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R S Lee

Publications and source records attributed to R S Lee.

At least 55 records · Page 3Linked to original sources
PubMed1996

The cytokine-activated tyrosine kinase JAK2 activates Raf-1 in a p21ras-dependent manner.

JAK2, a member of the Janus kinase superfamily was found to interact functionally with Raf-1, a central component of the ras/mitogen-activated protein kinase signal transduction pathway. Interferon-gamma and several other cytokines that are known to activate JAK2 kinase were also found to stimulate Raf-1 kinase activity toward MEK-1 in mammalian cells. In the baculovirus coexpression system, Raf-1 was activated by JAK2 in the presence of p21ras. Under these conditions, a ternary complex of p21ras, JAK2, and Raf-1 was observed. In contrast, in the absence of p21ras, coexpression of JAK2 and Raf-1 resulted in an overall decrease in the Raf-1 kinase activity. In addition, JAK2 phosphorylated Raf-1 at sites different from those phosphorylated by pp60v-src. In mammalian cells treated with either erythropoietin or interferon-gamma, a small fraction of Raf-1 coimmunoprecipitated with JAK2 in lysates of cells in which JAK2 was activated as judged by its state of tyrosine phosphorylation. Taken together, these data suggest that JAK2 and p21ras cooperate to activate Raf-1.

Animals↗
PubMed1996

Dielectric studies of intermolecular interactions in native DNA.

The concentration dependence of the high frequency dielectric dispersion exhibited by solutions of DNA and the sensitivity of this to the ionic strength of the solution are described. Dielectric data obtained are consistent with a model involving the fluctuation of counterions along short segments of the DNA chain. The concentration and ion strength dependencies are discussed in terms of intermolecular interactions which are considered responsible for the existence of structure in DNA solutions.

Animals↗
PubMed1996

Phenotypic and functional characterisation of tumour-infiltrating lymphocytes derived from thyroid tumours.

The natural history of thyroid tumours and the hyper-reactivity of the immune system in patients with thyroid cancer suggest that immune surveillance may play a role in the control of this disease. A study was therefore undertaken to analyse the phenotypic and functional features of tumour-infiltrating lymphocytes (TILs) derived from thyroid tumours. In a series of experiments, it was found that, in contrast to TILs derived from patients with melanoma or renal cell carcinoma, thyroid TILs could be efficiently expanded in vitro only in the presence of allogeneic EBV transformed B (B. EBV) cells. Indeed, only one of the seven thyroid-derived TILs grew in vitro without feeder cells, whereas all 16 thyroid-derived TILs could be expanded in the presence of allogeneic B. EBV feeder cells. Phenotypic analysis of these TILs revealed a frequent in vitro expansion of an unusual T cell population that expressed both the CD4 and CD8 markers. Indeed, it was demonstrated that in five of 14 TILs in short-term culture (< day 23) and four of 11 TILs in long-term culture (> day 40), a lymphocyte population that coexpressed CD4 and CD8 antigen accounted for more than 15% of the total TIL population. This double-positive T cell population was not observed in TILs derived from melanoma or renal cell carcinoma. Thyroid-derived TILs also displayed an intense cytolytic activity against NK-sensitive tumour targets with 10 of 11 TILs exhibiting significant cytotoxicity towards the NK-sensitive K562 cell line. Six of 11 TILs were also cytotoxic towards autologous tumour, but when cold target inhibition with K562 was performed with three cultures, unlabelled K562 completely inhibited lysis of autologous tumour cells. A significant expansion of CD3+CD56+ T cells in the different TIL populations may explain this high level of NK-like cytotoxicity. In conclusion, TILs derived from thyroid tumours could be efficiently expanded in vitro under certain culture conditions. Different strategies must be explored to enhance their specific tumour autologous specificity, however, before they can be used in immunotherapy protocols.

Adult↗
PubMed1995

Effects of systemic and local ethanol on responses of rat cerebellar Purkinje neurons to iontophoretically applied gamma-aminobutyric acid.

The goals of this study were: (1) to determine the effects of acute systemic or local application of ethanol (ETOH) on the response of cerebellar Purkinje cells (P-cells) to iontophoretically applied gamma-aminobutyric acid (GABA) and (2) to characterize the effects of Ro15-4513, a putative antagonist of ETOH-GABA interactions, on ETOH-induced changes in GABA responsiveness. Male Sprague-Dawley rats (230-370 g) were anesthetized with halothane and implanted with intraperitoneal catheters for administration of ETOH (1.0-2.0 g/kg), before the recording session. Extracellular activity of single P-cells was recorded with the central barrel of a five-barrel micropipette, the other barrels of which were used for microiontophoresis of GABA and electro-osmosis of ETOH at the recording site. Spontaneous discharge and response of P-cells to GABA were monitored during a pre-ETOH control and for 1-1.5 h after systemic or electro-osmotic administration of ETOH. Transient suppression of spontaneous P-cell discharge was usually observed within 4-8 min of systemic ETOH injection. This effect lasted 2-4 min in 10 out of 19 rats tested. GABA-mediated inhibitory responses of cerebellar P-cells were increased by 45-50% relative to pre-ETOH values at 10 and 90 min post-ETOH injection. Prior administration of the imidazobenzodiazepine Ro15-4513 (4-6 mg/kg) failed to antagonize either the ETOH-induced enhancement of GABA-mediated inhibition or the transient inhibition of spontaneous P-cell activity rat cerebellar P-cell produced by ETOH. In these studies, electro-osmotically applied ETOH produced a potent suppression of spontaneous P-cell activity which precluded further augmentation of unit responses to GABA. These results show that doses of systemically administered ETOH which are mildly intoxicating in the awake, behaving animal, enhance the inhibitory action of GABA on cerebellar P-cell discharge.

Action Potentials↗
PubMed1995

Effects of systemic and local ethanol on responses of rat cerebellar Purkinje neurons to iontophoretically applied norepinephrine and gamma-aminobutyric acid.

The goal of the present study was to determine the effect of acute ethanol (ETOH), administered intraperitoneally or electro-osmotically, on norepinephrine (NE) induced increases in gamma-aminobutyric acid (GABA) mediated inhibition of single cerebellar Purkinje neurons (P-cells). Male Sprague-Dawley rats (230-370g) were anesthetized with halothane and implanted with an intraperitoneal catheter for systemic administration of ETOH (1.0-1.5 g/kg) prior to the recording session. Extracellular activity of single P-cells was recorded before and after iontophoresis of GABA and NE using five-barrel glass micropipettes. GABA was administered at the recording site by microiontophoretic pulses before, during and after continuous iontophoretic application of NE. Spontaneous discharge, GABA responses and NE-GABA interactions in P-cells were monitored for each experiment before and 1-1.5 h following systemic administration of ETOH. As in our previous reports administration of NE, at low ejection currents (10-60 nA), augmented GABA mediated suppression of P-cell spontaneous discharge. Between 10 and 60 min after injection of ETOH, this NE induced augmentation of GABA inhibition was further potentiated. This potentiation involved increases in both the magnitude and the duration of the GABA inhibition observed after NE alone. NE-induced augmentation of GABA inhibition persisted for 2-13 min longer after ETOH administration than in the pre-ETOH control period. Local electro-osmotic application of ETOH, which resulted in strong depression of spontaneous activity and caused small increases in GABA-mediated inhibition, did not directly potentiate NE-induced augmentation of GABA action. These results indicate that NE-mediated augmentation of GABA inhibition of P-cell activity is potentiated following systemic, but not local, ETOH administration.

Action Potentials↗
PubMed1995

Specific unresponsiveness to a retrovirally-transferred class I antigen is controlled through the helper pathway.

To investigate the potential role for gene therapy in induction of tolerance to solid organ grafts, we used a murine model based on the introduction of an allogeneic MHC class I gene (H-2Kb) into hematopoietic cells of congenic animals differing only at the class I locus. The H-2Kb gene was placed into a retroviral vector under the control of regulatory sequences of the myeloproliferative sarcoma virus long terminal repeat. Transplantation of H-2Kb retrovirus-transduced autologous bone marrow into B10.AKM (Kk Ik Dq) recipients resulted in detectable levels of H-2Kb RNA and cell surface protein in lymphoid and myeloid lineages. H-2Kb expression was significant, although variable, in bone marrow Mac1+ cells, in splenic B cells, and in CD4+/CD8+ thymocytes 8 wk post-bone marrow transplantation. The recipients of the H-2Kb-transduced bone marrow showed specifically delayed rejection of B10.MBR (Kb Ik Dq) skin grafts disparate only for Kb. However, B10.MBR skin grafts were rapidly rejected when grafted simultaneously with skin grafts from B10 (Kb Ib Db) mice, a strain bearing additional third party alloantigens in association with Kb. Our experiments suggest that the hyporesponsive state induced by using the retrovirally mediated gene transfer model is characterized by the persistence of H-2Kb-specific cytolytic T cell precursors, which may be inactive because of deficient T cell help. Tolerance to Kb induced by this approach may therefore be restricted to T helper cell lineages.

3T3 Cells↗
PubMed1995

Sensitivity of nucleus accumbens neurons in vivo to intoxicating doses of ethanol.

The nucleus accumbens septi (NAcc) is considered an important component of the final common pathway involved in the reinforcing properties of ethanol. We studied the effects of intraperitoneal administration of ethanol on spontaneous, glutamate-activated, and fimbria-activated NAcc neurons in acute anesthetized and freely moving unanesthetized rats. Ethanol significantly reduced the firing rate of spontaneous and glutamate-activated NAcc neurons in both electrophysiological preparations. Stimulation of the ipsilateral fimbria evoked single-unit activity in NAcc neurons with two characteristic latencies (early, 7.21 +/- 0.74 msec; late, 18.24 +/- 0.66 msec). Intoxicating doses of ethanol inhibited the recruitment of late, but not of early, fimbria-activated NAcc neurons. These data demonstrate electrophysiological evidence for the existence of neurons in the core region of the NAcc that are sensitive and insensitive to acute systemic ethanol administration.

Alcoholic Intoxication↗
PubMed1995

Stability: an ICD detection criterion for discriminating atrial fibrillation from ventricular tachycardia.

INTRODUCTION: The purpose of this study was to review a new implantable cardioverter defibrillator (ICD) detection criterion, stability, to determine if it can effectively discriminate rapid rhythms of atrial fibrillation from ventricular tachycardia. Inappropriate shocks for rapid atrial fibrillation limit the acceptance of ICDs. The advent of an additional detection criterion, stability, has been postulated to be of value in discriminating rapid atrial fibrillation, which may not warrant treatment, from ventricular tachycardia, which obviously does warrant therapy deliver. METHODS AND RESULTS: Twenty-six patients were studied during 32 episodes of rapid atrial fibrillation and 24 episodes of monomorphic ventricular tachycardia below 220 beats/min. Each rhythm was repeatedly evaluated by the device at each of the seven stability values available (8, 16, 23, 31, 39, 47, and 55 msec) and then classified as stable or unstable. Upon completion of this acute study, 32 ICD patients had the stability feature activated and were followed for proper arrhythmia treatment by the device. Using stability windows from 8 to 47 msec, all atrial fibrillation rhythms were appropriately classified as unstable. Three of 6 were classified correctly for the 55-msec window. All ventricular tachycardia rhythms were appropriately classified as stable from all stability windows from 8 to 55 msec. Clinical follow-up confirmed appropriate therapy delivery when coupled with sustained rate duration (SRD). Thirty-two patients followed for 292 patient-months had no episodes of untreated ventricular tachycardia with 428 successfully classified as stable and treated. Only three episodes of suspected atrial fibrillation resulted in therapy delivery as the rhythm duration exceeded the SRD of 30 seconds. CONCLUSIONS: The CPI Ventak PRx ICD is highly reliable in appropriately classifying atrial fibrillation as unstable and monomorphic ventricular tachycardia as stable for most stability windows evaluation tachycardias below 220 beats/min. As a result, when testing of atrial fibrillation is not possible, we recommend the routing programming of this stability feature at the 31-msec window with an SRD of 30 seconds. The reliability of this device in discriminating atrial fibrillation from monomorphic ventricular tachycardia may have important clinical implications for other tiered therapy ICDs with this feature as well as for future ICDs in development.

Adult↗
PubMed1994

Indirect recognition by helper cells can induce donor-specific cytotoxic T lymphocytes in vivo.

In vitro studies have revealed that help for cytotoxic T lymphocyte (CTL) induction can be mediated through several pathways, including direct recognition of allogenic class II antigens by CD4+ cells, direct recognition of allogeneic class I antigens by "CD4-independent" CD8+ cells, and "indirect" recognition of peptides of alloantigens presented in association with self class II molecules. Whereas good evidence for the two direct pathways is available in vivo, there is relatively little evidence to show that indirect recognition can initiate graft rejection. This study examined the role of indirect allorecognition during the generation of CTLs in mice as they rejected major histocompatibility complex (MHC) class II-deficient skin after depletion of CD8+ T cells in vivo. Recipients were depleted of CD8+ T cells by in vivo treatment with anti-CD8 monoclonal antibody and then grafted with allogeneic skin lacking MHC class II antigens. The mice rejected the skin grafts rapidly. Although flow cytometry showed marked depletion of CD8+ T cells in these mice, we found that (a) CD8+ CTLs were generated and sensitized to MHC class I antigens of the donor; (b) the generation of the CD8+ CTLs required the help in vivo of CD4+ cells, as well as priming with the allogeneic skin graft; and (c) the CD4+ T helper cells were sensitized indirectly to donor peptides presented in association with class II antigens on recipient antigen-presenting cells. These results provide evidence that indirect recognition can provide effective help for CTL induction during graft rejection, even when the cytotoxic T cells are sensitized by determinants expressed only on the donor graft.

Animals↗
PubMed1994

Anti-cytokines: promising tools for diagnosis and immunotherapy.

Anti-cytokines belong to a new family of biological response modifiers which interfere with the biological functions of cytokines. An imbalance between cytokines and natural anti-cytokines may represent an important factor to explain the pathogenesis of some human diseases. Indeed, dysregulation of anti-cytokine serum levels was observed in cancers and auto-immune and infectious diseases. It was sometimes associated with disease activity and in some circumstances they may help to predict clinical response to treatment. Control of disease activity in septic shock and auto-immune diseases was demonstrated after administration of anti-cytokines such as soluble TNF receptors and IL1ra inhibitors. Therefore these agents are promising tools for diagnosis and immunotherapy.

Autoimmune Diseases↗
PubMed1994

Electrophysiological and pharmacological evidence for the role of the nucleus accumbens in cocaine self-administration in freely moving rats.

The goal of this study was to clarify the role of the nucleus accumbens septi (NAS) in the expression of cocaine self-administration behaviors. Rats were trained in a continuous reinforcement schedule to press a lever to activate a pump that provided an intravenous injection of cocaine. Once the rats were trained, neuronal activity in the NAS was monitored during cocaine self-administration with chronic recording techniques using permanently implanted microwires. In the NAS, 19% of 181 neurons exhibited either increased or decreased firing rates seconds prior to lever pressing (termed "anticipatory responses"), and 48% had altered, predominantly decreased, firing rates for a few minutes after lever pressing ("postcocaine responses"). Two-thirds of the neurons with anticipatory responses had postcocaine responses. Neurons with either of these response patterns were localized histologically to both core and shell regions of the NAS, with no statistically significant differences in the proportion of response types in either area. Analysis of videotaped cocaine self-administration behaviors revealed that anticipatory responses were specifically associated either with the animal orienting toward and pressing the lever or only with movements directly related to pressing the lever. Anticipatory-like phasic spike activity was not observed during similar movements unrelated to lever pressing. In some animals, D1 (SCH 23390) or D2 (pimozide) receptor antagonists were injected systemically prior to or during self-administration sessions to assess the effects of dopamine receptor blockade on anticipatory and postcocaine responses. Each antagonist, given separately, often produced extinction of lever pressing. Both antagonists blocked the post-cocaine inhibitory response of neurons that had anticipatory responses. Neither antagonist modified anticipatory unit responses, nor did they affect postcocaine inhibitory responses in neurons that did not exhibit anticipatory responses. Taken together, these results suggest that the role of the NAS in cocaine self-administration may consist of two different mechanisms: (1) An initiation or trigger mechanism, as represented by the anticipatory neuronal responses, in which the NAS participates in triggering or mediating the goal-directed behaviors (e.g., lever pressing) that lead to the acquisition of the reinforcing agent (e.g., cocaine).(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1993

Specific placement of tryptophan in the catalytic sites of Escherichia coli F1-ATPase provides a direct probe of nucleotide binding: maximal ATP hydrolysis occurs with three sites occupied.

Residue beta Y331 of Escherichia coli F1-ATPase is known from previous affinity labeling, mutagenesis, and lin-benzo-ADP binding experiments to interact directly with the adenine moiety of substrates bound in catalytic sites. Here we mutagenized beta Y331 to tryptophan. Mutant cells grew well on succinate or limiting glucose; purified mutant F1 had kappa cat/Km and lin-benzo-ADP binding characteristics similar to wild type. Fluorescence from beta W331 residues exhibited a maximum at 349 nm, indicating a polar environment in unoccupied sites. ATP, ADP, or AMPPNP caused virtually complete quenching of beta W331 fluorescence, so that the fluorescence of mutant F1 with occupied catalytic sites resembled that of wild-type enzyme. Therefore the beta W331 fluorescence provided a direct probe of nucleotide binding to catalytic sites under true equilibrium conditions. We measured ATP binding and hydrolysis in parallel experiments and found that occupancy of one or two catalytic sites per F1 molecule did not yield significant rates of hydrolysis while occupancy of all three sites yielded Vmax rates. Km(ATP) was similar to Kd3, the Kd for ATP binding to the third catalytic site. We also measured AMPPNP and ADP binding parameters. For ADP, the "on" rate at the first catalytic site was much faster (> or = 5 x 10(5) M-1 s-1) than seen previously by centrifuge column procedures, although the Kd was not much changed. For AMPPNP, the "on" rate at the first site was 2 orders of magnitude less than for ADP or ATP, and the Kd was similar to that for ADP.

Adenosine Diphosphate↗
PubMed1993

The cysteine introduced into the alpha subunit of the Escherichia coli F1-ATPase by the mutation alpha R376C is near the alpha-beta subunit interface and close to a noncatalytic nucleotide binding site.

Mutation of the alpha subunit of the Escherichia coli F1-ATPase to convert Arg-376 to a Cys (alpha R376C) lowers multisite ATPase activity 400-1,000-fold while affecting unisite catalysis only around 6-fold, suggesting that the mutation is in a region important for transmission of conformational changes between catalytic sites (Soga, S., Noumi, T., Takeyama, M., Maeda, M., and Futai, M. (1989) Arch. Biochem. Biophys. 268, 643-648; this study). To learn more of the structural features of the segment of the alpha subunit around Arg-376, mutant enzyme with a Cys at this position was modified with several maleimides. N-[14C]Ethylmaleimide reacted rapidly with this Cys in one of the three alpha subunits/F1 (2,500 M-1 s-1); more slowly with a second alpha subunit (390 M-1 s-1); and the same Cys in the third copy of the alpha subunit was completely unreactive to the reagent, indicating asymmetry of alpha subunits in the ECF1 complex. The photoactivatable cross-linker N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide++ +, when reacted via its maleimide to alpha Cys-376 of the mutant, covalently linked alpha to beta subunits upon photolysis, indicating that Cys-376 of alpha is close to an interface between the alpha and beta subunits. The EDTA-induced exchangeable noncatalytic site could be filled by TNP-ATP in both wild type and alpha R376C mutant ECF1. Occupancy of this site in the alpha R376C mutant altered the rate of reaction of the second-fastest reacting Cys-376 from 390 M-1 s-1 to below 130 M-1 s-1, suggesting that the two sites are on the same alpha subunit. TNP-ATP in the EDTA-induced exchangeable noncatalytic site was quenched by reacting Cys-376 with 4-maleimido-(2,2,6,6-tetramethylpiperidine-N(oxyl), indicating that the region around Cys-376, which is involved in transmission of conformational changes between alpha and beta, and noncatalytic sites are maximally 10-12 A from each other.

Adenosine Triphosphate↗
PubMed1993

Combined application of site-directed mutagenesis, 2-azido-ATP labeling, and lin-benzo-ATP binding to study the noncatalytic sites of Escherichia coli F1-ATPase.

Noncatalytic nucleotide sites of Escherichia coli F1-ATPase were studied by site-directed mutagenesis, covalent photolabeling with 2-azido-ATP, and lin-benzo-ATP binding. In wild-type, 89% of 2-azido-ATP label was bound to beta-subunit, whereas in the beta Y354F mutant, 95% of the label was bound to alpha-subunit. In the alpha R365Y mutant, label was seen on both alpha (38%) and beta (62%); whereas in the alpha R365F mutant, 93% was on beta. The fluorescence of noncatalytic site-bound lin-benzo-ATP was quenched markedly in F1 from wild-type (76% quench), alpha R365F (85%), alpha R365Y (90%), and alpha R365Y, beta Y354F (83%), but only by 28% in beta Y354F. These results together demonstrate that residues alpha R365 and beta Y354 lie close to the base moiety of adenine nucleotide bound in F1 noncatalytic sites. From comparison of sequences of alpha- and beta-subunits, it appears that residue alpha R365 in noncatalytic sites is equivalent to residue beta Y331 of the catalytic sites. Two unintended mutants were obtained in which alpha-subunit was increased in length by 17 amino acids due to repeat of residues alpha 361 to alpha 377, with either F or Y in the repeated alpha 365 position. Soluble F1 was obtained from both mutants, with novel properties.

Adenosine Triphosphate↗
PubMed1992

Investigation of the aurovertin binding site of Escherichia coli F1-ATPase by fluorescence spectroscopy and site-directed mutagenesis.

(1) Previous mutational analyses have shown that residue beta R398 of the beta-subunit is a key residue for binding of the inhibitory antibiotic aurovertin to Escherichia coli F1Fo-ATP synthase. Here, we studied purified F1 from the beta R398C and beta R398W mutants. ATPase activity in both cases was resistant to aurovertin inhibition. The fluorescence spectrum (lambda exc = 278 or 295 nm) of beta R398W F1 showed a significant red-shift as compared to wild-type and beta R398C enzymes, indicating that residue beta R398 lies in a polar environment. On the basis of this and previous evidence, we propose that aurovertin binding to F1-ATPase involves a specific charged donor-acceptor H-bond between residue beta R398 and the 7-hydroxyl group of aurovertin. (2) The fluorescent substrate analog lin-benzo-ADP was shown to bind to beta R398W F1 catalytic sites with the same Kd values as to wild-type F1, and with the same quenching of the fluorescence of the analog. Fluorescence energy transfer was seen between the beta R398W residue and bound lin-benzo-ADP. Analysis of transfer efficiency at varying stoichiometry of bound lin-benzo-ADP showed that interaction occurred between one beta R398W residue and one catalytic-site-bound analog molecule at a distance of approximately 23 A. The relationships of the aurovertin and catalytic sites in the primary and tertiary structure are discussed.

Adenosine Diphosphate↗