PARP-1 is required for retrieval of cocaine-associated memory by binding to the promoter of a novel gene encoding a putative transposase inhibitor.

Reward-related memory is an important factor in cocaine seeking. One necessary signaling mechanism for long-term memory formation is the activation of poly(ADP-ribose) polymerase-1 (PARP-1), via poly(ADP-ribosyl)ation. We demonstrate herein that auto-poly(ADP-ribosyl)ation of activated PARP-1 was significantly pronounced during retrieval of cocaine-associated contextual memory, in the central amygdala (CeA) of rats expressing cocaine-conditioned place preference (CPP). Intra-CeA pharmacological and short hairpin RNA depletion of PARP-1 activity during cocaine-associated memory retrieval abolished CPP. In contrast, PARP-1 inhibition after memory retrieval did not affect CPP reconsolidation process and subsequent retrievals. Chromatin immunoprecipitation sequencing revealed that PARP-1 binding in the CeA is highly enriched in genes involved in neuronal signaling. We identified among PARP targets in CeA a single gene, yet uncharacterized and encoding a putative transposase inhibitor, at which PARP-1 enrichment markedly increases during cocaine-associated memory retrieval and positively correlates with CPP. Our findings have important implications for understanding drug-related behaviors, and suggest possible future therapeutic targets for drug abuse.

Erythropoietin-driven signalling and cell migration mediated by polyADP-ribosylation.

BACKGROUND: Recombinant human erythropoietin (EPO) is the leading biotechnology engineered hormone for treatment of anaemia associated with chronic conditions including kidney failure and cancer. The finding of EPO receptors on cancer cells has raised the concern that in addition to its action in erythropoiesis, EPO may promote tumour cell growth. We questioned whether EPO-induced signalling and consequent malignant cell manifestation is mediated by polyADP-ribosylation. METHODS: Erythropoietin-mediated PARP (polyADP-ribose polymerase-1) activation, gene expression and core histone H4 acetylation were examined in UT7 cells, using western blot analysis, RT-PCR and immunofluorescence. Erythropoietin-driven migration of the human breast epithelial cell line MDA-MB-435 was determined by the scratch assay and in migration chambers. RESULTS: We have found that EPO treatment induced PARP activation. Moreover, EPO-driven c-fos and Egr-1 gene expression as well as histone H4 acetylation were mediated via polyADP-ribosylation. Erythropoietin-induced cell migration was blocked by the PARP inhibitor, ABT-888, indicating an essential role for polyADP-ribosylation in this process. CONCLUSIONS: We have identified a novel pathway by which EPO-induced gene expression and breast cancer cell migration are regulated by polyADP-ribosylation. This study introduces new possibilities regarding EPO treatment for cancer-associated anaemia where combining systemic EPO treatment with targeted administration of PARP inhibitors to the tumour may allow safe treatment with EPO, minimising its possible undesirable proliferative effects on the tumour.

Anaesthetic specialisation leads to improved early- and medium-term survival following major vascular surgery.

OBJECTIVE: Vascular surgical specialisation is associated with improved outcomes. We aimed to assess the effect of anaesthetic specialisation on outcome following major vascular surgery. DESIGN: Retrospective cohort study. METHODS: Patients undergoing major vascular surgery (lower limb revascularisation, elective and ruptured abdominal aortic aneurysm repair, endovascular aneurysm repair and carotid endarterectomy) over a five-year period were identified from a prospective database. The primary outcomes were death within 30 days and death within two years of surgery. Potential risk factors for mortality were assessed using multivariate logistic regression modelling. RESULTS: The analysis cohort comprised 1155 patients followed up for a median of 583 days. Mortality within two years of surgery was 16%. For the overall cohort, care from vascular anaesthetists was independently associated with reduced 30-day (odds ratio 0.22; 95% CI 0.12-0.62) and medium-term mortality (0.31; 95% CI 0.18-0.55). For elective patients (n=851), vascular anaesthesia reduced two-year mortality (odds ratio 0.29; 95% CI 0.15-0.58; P=0.0004) though not 30-day mortality (odds ratio 0.55; 95% CI 0.15-1.95; P=0.35). For emergency patients, care by a vascular anaesthetist influenced neither 30-day mortality (odds ratio 0.33; 95% CI 0.08-1.41; P=0.13) nor medium-term mortality (odds ratio 0.45; 95% CI 0.17-1.21; P=0.11). CONCLUSIONS: Anaesthetic specialisation reduced early- and medium-term mortality rates following major vascular surgery. If replicated by prospective studies, these results suggest that vascular surgery services would benefit from specialised anaesthetic support.

Randomized clinical trial of folate supplementation in patients with peripheral arterial disease.

BACKGROUND: The aim was to determine whether folate supplementation improved arterial function in patients with peripheral arterial disease (PAD). METHODS: Individuals with PAD were randomly assigned to receive 400 microg folic acid (45 patients) or 5-methyltetrahydrofolate (5-MTHF) (48) daily, or placebo (40) for 16 weeks. Primary endpoints were changes in plasma total homocysteine (tHcy), ankle : brachial pressure index (ABPI) and pulse wave velocity (PWV). Secondary outcomes were changes in plasma inflammatory markers. RESULTS: Plasma tHcy was significantly reduced in folic acid and 5-MTHF groups compared with controls: median difference: - 2.12 (95 per cent confidence interval - 3.70 to - 0.75) micromol/l (P = 0.002) and - 2.07 (-3.48 to - 0.54) micromol/l (P = 0.007) respectively. ABPI improved significantly: median difference 0.07 (0.04 to 0.11) (P < 0.001) and 0.05 (0.01 to 0.10) (P = 0.009) respectively. Brachial-knee PWV (bk-PWV) decreased significantly in individuals receiving 5-MTHF and tended to be reduced in those taking folic acid compared with controls: median difference: - 1.10 (-2.20 to - 0.20) m/s (P = 0.011) and - 0.90 (-2.10 to 0.00) m/s (P = 0.051) respectively. Plasma levels of inflammatory markers were not affected. CONCLUSION: Folate administration reduced plasma homocysteine, and slightly improved ABPI and bk-PWV.

A fast signal-induced activation of Poly(ADP-ribose) polymerase: a novel downstream target of phospholipase c.

We present the first evidence for a fast activation of the nuclear protein poly(ADP-ribose) polymerase (PARP) by signals evoked in the cell membrane, constituting a novel mode of signaling to the cell nucleus. PARP, an abundant, highly conserved, chromatin-bound protein found only in eukaryotes, exclusively catalyzes polyADP-ribosylation of DNA-binding proteins, thereby modulating their activity. Activation of PARP, reportedly induced by formation of DNA breaks, is involved in DNA transcription, replication, and repair. Our findings demonstrate an alternative mechanism: a fast activation of PARP, evoked by inositol 1,4,5,-trisphosphate-Ca(2+) mobilization, that does not involve DNA breaks. These findings identify PARP as a novel downstream target of phospholipase C, and unveil a novel fast signal-induced modification of DNA-binding proteins by polyADP-ribosylation.

Homocysteine and peripheral arterial disease: systematic review and meta-analysis.

OBJECTIVE: To evaluate homocysteine (Hcy) levels in patients with peripheral arterial disease (PAD) as compared to unaffected controls, and to review the clinical effects of therapy aimed at lowering homocysteine in PAD patients. METHODS: MEDLINE, EMBASE and Cochrane databases were searched from 1950 to December 2007. We selected observational studies and trials that evaluated Hcy levels in patients with PAD compared to unaffected controls. We also included trials on the effect of Hcy-lowering therapy (folate supplementation) in PAD patients. Continuous outcomes were pooled in a random effects meta-analysis of the weighted mean difference between comparator groups. RESULTS: We retrieved 33 potentially suitable articles from our search. Meta-analysis of 14 relevant studies showed that Hcy was significantly elevated (pooled mean difference +4.31micromoll; 95% C.I. 1.71, 6.31, p<0.0001 with significant heterogeneity) in patients with PAD compared to controls. As all 14 studies consistently demonstrated raised plasma Hcy levels in PAD patients, the significant heterogeneity in this meta-analysis probably arises from differences in the degree of Hcy elevation. The effect of folate supplementation on PAD was tested in eight clinical trials but clinically important end points were inconsistently reported. CONCLUSION: Patients with PAD have significantly higher Hcy levels than unaffected controls. However, we did not find any robust evidence on clinically beneficial effects of folate supplementation in PAD.

A PARP1-Erk2 synergism is required for stimulation-induced expression of immediate early genes.

A PARP1-Erk2 synergism was required to generate synaptic long-term potentiation in the CA3-CA1 hippocampal connections. This molecular mechanism was associated with the recently identified pivotal role of polyADP-ribosylation in learning. High frequency electrical stimulation of cortical and hippocampal neurons induced binding of phosphorylated Erk2 (transported into the nucleus) to the nuclear protein PARP1. PARP1-Erk2 binding induced PARP1 activation and polyADP-ribosylation of its prominent substrate, linker histone H1. A facilitated access of PARP1-bound phosphorylated Erk2 to its substrates, transcription factors Elk1 and CREB was attributed to the release of polyADP-ribosylated H1 from the DNA, causing local DNA relaxation. Erk-induced phosphorylation of transcription factors activating the HAT activity of CBP (CREB binding protein), recruited acetylated histone H4 to the promoters of immediate early genes (IEG) cfos, zif268 and arc, which are implicated in synaptic plasticity. In accordance, their induced expression was suppressed after PARP1 genetic deletion in PARP1-KO mice, or after PARP1 inhibition or silencing. Moreover, under these conditions, long-term synaptic potentiation (LTP) (indicating synaptic plasticity) was not generation in the hippocampal CA3-CA1 connections, and learning abilities were impaired. Furthermore, both IEG expression and LTP generation failed when cerebral neurons accumulated single strand DNA breaks, due to a predominant binding of PARP1 to nicked DNA, occluding its Erk binding sites. Thus, a declined synaptic plasticity is anticipated when aged cerebral neurons accumulate DNA single-strand breaks during life span.

A PARP1-ERK2 synergism is required for the induction of LTP.

Unexpectedly, a post-translational modification of DNA-binding proteins, initiating the cell response to single-strand DNA damage, was also required for long-term memory acquisition in a variety of learning paradigms. Our findings disclose a molecular mechanism based on PARP1-Erk synergism, which may underlie this phenomenon. A stimulation induced PARP1 binding to phosphorylated Erk2 in the chromatin of cerebral neurons caused Erk-induced PARP1 activation, rendering transcription factors and promoters of immediate early genes (IEG) accessible to PARP1-bound phosphorylated Erk2. Thus, Erk-induced PARP1 activation mediated IEG expression implicated in long-term memory. PARP1 inhibition, silencing, or genetic deletion abrogated stimulation-induced Erk-recruitment to IEG promoters, gene expression and LTP generation in hippocampal CA3-CA1-connections. Moreover, a predominant binding of PARP1 to single-strand DNA breaks, occluding its Erk binding sites, suppressed IEG expression and prevented the generation of LTP. These findings outline a PARP1-dependent mechanism required for LTP generation, which may be implicated in long-term memory acquisition and in its deterioration in senescence.

Enhancing effect of ATP on intracellular adriamycin penetration in human ovarian cancer cell lines.

Ovarian cancer has the highest mortality rate of all gynecological malignancies probably due to the evolution of clones resistant to cytotoxic drugs. Exploring possibilities to overcome such resistance constitutes a challenge in this study. We present the effect of adenosine triphosphate (ATP), serving as a chemosensitizer, in combination with adriamycin on three human ovarian cancer cell lines of epithelial origin, OC-109, OC-238 and OC-7-NU, obtained from malignant ascites of different patients, and were proven to be tumorigenic in nude mice. The three lines differ in their sensitivity to the ATP-induced increase in adriamycin accumulation. FACS analysis showed a pronounced increase in intracellular adriamycin accumulation after treatment with various concentrations of ATP. In the OC-238 line, a 50.1% increase was observed at a low ATP concentration (200 microM), whereas higher concentrations (400 microM and 500 microM) were needed to obtain an increase in ADR accumulation of 30% with the other two lines. Our study demonstrates that ATP improves the penetration of adriamycin at the neoplastic cellular level. Furthermore, our results may indicate that intratumoral ATP may serve as an alternative chemosensitizer which lacks the deleterious side effects of other chemosensitizing options.

Modulation of the voltage-dependent sodium channel by agents affecting G-proteins: a study in Xenopus oocytes injected with brain RNA.

The effects of agents known to affect G-proteins on voltage-dependent, tetrodotoxin-sensitive Na+ channels were studied in Xenopus oocytes injected with rat brain RNA, using two-electrode voltage-clamp technique. The non-hydrolysable analogue of GTP, GTP-gamma-S, known to activate G-proteins, inhibited the Na+ current (INa). The decrease in the amplitude of INa was not accompanied by changes in activation or inactivation characteristics of the channel. The non-hydrolysable analogue of GDP, GDP-beta-S, had no effect on INa. The responses to gamma-aminobutyric acid and kainate in the same oocytes were also attenuated by GTP-gamma-S. Pertussis toxin, which inactivates some G-proteins by catalyzing their ADP-ribosylation, enhanced INa, but did not prevent the inhibition of INa by GTP-gamma-S. We conclude that the Na+ channel, and possibly the GABA and kainate receptors and/or channels, are coupled to a G-protein. The activation of the G-protein modulates the channels either directly, or via activation of biochemical cascade possibly involving production of second messengers and channel phosphorylation.

Inhibition of pertussis toxin catalyzed ADP-ribosylation of G-proteins by membrane depolarization in rat brain synaptoneurosomes.

Rat brainstem synaptoneurosomes at resting and depolarization potentials were subjected to ADP-ribosylation in the presence of pertussis toxin (PTX). Subsequent [32P]ADP-ribosylation of synaptoneurosomal membranes revealed labeling of a 39-kDa protein band which reacted with antibodies to the alpha-subunit of G-proteins, mainly Go. ADP-ribosylation of the G-proteins was completely achieved in synaptoneurosomes at resting potential ( [K+] = 4.7 mM). In the depolarized synaptoneurosomes, however, the higher the membrane potential the lower the extent of ADP-ribosylation achieved (46% and 11% in K+ concentrations of 50 and 100 mM, respectively). A similar effect of membrane depolarization on PTX-catalyzed ADP-ribosylation was expressed in the functional coupling between G-protein activation and changes induced in the muscarinic receptor affinity. These findings may indicate a depolarization-induced inhibition of PTX-catalyzed ADP-ribosylation of G-proteins.

An ion-site interaction model for sodium currents in the giant axon.

The time and voltage dependence of sodium currents in the Myxicola giant axon were examined as functions of the external sodium concentration. The results were incompatible with a model of free diffusion through a gated channel, but lent themselves to analysis in terms of a model involving a positive cooperative homotropic reaction in which Na+ interacts with two allosteric sites - a regulatory site and a transfer site - at the 'sodium channel'. The time-dependent solution of the rate equations describing the kinetics of the transfer reaction was derived as an expression describing the sodium current as a function of time, membrane potential and external sodium concentration. This function was used to test the validity of the model by its ability to predict the nerve excitability properties. The predicted i-v and i-t curves fitted the experimental results (p less than 0.005 and p less than 0.05, respectively). The computed parameters of these functions are consistent with other experimental results. The possibility of a noncooperative reaction was rejected.

Thrombolysis for acute deep vein thrombosis.

BACKGROUND: Standard treatment for deep vein thrombosis (DVT) aims to reduce immediate complications. Use of thrombolysis or clot dissolving drugs could reduce the long-term complications of post-thrombotic syndrome (pain, swelling, skin discolouration, or venous ulceration) in the affected leg. OBJECTIVES: To determine the efficacy and safety of thrombolysis for DVT. SEARCH STRATEGY: Publications describing randomised controlled trials (RCTs) of thrombolysis versus anticoagulation for acute DVT were sought through electronic searches of the Cochrane Peripheral Vascular Diseases trials register (last searched April 2004) and the Cochrane Central Register of Controlled Trials (CENTRAL) (last searched The Cochrane Library Issue 2, 2004). Additional trials were identified by reviewing reference lists of papers. There were no restrictions for language. SELECTION CRITERIA: RCTs examining thrombolysis versus anticoagulation for acute DVT and/or calf vein thrombosis were considered. DATA COLLECTION AND ANALYSIS: One reviewer (LIW) selected trials, extracted data and assessed study quality, with checking at all stages by the other reviewer (MPA). Where required, additional information was sought from trialists. MAIN RESULTS: Twelve studies were included. Complete clot lysis occurred significantly more often in the treatment group in early follow up, (relative risk (RR) 0.24; 95% confidence interval (CI) 0.07 to 0.82), and in late follow up, (RR 0.37; 95 % CI 0.25 to 0.54). A similar effect was also seen for any degree of improvement in venous patency. Significantly less post-thrombotic syndrome occurred in those receiving thrombolysis, (RR 0.66; 95 % CI 0.47 to 0.94). Leg ulceration was reduced although the data were limited by small numbers, (RR 0.53; 95 % CI 0.12 to 2.43). Venous function was improved at late follow up, but not significantly (RR 0.43; 95 % CI 0.06 to 3.17)Out of 668 patients, those receiving thrombolysis had significantly more bleeding complications, (RR 1.73; 95 % CI 1.04 to 2.88). Two strokes occurred in the treatment group (RR 1.70; 95 % CI 0.21 to 13.70). The incidence of bleeding appears to have reduced over time with the introduction of stricter selection criteria. There was no significant effect on mortality detected in either early or late follow up. Data on occurrence of pulmonary embolism (PE) and recurrent DVT were inconclusive. REVIEWERS' CONCLUSIONS: Thrombolysis appears to offer advantages in terms of reducing post-thrombotic syndrome and maintaining venous patency after DVT. Use of strict eligibility criteria has improved the safety and acceptability of this treatment. The optimum drug, dose and route of administration have yet to be determined.

Laparoscopic cholecystectomy in Leicester: an audit of 555 patients.

Laparoscopic techniques have revolutionised the surgical approach to cholecystectomy, even though there have been no published randomised controlled trials to demonstrate the safety of this approach. We present an audit of 555 patients offered laparoscopic cholecystectomy. In all, 54 patients (9.7%) were converted to an open procedure. Peroperative cholangiography (POC) was attempted in 190 cases (34.2%) and achieved in 141 (25.4%). Major complications occurred in 26 cases (4.7%) including 5 (0.9%) deaths, two of whom had major pre-existing morbidity. There was one common bile duct (CBD) injury (0.18%). There were 30 patients (5.4%) found to have CBD stones, 27 of which were cleared at ERCP, and three converted to open exploration. Cholecystectomy by any route is a major operation and we conclude that careful case selection remains imperative. However, morbidity is favourable compared with open cholecystectomy, and comparable with other reports using the laparoscopic technique. Our experience of CBD injury (0.18%) is also acceptable compared with the risk of injury during open cholecystectomy. There were 312 patients (56.2%) who did not undergo perioperative CBD imaging with ERCP or POC and three of these developed early symptomatic retained stones. This group requires further follow-up.

Neonatal mycotic internal iliac aneurysm due to methicillin-resistant Staphylococcus aureus (MRSA) septicaemia successfully treated by coil embolisation.

A 12-day-old term male neonate presented with septic arthritis, multiple skin and intrabdominal abscesses and a mycotic aneurysm of the right internal iliac artery. He was diagnosed as having methicillin resistant staphylococcus aureus (MRSA) septicaemia and deemed unsuitable for surgical treatment of the aneurysm. Coil embolisation of the internal iliac artery was performed, followed by a successful recovery and with no evidence of residual or recurrent infection. The authors describe a method of treating internal iliac mycotic aneurysms in high-risk patients by endovascular means, which we believe has not been attempted in this precise scenario before.

Depolarization-induced changes in the muscarinic receptor in rat brain and heart are mediated by pertussis-toxin-sensitive G-proteins.

Muscarinic receptor properties in rat cortical and brain stem synaptoneurosomes and in heart myocytes were examined at resting potential and at depolarization. Depolarization induced the conversion of agonist-binding sites of the receptor from a high to a low affinity state, which could be reversed by a return to resting potential. No effect was observed on the affinity of the receptor for antagonists. Pertussis-toxin (PTX)-catalyzed ADP-ribosylation of all substrates in both synaptoneurosomal and myocyte membranes, when conducted at resting potential, prevented depolarization-induced conversion of the receptor affinity in these preparations. The target substrates were identified by [32P]ADP-ribosylation of membranes prepared from brain stem synaptoneurosomes. Autoradiography revealed labeling of a 39-kDa protein band, which reacted mainly with antibodies to the alpha-subunit of Go-proteins. The possible involvement of G-proteins in depolarization-induced changes in the receptor activity was further investigated by examining the effect of membrane potential on the PTX-sensitive binding of di- and triphosphated guanine nucleotides to synaptoneurosomal membranes. Brain stem synaptoneurosomes were made permeable to guanine nucleotides ([3H]GTP, [3H]GDP, [3H]5'-guanylyl imidodiphosphate) by treatment with ATP. After the synaptoneurosomes had been loaded with labeled GTP/GDP, resealed, and then subjected to either resting potential of short depolarization, binding of [3H]GDP to the membranes of depolarized synaptoneurosomes was 4.0 +/- 0.3 (n = 20) times higher than to the membranes of synaptoneurosomes at resting potential. Repolarization reversed this effect. Enhancement of [3H]GDP binding to the synaptoneurosomal membranes was induced also by muscarinic activation, although the increase obtained was only 30-40% (n = 5) relative to [3H]GDP binding at resting potential. Both the depolarization-induced and the muscarinically-induced enhancement of [3H]GDP binding were prevented following PTX-catalyzed ADP-ribosylation of G-proteins in the synaptoneurosomal membrane. Our results suggest that the depolarization-induced enhancement in the binding of [3H]GTP/[3H]GDP may be attributable to activation of PTX-sensitive G-proteins, which mediate the depolarization-induced alteration of the affinity of the muscarinic receptor for agonists.

Interactions between the muscarinic receptors, sodium channels, and guanine nucleotide-binding protein(s) in rat atria.

The effects of the voltage-sensitive sodium channel activator batrachotoxin (BTX) on the binding properties of muscarinic receptors were studied in homogenates of rat atria. Also studied were the effects of muscarinic ligands on the binding of tritium-labeled batrachotoxin ([3H]BTX) to the same preparation. BTX (1 microM), which induces an open state in sodium channels, enhanced the affinity of binding of several agonists to the muscarinic receptors. Analysis of the data indicated that the effect of BTX was to increase the affinity of the agonists toward the high-affinity sites. Binding of antagonists was not affected by BTX. At higher concentrations of toxin, the density of the high affinity muscarinic sites was also affected. The binding of agonists (but not of antagonists) to muscarinic receptors in turn enhanced the specific binding of [3H]BTX to sodium channels. These effects on the muscarinic receptors and on the sodium channels were inhibited in the presence of Gpp(NH)p at concentrations lower than those bringing about conversion of binding sites from the high affinity to the low affinity conformation. On the basis of these findings we suggest that the opening of sodium channels and the binding of agonists to muscarinic receptors in rat atrial membranes are coupled events which are mediated by guanine nucleotide-binding protein(s). Such a hypothesis is consistent with previously proposed models for signal transduction in the membrane.

Evidence for involvement of the voltage-dependent Na+ channel gating in depolarization-induced activation of G-proteins.

Evidence for activation of pertussis-toxin-sensitive G-proteins by membrane depolarization in rat brainstem synaptoneurosomes was recently reported (Cohen-Armon, M., and Sokolovsky, M. (1991) J. Biol. Chem. 266, 2595-2605; (1991) Neurosci. Lett. 126, 87-90) and is further supported in this study by the observation that the depolarization-induced effect is inhibited when G-proteins are stabilized in the non-activated state with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which was introduced into synaptoneurosomes during the process of permeabilization and resealing. In the present study, agents that either keep the voltage-dependent Na+ channel in persistently activated state (while Na+ currents are blocked) or prevent it from activation were used in an attempt to determine whether the voltage-dependent Na+ channels are involved in the depolarization-induced activation of pertussis-toxin-sensitive G-proteins. The main probe employed was the cardiotonic and antiarrhythmic agent DPI, which is a racemic mixture of two enantiomers, one of which (the R enantiomer) reportedly prevents depolarization-induced activation of the Na+ channel while the other (the S enantiomer) inhibits Na+ channel inactivation. The results suggest that while inactivation of the voltage-dependent Na+ channel does not interfere with the putative depolarization-induced activation of G-proteins, membrane depolarization affects G-proteins and the coupled muscarinic receptors only if the voltage-dependent Na+ channels are capable of being activated. Thus, inhibition of the depolarization-induced activation of Na+ channels was accompanied by inhibition of the depolarization-induced activation of pertussis-toxin-sensitive G-proteins and by modifications of both the coupling of G-proteins to muscarinic receptors and the ADP-ribosylation of Go-proteins. These effects could be counteracted by persistent activation of the voltage-dependent Na+ channels (while Na+ current was blocked). Our observations may suggest that the voltage-dependent Na+ channel gating is involved in the depolarization-induced activation of pertussis toxin-sensitive G-proteins and may provide evidence for a possible mechanism of membrane depolarization signal transduction in excitable cells.

Numerical method for correcting the series resistance error in voltage clamp experiments.

There is an inherent error in the voltage clamp, which is a major electrophysiological research tool. Current (I) flowing across the voltage-clamped membrane generates a drop in the resistance potential in series with the membrane (Rs) and thus an error in control of the potential. This error, which may be very significant, can only be partially corrected by electronic compensation during the experiment. Subsequent correction by computation is generally believed impossible for membrane potentials (Vm) for which the conductance parameters are voltage dependent. In the proposed method, which is based on the assumption that dI/dt is a function of I and Vm only, the reconstruction begins with very small sodium or potassium currents (for which the Rs error is negligible). The subsequent I value is calculated using a dI/dt value obtained under any set of conditions where Vm is known to have the desired value (as ensured by the experimental protocol) and where I equals the initial I value. By iterations, the correct I vs. t curve is reconstructed for the chosen potential. It is shown analytically that for squid giant axons, with an uncompensated Rs of 5 omega . cm2 the maximal error in INa is reduced from about 30% to under 3%. The validity of the reconstruction is demonstrated experimentally by corrected INa generated in artificial seawater and comparing it with INa obtained in a solution containing a low concentration of tetrodotoxin.

Lymphocyte phenotypes and infection incidence in transfused preterm neonates.

The immunomodulating effects of repeated exposure to blood from multiple donors coupled with an immature immune system may predispose the preterm neonate to an increased incidence of infection in his first few months of life. To test this hypothesis, we compared lymphocyte phenotypes, serum IgG concentrations, and histories of infection and rehospitalization in neonates at 4 months corrected age. Two of the study groups were preterm infants who had been transfused with either frozen, deglycerolized or CMV-negative, gamma-irradiated blood. Control groups consisted of nontransfused term and preterm infants. There were no differences found in lymphocyte phenotypes or serum IgG concentrations of controls or transfused infants. No differences were found in the infection or rehospitalization incidence in the transfused infants as compared with nontransfused preterm neonates. We failed to show differences in immune parameters or in infection and rehospitalization rates of the preterm infants analysed. Alongside previously published reports, our data suggest that red cell transfusions have a minimal impact on the immature immune system of the neonate.