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Biomedical subjects

R Levy

Publications and source records attributed to R Levy.

At least 109 records · Page 6Linked to original sources
PubMed2000

Acute and chronic activation of the mu-opioid receptor with the endogenous ligand endomorphin differentially regulates adenylyl cyclase isozymes.

While acute activation of G(i/o)-coupled receptors leads to inhibition of adenylyl cyclase, chronic activation of such receptors produces an increase in cyclic AMP accumulation, particularly evident upon withdrawal of the inhibitory agonist. This phenomenon has been referred to as adenylyl cyclase superactivation and is believed to play an important role in opiate addiction. Nine adenylyl cyclase isozymes have been recently identified and shown by us to be differentially regulated by acute and chronic inhibitory receptor activation. Using COS-7 cells cotransfected with various adenylyl cyclase isozymes, we examined here whether the endomorphins (the most recently discovered of the four classes of endogenous opioid peptides, and which interact selectively with the mu receptor) are able to induce inhibition/superactivation of representatives from the various adenylyl cyclase isozyme classes. Here, we show that adenylyl cyclase types I and V were inhibited by acute endomorphin application and superactivated upon chronic exposure, while adenylyl cyclase type II was stimulated by acute and "superinhibited" by chronic endomorphin exposure. These results show that the endomorphins are capable of regulating adenylyl cyclase activity and that different adenylyl cyclase isozymes respond differently to these endogenous ligands.

Adenylyl Cyclases↗
PubMed2000

Elevated cytosolic phospholipase A(2) expression and activity in human neutrophils during sepsis.

Sepsis is defined as the systemic inflammatory response to infection. Phospholipase A(2) (PLA(2)) plays an important role in inflammation processes by initiating the production of inflammatory mediators. The role of cytosolic PLA (cPLA(2)) has not yet been identified in inflammatory and infectious disease clinical settings. The aim of the present research was to determine whether cPLA(2) activity has a role during sepsis. Since neutrophil activation has been documented during sepsis, these cells were chosen as a model to evaluate the function of cPLA(2) in this clinical setting. cPLA(2 )was studied at 3 levels: activity, protein expression, and messenger RNA (mRNA). Neutrophils from 32 septic patients with and without bacteremia were examined. cPLA(2) activity was measured using labeled phosphatidyl choline vesicles as a substrate, and total PLA(2) was determined by the release of labeled arachidonic acid from prelabeled cells. A significant increase in cPLA(2) activity, protein expression, and total PLA(2) activity in neutrophils was detected during sepsis. mRNA levels, detected by reverse transcriptase-polymerase chain reaction, were significantly higher during sepsis, indicating that the increase in the amount of cPLA(2) is regulated on the mRNA level. The significant elevation of cPLA(2) activity and expression in neutrophils during sepsis suggests that this enzyme plays a major role in neutrophil function in this clinical setting. (Blood. 2000;95:660-665)

Adult↗
PubMed2000

Cognitive function in UK community-dwelling African Caribbean and white elders: a pilot study.

In recent years there has been interest in risk of cognitive impairment and dementia in populations of African origin. Little is known about this risk in older African Caribbean residents in the UK. One difficulty is lack of consensus over an adequate cognitive test battery for this community. Forty-five African Caribbean and 45 age and gender matched white community residents were recruited by household enumeration of an inner London electoral ward. These participants were administered the MMSE during a screening interview. Where possible, this was followed up by tests from the CERAD and CAMCOG neuropsychological batteries, a medical examination, and a structured interview with an informant. Based on these data, a psychiatrist blind to ethnicity independently rated 86 of these participants (41 of the African Caribbeans, all 45 of the whites) as cognitively normal, cognitively impaired, or demented. Of 41 African Caribbeans, 18 (44%) were rated as cognitively normal, 9 (22%) were rated as cognitively impaired, and 14 (34%) were rated as demented. Of the 45 whites, 39 (87%) were rated as cognitively normal, 4 (9%) were rated as cognitively impaired, and 2 (4%) were rated as demented. African Caribbeans scored significantly lower than whites in most cognitive test scores, which was not accounted for by their lower educational and occupational attainment, or their higher frequency of cardiovascular risk factors. African Caribbean elders in the UK appear to be at high risk of cognitive impairment and dementia. However, the influence of potential confounding factors such as socio-economic position and ill-health, and the effect of cultural test bias, cannot be ruled out.

Aged↗
PubMed2000

Activation of cytosolic phospholipase A2 by opsonized zymosan in human neutrophils requires both ERK and p38 MAP-kinase.

The present study demonstrates that stimulation of human neutrophils with opsonized zymosan (OZ), which binds to Fc gamma receptors (Fc gamma Rs) and C3b receptors, activates both ERK and p38 MAP-kinase. Thus, the relative role of both types of MAP-kinase, ERK and p38, in activation of cPLA2 by OZ was studied. cPLA2 activation by OZ was detected 15 sec after stimulation, maintained a plateau for 10 min and decreased thereafter. p38 MAP-kinase activation exhibited kinetics similar to that of cPLA2, while ERK activation was detected within 15 sec but decreased significantly in less than 5 min after stimulation. Pretreatment of the cells with the MEK inhibitor, PD-098059, or the p38 MAP-kinase inhibitor, SB-203580 resulted in total inhibition of ERK or p38 MAP-kinase activity, respectively. Each inhibitor caused a partial inhibition during the time course of cPLA2 activity, while their combination caused a total inhibition. Compared to OZ, inactivated OZ, which does not contain the complement proteins, induced an identical time-dependent stimulation of ERK and p38 MAP-kinase as well as a similar cPLA2 activity, suggesting that the role of the C3b receptors in this system is negligible. It is concluded that OZ activates both ERK and p38 MAP-kinase and that the two isotypes are required for the onset and maintenance of cPLA2 activity.

Cytosol↗
PubMed2000

Cytosolic phospholipase A2 is required for the activation of the NADPH oxidase associated H+ channel in phagocyte-like cells.

The NADPH oxidase producing-superoxide is the major mechanism by which phagocytes kill invading pathogens. The human myeloid cell line PLB-985 was transfected to express p85 cytosolic phospholipase A2 (cPLA2) antisense mRNA and stable clones were selected which lack detectable cPLA2. cPLA2-deficient PLB-985 cells differentiate similarly to control PLB-985 cells in response to retinoic acid, DMSO or 1,25 dihydroxyvitamin D3 indicating that cPLA2 is not involved in the differentiation process. Despite the normal synthesis of NADPH oxidase subunits during differentiation of cPLA2-deficient PLB-985 cells, these cells fail to activate NADPH oxidase in response to a variety of soluble and particulate stimuli, but addition of exogenous arachidonic acid (AA) fully restores oxidase activity. This establishes an essential requirement of cPLA2 generated AA for activation of phagocyte NADPH oxidase. In order to elucidate the mechanism by which cPLA2 regulates the oxidase, the role of cPLA2 in NADPH oxidase associated H+ channel was studied. Activation of differentiated PLB cells resulted in a Zn+2 sensitive alkalization, indicating H+ channel activity. In contrast, differentiated PLB-D cells failed to activate the H+ channel, but addition of exogenous AA fully restored this activity, indicating an essential and specific physiological requirement of cPLA2-generated AA for activation of the H+ channel. The presence of the H+ channel inhibitor, Zn+2, caused significant inhibition of NADPH oxidase activity, suggesting a role of the NADPH oxidase associated H+ channel in regulating oxidase activity.

Arachidonic Acid↗
PubMed2000

The NADPH oxidase diaphorase activity in permeabilized human neutrophils and granulocytic like PLB-985 cells.

The phagocyte NADPH oxidase is a multicomponent transport chain that generates superoxide, a precursor of microbicidal oxidants, important for host defense. This transport chain is contained mainly in the large membrane subunit of the oxidase (gp91phox), and transfers electrons from cytosolic NADPH, through FAD binding and heme centers, to molecular oxygen (Babior, 1999; Fujii and Kakinuma, 1991; Rotrosen et al., 1992; Segal and Abo, 1993). Cross et al. have recently described a novel NADPH oxidase diaphorase activity present in the membrane fraction of activated neutrophils, using a cell free model (Cross et al., 1994). This diaphorase activity is measured by the artificial electron acceptor 4-iodonitrotetrazolium violet (INT) and is attributed to the reduction of the flavin center of the flavocytochrome (Cross et al., 1994; Li and Guillory, 1997). In the present study we establish a system for detecting diaphorase activity in intact cells. Neutrophils and PLB-985 cells, that were differentiated using 1.25% dimethyl sulfoxide (DMSO) to granulocyte phenotype, were permeabilized by electroporation, and diaphorase activity was determined using INT. Neutrophils and differentiated PLB-985 cells stimulated by PMA or GTP gamma S showed a diaphorase activity that was not present in unstimulated differentiated cells. The diaphorase activity could not be detected in undifferentiated cells and was developed during differentiation. The pattern of diaphorase activity in stimulated parent differentiated PLB cells was similar to that observed in stimulated human neutrophils. The permeabilized-INT cell system offers a unique tool for the evaluation of NADPH oxidase diaphorase activity, in whole cells.

Cell Line↗
PubMed2000

Segregation of working memory functions within the dorsolateral prefrontal cortex.

It is now widely accepted that the prefrontal cortex (PFC) plays a critical role in the neural network subserving working memory (WM). At least three related questions are still under debate: (1) is the PFC critical for all constituent processes of WM (i.e., short-term storage, manipulation, and utilization of mental representations) or only in one or a few of them? (2) Is there segregation of function among different cytoarchitectonic subdivisions of the PFC? (3) If this be the case, is this segregation based on the nature of the information being processed or on the type of cognitive operation performed? The present review article describes findings in the monkey supporting a modular "domain-specific" model of PFC functional organization with respect to WM operations. In this model, the dorsolateral prefrontal cortex (DLPFC) is composed of several subregions, based primarily on the nature of the information being processed in WM. Storage and processing functions are integrally related in each area. Future studies designed to map as yet uncharted areas of prefrontal cortex with refined anatomical and physiological approaches may provide a critical test of the model and evaluate the extent to which it applies generally or, instead, mainly to visual domains or only to dorsolateral convexity areas.

Animals↗
PubMed2000

A perspective on monoclonal antibody therapy: where we have been and where we are going.

The history of monoclonal antibody therapy for cancer includes many promising beginnings, a vast number of detours, and now a major success--perhaps soon to be joined by several more. Years of research by hundreds of investigators worldwide have led to a growing understanding of the principles that underlie successful monoclonal antibody therapy and that can be used to create new antibody-based agents. However, there are many remaining uncertainties about this mode of therapy, even for those agents that have already joined our therapeutic armamentarium. Several key questions will be identified and used to suggest avenues for future research.

Antibodies, Monoclonal↗
PubMed2000

Idiotype vaccination using dendritic cells after autologous peripheral blood progenitor cell transplantation for multiple myeloma.

The idiotype (Id) determinants on the multiple myeloma immunoglobulin can serve as tumor-specific antigens. An anti-Id immune response may stem the growth of the malignant clone. We report on 26 patients treated at our institution with high-dose chemotherapy and peripheral blood progenitor cell transplantation (PBPCT) and vaccinated with the Id protein. The patients received chemotherapy and PBPCT to establish a minimal residual disease state. After high-dose therapy, the patients received a series of monthly immunizations consisting of 2 intravenous infusions of dendritic cells (DCs) pulsed with either Id protein or Id coupled with keyhole limpet hemocyanin (KLH) as an immunogenic carrier protein, followed by subcutaneous boosts of Id-KLH conjugates. DCs were obtained in all patients from a leukapheresis product 3 to 9 months after PBPCT. Patients were observed for toxicity, immune responses, and tumor status. The DC infusions and the administration of Id-KLH boosts were well tolerated, with patients experiencing only minor and transient side effects. Of the patients, 24 of 26 generated a KLH-specific cellular proliferative immune response. Only 4 patients developed an Id-specific proliferative immune response. Three of these immune responders were in complete remission at the time of vaccination. A total of 17 patients are alive at a median follow-up of 30 months after transplantation. Id vaccination with autologous DCs is feasible for myeloma patients after transplantation. Id-specific cellular responses can be induced in patients who are in complete remission. Further studies are needed to increase the rate of anti-Id immune responses in patients who do not achieve complete remission.

Adult↗
PubMed2000

Metabolic effects of nigrostriatal denervation in basal ganglia.

In the past, functional changes in the circuitry of the basal ganglia that occur in Parkinson's disease were primarily analyzed with electrophysiological and 2-deoxyglucose measurements. The increased activity of the subthalamic nucleus (STN) observed has been attributed to a reduction in inhibition mediated by the external segment of the globus pallidus (GPe), secondary to the loss of dopaminergic-neuron influence on D2-receptor-bearing striato-pallidal neurons. More recently, in situ hybridization studies of cytochrome oxidase subunit I have confirmed the overactivity of the STN in the parkinsonian state. In addition, this technique has provided evidence that the change in STN activity is owing not only to decreased inhibition from the GPe but to hyperactivity of excitatory inputs from the parafascicular nucleus of the thalamus and the pedunculopontine nucleus in the brainstem.

Basal Ganglia↗
PubMed2000

Phagocytic killing and antibody response during the first year after tetravalent meningococcal vaccine in complement-deficient and in normal individuals.

Seven individuals with late complement component (LCC) deficiency and seven control subjects were vaccinated with tetravalent meningococcal vaccine. The response to vaccination was evaluated by measuring the antibody titer and the phagocyte killing of the bacteria, before, 5-7 weeks, and 12-14 months after vaccination. Prior to vaccination, no phagocytic killing and a low titer of antibody was found in the LCC-deficient group and a low killing (mean of 40-58%, according to the serogroup) in normal controls. The phagocytic killing increased significantly 5-7 weeks after vaccination. However, while in normal controls the phagocytic killing was close to 100% after 5-7 weeks and decreased only slightly during the first year, the mean killing of the various meningococcal subgroups in LCC-deficient individuals was 70-89% and dropped to only 53-71% one year after vaccination. Six weeks after vaccination the mean antimeningococcal antibody titer increased similarly in the sera of LCC-deficient patients and controls. One year after vaccination the controls maintained the high concentration, while the LCC-deficient patients had tendency toward a decrease. In addition, the interpersonal variability of the antibody concentration, both in LCC-deficient individuals and in normal controls, was much higher than the phagocytic killing, with only a very mild increase in some individuals. Thus, it is possible that in spite of adequate increase of antimeningococcal antibody titer after vaccination of LCC-deficient individuals their immunity against the bacteria may not be optimal. Our data show also that phagocytic killing of meningococci is probably a more consistent assay than antibody titer levels for antimeningococcal immunity, especially in LCC-deficient patients.

Antibodies, Bacterial↗
PubMed2000

Superantigen antagonist protects against lethal shock and defines a new domain for T-cell activation.

Superantigens trigger an excessive cellular immune response, leading to toxic shock. We have designed a peptide antagonist that inhibits superantigen-induced expression of human genes for interleukin-2, gamma interferon and tumor necrosis factor-b, which are cytokines that mediate shock. The peptide shows homology to a b-strand-hinge-a-helix domain that is structurally conserved in superantigens, yet is remote from known binding sites for the major histocompatibility class II molecule and T-cell receptor. Superantigens depend on this domain for T-cell activation. The peptide protected mice against lethal challenge with staphylococcal and streptococcal superantigens. Moreover, it rescued mice undergoing toxic shock. Surviving mice rapidly developed protective antibodies against superantigen that rendered them resistant to further lethal challenges, even with different superantigens. Thus, the lethal effect of superantigens can be blocked with a peptide antagonist that inhibits their action at the beginning of the toxicity cascade, before activation of T cells takes place.

Amino Acid Sequence↗
PubMed2000

To be, or not to be, that is the question. Apoptosis in human trophoblast.

Apoptosis, the morphology of cell suicide, may result from programmed cell death or may be a response to exogenous stimuli. Apoptosis can be induced in cultured trophoblast and can be identified in the trophoblast of placental villi. The trophoblast regulates maternal-fetal gas, nutrient and waste product exchange; therefore, the presence of apoptosis in this key cellular interface highlights the importance of understanding what controls apoptosis in the placenta. In this review, we describe the signal transduction pathways that trigger apoptosis in other systems, identify key genetic controls for the process and outline the final common pathway which effects execution in cells committed to suicide. Multiplicity, redundancy and cross talk among pathways characterize the surface membrane signals and exogenous stimuli that trigger apoptosis in other cells. As each step in the apoptotic process is discussed, we describe what is known about the step in human placental villi. Recent studies suggest that a further understanding of receptor-mediated signalling pathways, the Bcl-2 regulators and the caspases and substrates involved in placental apoptosis will surely provide insights into both normal placental development and the placental dysfunction associated with some abnormal pregnancies.

Apoptosis↗
PubMed2000

Localization of ribophorin II to the endoplasmic reticulum involves both its transmembrane and cytoplasmic domains.

Proteins that are concentrated in specific compartments of the endomembrane system in order to exert their organelle-specific function must possess specific localization signals that prevent their transport to distal regions of the exocytic pathway. Some resident proteins of the endoplasmic reticulum (ER) that are known to escape with low efficiency from this organelle to a post ER compartment are recognized by a recycling receptor and brought back to their site of residence. Other ER proteins, however, appear to be retained in the ER by mechanisms that operate in the organelle itself. The mammalian oligosaccharyltransferase (OST) is a protein complex that effects the cotranslational N-glycosylation of newly synthesized polypeptides, and is composed of at least four rough ER-specific membrane proteins: ribophorins I and II (RI and RII), OST48, and Dadl. The mechanism(s) by which the subunits of this complex are retained in the ER are not well understood. In an effort to identify the domains within RII responsible for its ER localization we have studied the fate of chimeric proteins in which one or more RII domains were replaced by the corresponding ones of the Tac antigen, the latter being a well characterized plasma membrane protein that lacks intrinsic ER retention signals and serves to provide a neutral framework for the identification of retention signals in other proteins. We found that the luminal domain of RII by itself does not contain retention information, while the cytoplasmic and transmembrane domains contain independent ER localization signals. We also show that the retention function of the transmembrane domain is strengthened by the presence of a flanking luminal region consisting of 15 amino acids.

Animals↗
PubMed2000

Transmission risk of hepatitis C virus in assisted reproductive techniques.

Medical assistance for procreation in a couple where one or both parents has hepatitis C viral infection (HCV) raises the issue of the transmission of the infection to the baby and/or of possible contamination of both the technicians and the gametes or embryos from virus-free parents in the laboratory. It becomes essential to assess transmission risk in assisted reproductive techniques in order to define clearly the management of couples according to their viral status. To define the HCV transmissibility risk in assisted reproduction related to the presence of virus in semen from infected infertile men, HCV RNA detection was performed in sera, and semen and sperm fractions obtained after Percoll gradient centrifugation. HCV RNA was detected in 5% (2/39) of the semen samples tested: in the raw semen, in the seminal fluid and in the cell pellet but never after Percoll selection. According to these results, we suggest a strategy for HCV-infected infertile men who need assistance for procreation.

Adult↗
PubMed2000

Differential superactivation of adenylyl cyclase isozymes after chronic activation of the CB(1) cannabinoid receptor.

Many types of cells exhibit increased adenylyl cyclase (AC) activity after chronic agonist treatment of G(i/o)-coupled receptors. This phenomenon, defined as AC superactivation or sensitization, has mostly been studied for the opioid receptors and is implicated in opiate addiction. Here we show that this phenomenon is also observed on chronic activation of the CB(1) cannabinoid receptor. Moreover, using COS-7 cells cotransfected with CB(1) receptor and individual AC isozymes, we could show selective superactivation of AC types I, III, V, VI, and VIII. The level of superactivation was dependent on the concentration of agonist and time of agonist exposure and was not dependent on the AC stimulator used. No superactivation of AC types II, IV, or VII was observed in COS-7 cells cotransfected with CB(1). The superactivation of AC type V was abolished by pretreatment with pertussis toxin and by cotransfection with the carboxy terminus of beta-adrenergic receptor kinase, which serves as a scavenger of G(betagamma) dimers, implying a role for the G(i/o) proteins and especially G(betagamma) dimers in the cannabinoid-induced superactivation of AC.

Adenylyl Cyclases↗
PubMed2000

Alterations in detergent solubility of heterotrimeric G proteins after chronic activation of G(i/o)-coupled receptors: changes in detergent solubility are in correlation with onset of adenylyl cyclase superactivation.

Prolonged G(i/o) protein-coupled receptor activation has been shown to lead to receptor internalization and receptor desensitization. In addition, it is well established that although acute activation of these receptors leads to inhibition of adenylyl cyclase (AC), long-term activation results in increased AC activity (especially evident on removal of the inhibitory agonist), a phenomenon defined as AC superactivation or sensitization. Herein, we show that chronic exposure to agonists of G(i)-coupled receptors also leads to a decrease in cholate detergent solubility of G protein subunits, and that antagonist treatment after such chronic agonist exposure leads to a time-dependent reversal of the cholate insolubility. With Chinese hamster ovary and COS cells transfected with several G(i/o)-coupled receptors (i.e., mu- and kappa-opioid, and m(4)-muscarinic), we observed that although no overall change occurred in total content of G(alphai)- and beta(1)-subunits, chronic agonist treatment led to a marked reduction in the ability of 1% cholate to solubilize G(betagamma) as well as G(alphai). This solubility shift is exclusively observed with G(alphai), and was not seen with G(alphas). The disappearance and reappearance of G(alphai) and G(betagamma) subunits from and to the detergent-soluble fractions occur with similar time courses as observed for the onset and disappearance of AC superactivation. Lastly, pertussis toxin, which blocks acute and chronic agonist-induced AC inhibition and superactivation, also blocks the shift in detergent solubility. These results suggest a correlation between the solubility shift of the heterotrimeric G(i) protein and the generation of AC superactivation.

Adenylate Cyclase Toxin↗