Long-term balancing selection maintains trans-specific polymorphisms in the human TRIM5 gene.

The human TRIM5 genes encodes a retroviral restriction factor (TRIM5α). Evolutionary analyses of this gene in mammals have revealed a complex and multifaceted scenario, suggesting that TRIM5 has been the target of exceptionally strong selective pressures, possibly exerted by recurrent waves of retroviral infections. TRIM5 displays inter-individual expression variability in humans and high levels of TRIM5 mRNA have been associated with a reduced risk of HIV-1 infection. We resequenced TRIM5 in chimpanzees and identified two polymorphisms in intron 1 that are shared with humans. Analysis of the gene region encompassing the two trans-specific variants in human populations identified exceptional nucleotide diversity levels and an excess of polymorphism compared to fixed divergence. Most tests rejected the null hypothesis of neutral evolution for this region and haplotype analysis revealed the presence of two deeply separated clades. Calculation of the time to the most recent common ancestor (TMRCA) for TRIM5 haplotypes yielded estimates ranging between 4 and 7 million years. Overall, these data indicate that long-term balancing selection, an extremely rare process outside MHC genes, has maintained trans-specific polymorphisms in the first intron of TRIM5. Bioinformatic analyses indicated that variants in intron 1 may affect transcription factor-binding sites and, therefore, TRIM5 transcriptional activity. Data herein confirm an extremely complex evolutionary history of TRIM5 genes in primates and open the possibility that regulatory variants in the gene modulate the susceptibility to HIV-1.

Genetic correlates of protection against HIV infection: the ally within.

Repeated exposure to HIV does not necessarily result in infection and HIV infection does not inevitably lead to the development of the AIDS. Multiple immunological and genetic features can confer resistance to HIV acquisition and progression at different steps in viral infection; a full understanding of these mechanisms could result in the development of novel therapeutic and vaccine approaches for HIV infection. In this review, we focus on the genetic mechanisms associated with resistance to HIV infection and to the progression to AIDS.

Stable GDP analog-induced inactivation of G(i) proteins promotes cardiac adenylyl cyclase inhibition by guanosine 5'-(beta gamma-imino)triphosphate and muscarinic acetylcholine receptor.

Low concentrations of GDP and its stable analog guanosine 5'-O-(2-thio)diphosphate (GDP beta S) have been shown to stimulate adenylyl cyclase activity in canine cardiac sarcolemmal membranes independent from a phosphate transfer reaction. The mechanism of this stimulation was further examined. The stable GTP analog guanosine 5'-(beta gamma-imino)triphosphate (Gpp(NH)p) increased basal adenylyl cyclase activity and inhibited forskolin-stimulated activity with EC50 (half-maximal effective concentration) values of 0.7 mumol/l and 10 nmol/l, respectively. In the presence of GDP beta S (5 mumol/l), which increased basal activity by about 150%, addition of Gpp(NH)p inhibited adenylyl cyclase activity by up to 50% with an EC50 value of 40 nmol/l. Activation of cardiac muscarinic acetylcholine receptors by carbachol amplified this Gpp(NH)p-induced inhibition of GDP beta S-stimulated adenylyl cyclase activity. The stimulatory effect of GDP beta S and the inhibitory effect of Gpp(NH)p on GDP beta S-stimulated adenylyl cyclase activity were both attenuated by increasing the Mg2+ concentration or substituting Mn2+ for Mg2+ in the assay. Furthermore, both effects were strongly reduced or abolished upon pretreatment of the sarcolemmal membranes with a low concentration of the SH reagent N-ethylmaleimide (10 mumol/l). These results suggest that the stimulatory effect of GDP beta S on basal adenylyl cyclase activity in canine cardiac sarcolemmal membranes is caused by inactivation of G(i) proteins, which are then rendered susceptible to activation by Gpp(NH)p and inhibitory receptors.

Altered gene expression during hypoxia and reoxygenation of the heart.

The heart is exposed to alterations in oxygen tension under different pathophysiological conditions. In order to maintain function, changes in the pattern of cardiac gene expression arise. Through the activity of multiple transcription factors, which include activating protein-1, hypoxia-inducible factor-1, and nuclear factor kappaB, there is up-regulation of mRNA encoding factors that enable the cardiomyocyte to adapt to the new environment. In the case of hypoxia or anoxia, there is an increased expression of growth factors, glucose transporters, enzymes associated with anaerobic glycolysis, and stress proteins. When the cardiomyocyte is reoxygenated after hypoxia, there is a rapid increase in antioxidants, pro-inflammatory cytokines, and stress proteins.

The antiarrhythmic effect of ischaemic preconditioning in isolated rat heart involves a pertussis toxin sensitive mechanism.

OBJECTIVE: The aims were to examine the effect of pretreatment with Bordetella pertussis toxin on the antiarrhythmic effect of ischaemic preconditioning in order to determine the possible involvement of inhibitory G proteins in this phenomenon; and (2) to characterise the model used by varying the duration of a single preconditioning occlusion of the left coronary artery, the reperfusion time, and the duration of the subsequent prolonged coronary artery occlusion. METHODS: Isolated rat hearts perfused with Krebs Henseleit solution at constant flow (8-10 ml.min-1) were subjected to a single preconditioning occlusion of the left coronary artery (either 1 or 3 min) followed, up to 60 min later, by a prolonged occlusion of 30 or 60 min (n = 56). The ventricular arrhythmias during occlusion were compared to those from control rats in which the artery was occluded for 30 or 60 min but without preconditioning (n = 29 and 14 respectively). RESULTS: Protection against ventricular arrhythmias was most pronounced when a 3 min preconditioning occlusion was used followed by a 10 min reperfusion period: reduction in ventricular premature beats (VPB) during the 30 min occlusion from 514(SEM 119) in control hearts to 79(29) in preconditioned hearts (p < 0.01). This protection was still apparent when the reperfusion time was extended to 30 min [VPB 52(16); p < 0.01] but lost when reperfusion was extended to 1 h. Rendering Gi proteins non-functional (ascertained by responses to acetylcholine) resulted in loss of this antiarrhythmic effect of preconditioning [VPB 241(93) v 226(120) for non-preconditioned hearts]. CONCLUSIONS: The antiarrhythmic effects of preconditioning can be demonstrated in isolated rat hearts perfused at constant flow with an artificial medium and this protection is lost following treatment with Bordetella pertussis toxin 48 h previously.

Effects of Bordetella pertussis toxin pretreatment on the antiarrhythmic action of ischaemic preconditioning in anaesthetized rats.

1. Bordetella pertussis toxin, which catalyses the ADP-ribosylation of certain guanine nucleotide binding proteins (G proteins), thus functionally uncoupling them from associated receptors, was examined to determine whether it modified the antiarrhythmic effect of ischaemic preconditioning in anaesthetized rats. 2. Pertussis toxin (25 micrograms kg-1, i.p., 48 h prior to heart isolation) attenuated the negative chronotropic effect of acetylcholine (ACh) in rat isolated Langendorff perfused hearts. ACh (10 microM) reduced heart rate by 4% in hearts taken from pertussis toxin-treated animals, compared to a reduction of 57% in hearts taken from animals treated only with vehicle. 3. In anaesthetized rats, ischaemic preconditioning (a single 3 min occlusion of the left main coronary artery followed by 10 min reperfusion) had a pronounced antiarrhythmic effect during a subsequent 30 min period of regional myocardial ischaemia. Compared to hearts receiving only a 30 min period of left coronary occlusion, there was a reduced mortality (67% and 0% for control and preconditioned groups, respectively; P < 0.01) and decreased incidences of ventricular tachycardia (VT) and ventricular fibrillation (VF). Pretreatment with pertussis toxin (25 micrograms kg-1, i.p., 48 h previously) did not modify the arrhythmias associated with a 30 min period of regional myocardial ischaemia, neither did it modify the reduction in mortality (from 56% to 0%; P < 0.05) associated with preconditioning. Furthermore, the decrease in total ventricular premature beat count induced by preconditioning seen in controls (from 427 +/- 130 to 95 +/- 45) was also seen in pertussis toxin-treated rats (from 252 +/- 190 to 57 +/- 25). 4. These results suggest that receptor coupling to pertussis toxin-sensitive G proteins is not necessary for the antiarrhythmic effect of ischaemic preconditioning in this model.

Opioid receptor agonists activate pertussis toxin-sensitive G proteins and inhibit adenylyl cyclase in canine cardiac sarcolemma.

Although both opioid receptors and endogenous opioids are abundant in cardiac tissues, the signal transduction pathways of opioids in cardiac sarcolemmal membranes have yet to be identified. In highly purified canine cardiac sarcolemmal membranes, binding of the opioid receptor antagonist [3H]diprenorphine and effects of mu, delta and kappa agonists on low Km GTPase and adenylyl cyclase were measured. Equilibrium binding of [3H]diprenorphine revealed a maximal binding capacity of 7.2 pmol/mg protein and a Kd of 1.3 nmol/l. In the presence of GTP, (D-Pen2,5, p-Cl-Phe4) enkephalin and (D-Arg6) dynorphin A 1-13 fragment both inhibited adenylyl cyclase by 20-25% (from 206 +/- 30 to 164 +/- 28 pmol.min-1.mg protein-1, EC50 6 mumol/L, and from 254 +/- 109 to 204 +/- 90 pmol.min-1.mg protein-1, EC50 8 mumol/L, respectively; P < 0.001). Both substances stimulated low Km GTPase by 20% and 13%, respectively (from 12.7 +/- 3.0 to 15.2 +/- 3.7 pmol.min-1.mg protein-1, EC50 12 mumol/L, P < 0.01, and from 9.1 +/- 2.8 to 10.4 +/- 3.2 pmol.min-1.mg protein-1, EC50 6 mumol/L, P < 0.05, respectively). These effects were blocked by the opioid receptor antagonist naltrexone and by pretreatment of sarcolemmal membranes with pertussis toxin. The mu opioid receptor agonists (D-Ala2, Me Phe4, Gly-[ol]5)enkephalin and morphiceptin had no effect on either adenylyl cyclase or low Km GTPase activities. These data suggest that in cardiac sarcolemma, opioid receptors are coupled to pertussis toxin sensitive G proteins and mediate inhibition of adenylyl cyclase activity.

Evidence against a regulation of Na+/K(+)-ATPase by Gi proteins. Failure to detect an influence of G proteins on Na+/Ca(2+)-exchange in cardiac sarcolemmal membranes.

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (Gi proteins) mediate negative chronotropic and negative inotropic effects by activation of K+ channels and inhibition of adenylyl cyclase. The concept of a uniform inhibitory action of Gi proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na+/Ca(2+) exchange and a carbachol-induced inhibition of the Na+/K(+)-ATPase activity in cardiac sarcolemmal membranes. The aim of the present study, therefore, was to reinvestigate the putative regulation of Na+/Ca(2+) exchange and Na+/K(+)-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A1 (Maximum number of receptors, Bmax 0.033 pmol/mg) and muscarinic M2 (Bmax 2.9 pmol/mg) receptors and contained Gi2 and Gi3, two Gi protein isoforms, and G0, another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A1-selective agonist, (-)-N6-(2-phenylisopropyl)-adenosine, and the muscarinic agonist, carbachol, both inhibited isoprenaline-stimulated adenylyl cyclase activity by 25% and 35% respectively, and the stable GTP analogue 5'-guanylylimidodiphosphate inhibited forskolin-stimulated adenylyl cyclase activity by 35% in these membranes. The characteristics of Na+/Ca(2+) exchange and Na+/K(+)-ATPase activity as well as those of the ouabain-sensitive, K(+)-activated 4-nitrophenylphosphatase, an ATP-independent, partial reaction of the Na+/K(+)-ATPase, were in agreement with published data with regard to specific activity, time course of activity and substrate dependency. However, none of these activities were influenced by adenosine, (-)-N6-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na+/Ca(2+) exchange and Na+/K(+)-ATPase are not regulated by Gi proteins in canine cardiac sarcolemmal membranes.

[New findings regarding neuroreceptors in the lower urinary tract]. / Nuove acquisizioni sull'assetto neurorecettoriale del tratto urinario inferiore.

In the field of the neuroreceptors of the lower urinary tract, knowledge is being increased by some recent experimental findings, such as muscarinic subtypes, adrenergic and serotoninergic selective receptors. Moreover, the role of peptidergic receptors (VIP, CGRP, TKs, SP, ecc.; neuropeptide release from peripheral endings of capsaicin-sensitive nerves) has been suggested and investigations are in progress.

CC chemokine receptor expression in childhood asthma is influenced by natural allergen exposure.

Chemokines and their receptors may play an important role for leukocyte trafficking in allergic inflammation. Aim was to evaluate whether expression of chemokine receptors CCR4 and CCR8 on cells obtained by sputum induction from asthmatic allergic children may be influenced by house dust mite (HDM) allergen natural exposure. Twenty-one children (7-13 yr) with moderate asthma and sensitized to HDM were evaluated during a prolonged period of allergen avoidance (T0) and after a period of natural allergen exposure (T1). At each time point of sputum induction, lung function evaluation, exhaled nitric oxide (eNO) measurements were performed. At T1, CCR4 and CCR8 expression on sputum-induced cells increased from 28.4% +/- 2.9% and 25.8% +/- 1.9%, to 41.1% +/- 4.2% and 37.5% +/- 2.0%, respectively (p < 0.05 and p = 0.01). After allergen exposure, both sputum eosinophils (from 5.2% +/- 2.0% to 12.1% +/- 4.1%, p < 0.01) and eNO (from 15.1 +/- 2.2 ppb to 24.2 +/- 5.8 ppb, p < 0.05) showed significant increase. Lung function tests presented significant deterioration of Forced Expiratory Flow at 25-75% of Vital Capacity (FEF(25--75)) (p < 0.05) and increase of residual volume (p = 0.002). Significant changes in CC chemokine receptor expression in sputum-induced cells in asthmatic children in response to HDM exposure have been observed leading to consider the relevance of CCR4 and CCR8 in allergic asthmatic inflammation.

Phosphotransfer reactions as a means of G protein activation.

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) serve to transduce information from agonist-bound receptors to effector enzymes or ion channels. Current models of G protein activation-deactivation indicate that the oligomeric GDP-bound form must undergo release of GDP, bind GTP and undergo subunit dissociation, in order to be in active form (GTP bound alpha subunits and free beta gamma dimers) and to regulate effectors. The effect of receptor occupation by an agonist is generally accepted to be promotion of guanine nucleotide exchange thus allowing activation of the G protein. Recent studies indicate that transphosphorylation leading to the formation of GTP from GDP and ATP in the close vicinity, or even at the G protein, catalysed by membrane-associated nucleoside diphosphate kinase, may further activate G proteins. This activation is demonstrated by a decreased affinity of G protein-coupled receptors for agonists and an increased response of G protein coupled effectors. In addition, a phosphorylation of G protein beta subunits and consequent phosphate transfer reaction resulting in G protein activation has also been demonstrated. Finally, endogenously formed GTP was preferentially effective in activating some G proteins compared to exogenous GTP. The aim of this report is to present an overview of the evidence to date for a transphosphorylation as a means of G protein activation (see also refs [1 and 2] for reviews).

The response of scene call volume to prehospital education.

INTRODUCTION: Transport of injured patients directly from a scene to a trauma center improves survival of patients and shortens their length of stay in the hospital. This paper studies the relationship between education presentations to prehospital personnel and scene call volume. The education sessions emphasize safety issues and how, when, and why to call for air medical transport. METHODS: The town and date of scene flights were compared to the town and date of flight nurse presentations and aircraft demonstrations. The length of time from a presentation to a scene call for each town was determined, and a cumulative frequency graph was drawn. Epidemiologic curves of presentations and calls were drawn for each town. Based on these graphs, observations of a relationship were obtained. RESULTS: There were 65 scene calls to 27 towns that had no education programs. There were 880 scene calls to 90 towns that had 235 education programs. There were 21 towns that received a total of 41 presentations and never initiated a scene call. The results show that scene call requests are more likely to occur within three months of a presentation. Individual town analysis shows variability of response to education programs. CONCLUSION: Prehospital provider education programs increase scene call volume, but this effect seems to last for three months. On a town-by-town basis there are many other determinants of scene call volume.

Endothelin-1 stimulates cardiac fibroblast proliferation through activation of protein kinase C.

After myocardial ischemia, circulating levels of the mitogen endothelin-1 (ET-1) increase. The effects of ET-1 on cardiac fibroblasts are poorly characterized. Therefore we examined the influence of ET-1 on cardiac fibroblast proliferation with a view to elucidating the signal transduction mechanisms underlying this effect. ET-1 (10 n m) stimulated [(3)H]thymidine incorporation and cell proliferation in cultured neonatal rat cardiac fibroblasts, consistent with its activity as a mitogen. We examined the role of protein kinase C (PKC) on this function. Inhibition of PKC activation with either chelerythrine (1 microm) or staurosporine (1 n m) attenuated ET-1-induced increases in DNA synthesis and cell number. Downregulation of PKC by chronic pretreatment with 10 n m phorbol 12-myristate 13-acetate (PMA) also prevented ET-1-induced mitogenesis. In contrast to previous reports that cardiac fibroblast proliferation stimulated by angiotensin II acts independently of PKC, the ET-1 mediated mitogenic effect requires activation of PKC in these cells. Findings in adult rat cardiac fibroblasts were identical. In addition, we noted that concurrent treatment with the pro-inflammatory cytokine interleukin 1 beta which, like ET-1, is released after myocardial ischemia, attenuated the ET-1-induced increases in DNA synthesis and cell number. This effect was not mediated through a nitric oxide synthase pathway.

Screening of HBV-DNA in chronic HBsAg carriers.

A series of 52 serum samples from chronic HBsAg carriers was tested for the presence of HBV-DNA by means of the Polymerase Chain Reaction (PCR) and Liquid Phase Hybridization (LPH). The samples were obtained from two groups of patients: group A included 34 chronic HBsAg carriers ("healthy" individuals) without hepatocytolysis or viral replication; group B included 18 chronic HBsAg carriers with signs of hepatocytolysis (ALT levels at least twice the normal value) and activated markers of viral replication. PCR was superior to LPH in group A, with 7/34 versus 5/34 positive samples being detected, respectively. No difference in sensitivity was found between the two techniques in group B, since 9/18 samples were positive both cases. The data stress the need to adopt PCR for the HBV-DNA screening of HBeAg-/HBsAg+-carriers.