendo un cuestionario válido para analizar la factibilidad del nuevo formato durante la transición curricular

Introduction

Although an objective structured clinical examination (OSCE) format has been applied in Uruguay since 2004, and providing reliable performance measures, perceptions of it properties and level of student satisfaction have not been determined.

Chemoarchitecture and afferent connections of the "olfactostriatum": a specialized vomeronasal structure within the basal ganglia of snakes

The olfactostriatum, a portion of the striatal complex of snakes, is the major tertiary vomeronasal structure in the ophidian brain, receiving substantial afferents from the nucleus sphericus, the primary target of accessory olfactory bulb efferents. In the present study, we have characterized the olfactostriatum of garter snakes (Thamnophis sirtalis) on the basis of chemoarchitecture (distribution of serotonin, neuropeptide Y and tyrosine hydroxylase) and hodology (afferent connections). The olfactostriatum is densely immunoreactive for serotonin and neuropeptide Y and shows moderate-to-weak immunoreactivity for tyrosine hydroxylase. In addition to afferents from the nucleus sphericus, the olfactostriatum receives inputs from the dorsal and lateral cortices, nucleus of the accessory olfactory tract, external and dorsolateral amygdalae, dorsomedial thalamic nucleus, ventral tegmental area and raphe nuclei. Double labeling experiments demonstrated that the distribution of serotonin and neuropeptide Y in this area almost completely overlaps the terminal field of projections from the nucleus sphericus. Also, serotonergic and dopaminergic innervation of the olfactostriatum likely arise, respectively, from the raphe nuclei and the ventral tegmental area, whereas local circuit neurons originate the neuropeptide Y immunoreactivity. These results indicate that the olfactostriatum of snakes could be a portion of the nucleus accumbens, with features characteristic of the accumbens shell, devoted to processing vomeronasal information. Comparative data suggest that a similar structure is present in the ventral striatum of amphibians and mammals.     

A novel fungal prenyl diphosphate synthase in the dimorphic zygomycete Mucor circinelloides

Two Mucor circinelloides structural genes involved in isoprenoid biosynthesis were isolated and characterised. The isoA gene encodes a typical eukaryotic farnesyl diphosphate synthase (EC 2.5.1.10), whereas the isoB gene deduced amino acid sequence shows similarity to fungal medium-chain prenyl diphosphate synthases. By functional complementation in Escherichia coli, the isoB gene product was shown to be a solanesyl diphosphate synthase (EC 2.5.1.11), which is the first fungal enzyme reported having this specificity. In addition, a M. circinelloides one-marker-per-chromosome map was completed by contour-clamped homogeneous electric field localisation of isoA, isoB and three other isoprenoid biosynthesis genes to individual chromosomes.Abbreviations FPP farnesyl diphosphate (or pyrophosphate) - GGPP geranylgeranyl diphosphate - PrenylPP prenyl diphosphate - DPP decaprenyl diphosphate - HPP hexaprenyl diphosphate - SPP solanesyl diphosphate     

Allele-Specific PCR Method Based on pncA and oxyR Sequences for Distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: Intraspecific M. bovis pncA Sequence Polymorphism

An allele-specific amplification method based on two genetic polymorphisms to differentiate Mycobacterium tuberculosis from Mycobacterium bovis was tested. Based on the differences found at position 169 in the pncA genes from M. tuberculosis and M. bovis, a PCR system which was able to differentiate most of the 237 M. tuberculosis complex isolates tested in one of the two species was developed. All 121 M. tuberculosis strains showed the expected base (cytosine) at position 169. Most of the M. bovis isolates had a guanine at the cited position. Nevertheless, 18 of the 116 M. bovis isolates, all of them goat isolates, showed the pncA polymorphism specific to M. tuberculosis. These results suggest that goat M. bovis may be the nicotinamidase-missing link at the origin of the M. tuberculosis species. Based on the polymorphism found at position 285 in the oxyR gene, the same system was used to differentiate M. tuberculosis from M. bovis. In this case, DNAs from all 121 M. tuberculosis isolates had the expected base (guanine) at this position. In addition, all 116 M. bovis isolates, including those from goats, showed the identical polymorphism (adenine). The oxyR allele-specific amplification method can differentiate M. bovis from M. tuberculosis, is rapid (results can be obtained in less than 3 h), and is easy to perform.     

Histamine H3 receptor activation inhibits dopamine D1 receptor-induced cAMP accumulation in rat striatal slices

In striatal membranes bearing significant levels of histamine H3 receptors (72 +/- 14 fmol/mg protein), the H3 agonist immepip (1 microM) increased [35S]GTPgammaS binding to 119 +/- 2% of basal, an effect prevented by the H3 antagonist clobenpropit and by pre-treatment with pertussis toxin. In slices labelled with [3H]adenine and in the presence of 1 mM isobutylmethylxantine (IBMX), the selective dopamine D1-like (D1/D5) receptor agonist SKF-81297 stimulated cyclic [3H]AMP ([3H]cAMP) accumulation (maximal stimulation 205 +/- 24% of basal, EC50 113 +/- 12 nM), an effect fully blocked by the D1/D5 antagonist SCH-23390. The accumulation of [3H]cAMP induced by 1 microM SKF-81297 was inhibited in a concentration-dependent manner by the selective H3 receptor agonist immepip (maximal inhibition 60+/-5%, IC50 13 +/- 5 nM). The inhibitory action of 100 nM immepip was reversed in a concentration-dependent manner by the H3 antagonist thioperamide (EC50 13 +/- 3 nM, Ki 1.4 +/- 0.3 nM). Forskolin-induced [3H]cAMP accumulation (726 +/- 57% of basal) was also reduced by H3 receptor activation, although to a lesser extent (19.1 +/- 3.2% inhibition), an action not affected by the absence of either IBMX or Ca2+ ions in the incubation medium. Neither the density of [3H]SCH-23390 binding sites (D1 receptors) nor the inhibition by SKF-81297 were affected by 1 microM immepip, ruling out a direct interaction between D1 and H3 receptors. These results indicate that through H3 receptors coupled to Galphai/o proteins, histamine modulates cAMP formation in striatal neurones that possess D1 receptors, most probably GABAergic striato-nigral neurones.