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Objectives: Emergency Medicine (EM) provider experiences consulting telestroke (TS) are poorly studied. In this qualitative study, we aimed to determine how TS changes patient management and to measure TS effects on EM provider confidence with acute ischemic stroke (AIS) treatment., Materials and Methods: We designed a survey for EM providers querying perceptions of TS value, confidence with treating AIS, and counterfactuals regarding what EM providers would have done without TS. Eligible EM providers participated in an audio-visual TS consult within a 6-state TS network between 11/2016-11/2017., Results: We received 48 surveys (response rate 43%). The most common reason (71%) for using TS was tPA eligibility expert opinion. Most EM providers (94%) thought the patient/family were satisfied with TS and none felt their medical knowledge was doubted because of using TS. EM providers had high confidence in diagnosing AIS (95%) and tPA decision-making (86%), but not in determining thrombectomy eligibility (10%). Among EM providers who administered tPA, 85% said tPA would not have been given without TS consultation. TS consultation changed patient diagnosis in 60% of all patients and treatment plans in 56% of non-stroke patients. Most EM providers (86%) had increased confidence in their knowledge of future stroke patient management. Nearly all TS consults (93%) resulted in EM providers being more likely to use TS again., Conclusions: TS consult frequently results in both patient management change and increased EM knowledge of stroke management with increased likelihood of repeat usage. Discomfort in determining eligibility for thrombectomy points to educational opportunities., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Endothelial diaphragms are subcellular structures critical for mammalian survival with poorly understood biogenesis. Plasmalemma vesicle associated protein (PLVAP) is the only known diaphragm component and is necessary for diaphragm formation. Very little is known about PLVAP regulation. Phorbol esters (PMA) are known to induce de novo PLVAP expression and diaphragm formation. We show that this induction relies on the de novo production of soluble factors that will act in an autocrine manner to induce PLVAP transcription and protein expression. We identified vascular endothelial growth factor-A (VEGF-A) signalling through VEGFR2 as a necessary but not sufficient downstream event as VEGF-A inhibition with antibodies and siRNA or pharmacological inhibition of VEGFR2 only partially inhibit PLVAP upregulation. In terms of downstream pathways, inhibition of MEK1/Erk1/2 MAP kinase blocked PLVAP upregulation, whereas inhibition of p38 and JNK MAP kinases or PI3K and Akt had no effect on PMA-induced PLVAP expression. In conclusion, we show that VEGF-A along with other secreted proteins act synergistically to up-regulate PLVAP in MEK1/Erk1/2 dependent manner, bringing us one step further into understanding the genesis of the essential structures that are endothelial diaphragms., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The current research examined whether carriers of the short 5-HTTLPR allele (in SLC6A4), who have been shown to selectively attend to negative information, exhibit a bias towards negative self-referent processing. The self-referent encoding task (SRET) was used to measure self-referential processing of positive and negative adjectives. Ratcliff's diffusion model isolated and extracted decision-making components from SRET responses and reaction times. Across the initial (N = 183) and replication (N = 137) studies, results indicated that short 5-HTTLPR allele carriers more easily categorized negative adjectives as self-referential (i.e., higher drift rate). Further, drift rate was associated with recall of negative self-referential stimuli. Findings across both studies provide further evidence that genetic variation may contribute to the etiology of negatively biased processing of self-referent information. Large scale studies examining the genetic contributions to negative self-referent processing may be warranted., Competing Interests: The authors have declared that no competing interests exist.

Rituximab (RTX) has been the hallmark anti-CD20 mAb for the treatment of B cell neoplasms, including B cell chronic lymphocytic leukemia (B-CLL). Recently, a novel humanized anti-CD20 mAb obinutuzumab (GA101) has been implemented as first-line CLL therapy. Treatment of CLL patients with RTX is associated with CD20 loss via an FcγR-mediated process, trogocytosis. RTX-induced trogocytosis has been characterized as both the means of resistance to therapy, via loss of cell surface target proteins (antigenic modulation), as well as a process that alters B cell phenotype and function. This study investigates the nature and clinical relevance of GA101-mediated trogocytosis. In this study, we demonstrate that GA101 is a more potent mediator of trogocytosis than RTX in vitro in both normal B cells and B-CLL cells. Qualitative differences in the effector function of these anti-CD20 Abs appear specific to B-CLL cells. GA101-mediated CD19 and CD20 trogocytosis from B-CLL cells is associated with its ability to induce homotypic adhesion (HA). The degree of HA varies between CLL patients and positively correlates with the expression of ZAP-70, a BCR-associated kinase. Deregulation of ZAP-70 using tyrosine kinase inhibitors, gefitinib or ibrutinib, diminishes HA formation and trogocytosis by GA101. Taken together, these findings elucidate the differences in trogocytosis and HA formation mediated by anti-CD20 mAbs RTX and GA101, as well as provide a novel link between ZAP-70 expression and these effector functions., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Objective: Anti-carbamylated protein (anti-CarP) antibodies are associated with the risk and severity of rheumatoid arthritis (RA) and are primarily directed against fibrinogen. The lack of understanding of anti-CarP antibody reactivity has limited analysis of the immunopathogenic associations in RA. To address this shortcoming, we mapped anti-CarP antibody epitope reactivity in RA patient sera., Methods: Immunoblotting identified a patient serum sample with specific reactivity to the carbamylated human fibrinogen β-chain. Liquid chromatography mass spectrometry (LC-MS) identified sites of homocitrullines (carbamylated lysines) present in the human fibrinogen β-chain. The reactivity of an anti-CarP antibody-positive cohort to specific peptides containing carbamylated lysines was determined by enzyme-linked immunosorbent assay, through direct binding (n = 63 sera) and by competition assays (n = 40 sera)., Results: Serum with specific reactivity to carbamylated, but not citrullinated, fibrinogen β-chain was identified in a specimen obtained from an RA patient. LC-MS identified carbamylation of 9 of 34 lysines in the human fibrinogen β-chain. Mapping of immunoreactivity to tryptic peptide fragments demonstrated several candidate carbamylated epitopes that were confirmed by competition experiments. Peptides containing a homocitrulline at position 83 appeared to be an immunodominant epitope in some RA patient sera, with additional reactivity to peptides containing homocitrullines at positions 52, 264, 351, 367, and 374., Conclusion: Anti-CarP antibodies appear to preferentially target specific regions of the human fibrinogen β-chain that contain homocitrullines. Interestingly, humoral immunoreactivity appears to be relatively restricted in some patients, which may enable detection of specific relationships with disease phenotype., (© 2017, American College of Rheumatology.)

While respiratory failure in cystic fibrosis (CF) frequently associates with chronic infection by Pseudomonas aeruginosa , no single factor predicts the extent of lung damage in CF. To elucidate other causes, we studied the autoantibody profile in CF and rheumatoid arthritis (RA) patients, given the similar association of airway inflammation and autoimmunity in RA. Even though we observed that bactericidal permeability-increasing protein (BPI), carbamylated proteins, and citrullinated proteins all localized to the neutrophil extracellular traps (NETs), which are implicated in the development of autoimmunity, our study demonstrates striking autoantibody specificity in CF. Particularly, CF patients developed anti-BPI autoantibodies but hardly any anti-citrullinated protein autoantibodies (ACPA). In contrast, ACPA-positive RA patients exhibited no reactivity with BPI. Interestingly, anti-carbamylated protein autoantibodies (ACarPA) were found in both cohorts but did not cross-react with BPI. Contrary to ACPA and ACarPA, anti-BPI autoantibodies recognized the BPI C-terminus in the absence of posttranslational modifications. In fact, we discovered that P . aeruginosa -mediated NET formation results in BPI cleavage by P . aeruginosa elastase, which suggests a novel mechanism in the development of autoimmunity to BPI. In accordance with this model, autoantibodies associated with presence of P . aeruginosa on sputum culture. Finally, our results provide a role for autoimmunity in CF disease severity, as autoantibody levels associate with diminished lung function.

Objective: The presence of anticitrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) indicates a breach in immune tolerance. Recent studies indicate that this breach extends to homocitrullination of lysines with the formation of anti-carbamylated protein (anti-CarP) antibodies. We analyzed the clinical and serologic relationships of anti-CarP in 2 RA cohorts., Methods: Circulating levels of immunoglobulin G anti-CarP antibodies were determined by ELISA in established (Dartmouth-Hitchcock Medical Center) and early (Sherbrooke University Hospital Center) cohorts and evaluated for anticyclic citrullinated peptide antibodies (anti-CCP), specific ACPA, and rheumatoid factor (RF) levels using the Student t test and correlation analysis., Results: We identified elevated anti-CarP antibodies titers in 47.0% of seropositive patients (Dartmouth, n = 164), with relationships to anti-CCP (p < 0.0001) and IgM-RF (p = 0.001). Similarly, 38.2% of seropositive patients from the Sherbrooke cohort (n = 171) had elevated anti-CarP antibodies; titers correlated to anti-CCP (p = 0.01) but not IgM-RF (p = 0.09). A strong correlation with anti-Sa was observed: 47.9% anti-Sa+ patients were anti-CarP antibodies+ versus only 25.4% anti-Sa- in the Sherbrooke cohort (p = 0.0002), and 62.6% anti-Sa+ patients versus 26.9% anti-Sa- were anti-CarP antibodies+ in Dartmouth (p < 0.0001). We found a more variable response for reactivity to citrullinated fibrinogen or to citrullinated peptides from fibrinogen and α enolase., Conclusion: In 2 North American RA cohorts, we observed a high prevalence of anti-CarP antibody positivity. We also describe a surprising and unexpected association of anti-CarP with anti-Sa antibodies that could not be explained by cross-reactivity. Further, considerable heterogeneity exists between anti-CarP reactivity and other citrullinated peptide reactivity, raising the question of how the pathogenesis of antibody responses for carbamylated proteins and citrullinated proteins may be linked in vivo.

Objective: Rituximab (RTX), an anti-CD20 monoclonal antibody, is highly effective in the treatment of several autoimmune diseases. The mechanism by which RTX treatment improves rheumatoid arthritis and antineutrophil cytoplasmic antibody-associated vasculitis is not easily related to B cell depletion alone. Prior studies have shown that RTX mediates a rapid stripping of CD20 and CD19 from the human B cell through a process known as trogocytosis. The aim of the present study was to investigate whether changes in B cell phenotype resulting from trogocytosis would diminish the ability of B cells to promote autoimmune disease., Methods: Human peripheral blood mononuclear cells were cultured with RTX under conditions that permitted trogocytosis. Changes in B cell phenotype and cytokine production were measured in the basal state and under conditions of activation with interleukin-4 (IL-4)/anti-CD40. The effects of RTX were characterized in terms of a requirement for interaction with the Fcγ receptor (FcγR) and other Fc-dependent interactions., Results: Trogocytosis induced a marked loss of surface CD19, IgD, CD40, and B cell-activating factor receptor, but did not alter induction of CD86 expression on purified B cells following IL-4/anti-CD40 treatment. Unexpectedly, RTX-dependent trogocytosis of normal human B cells in vitro led to a rapid up-regulation of IL-6 production, with no effect on the production of tumor necrosis factor α, IL-1β, interferon-γ, or IL-10. This effect was Fc-dependent and required the presence of an FcγR-bearing cell. Moreover, this effect involved the release of preformed intracellular IL-6 protein, as well as marked increases in IL-6 messenger RNA levels., Conclusion: RTX-mediated trogocytosis of B cells in vitro results in acute production and release of IL-6. The nature of this effect and how it is related to the acute infusion reactions seen with RTX administration remain to be determined., (Copyright © 2014 by the American College of Rheumatology.)

Objectives: The study's purpose was to examine the impact of somatic symptoms and perceived neighborhood environment on depression using a comparison among US-born and non-US-born free clinic patients., Methods: US-born English speakers (n = 99), non-US-born English speakers (n = 89), and non-US-born Spanish speakers (n = 158) 18 years old or older (N = 346) were surveyed at a free clinic that provides primary care to people without health insurance. Depression, somatic symptoms, and perceived neighborhood environment were measured using standardized instruments., Results: US-born English speakers reported higher levels of depression and a greater number of somatic symptoms than non-US-born Spanish speakers and non-US-born English speakers. Non-US born English speakers reported lower levels of depression and fewer somatic symptoms than Spanish speakers. Somatic symptoms and perceived neighborhood satisfaction were related to depression., Conclusions: Developing mental health services for patients in a free clinic setting is needed; however, because of limited financial and human resources, providing mental health services in a free clinic setting often is difficult. Community-based health promotion programs as supplements to efforts within clinical settings would be valuable in improving the mental health of free clinic patients. Future studies should implement collaborative pilot programs and evaluate health outcomes.

Free clinics provide free or reduced fee healthcare to individuals who lack access to primary care and are socio-economically disadvantaged. The purpose of this study is to examine health-related quality of life (HRQoL) among free clinic patients and its association with somatic symptoms, depression, and perceived neighborhood environment. Free clinic patients (n = 186) aged 18 years or older completed a self-administered survey. HRQoL, depression, somatic symptoms, and perceived neighborhood environment were measured using standardized instruments. Overall, the participants reported low level of HRQoL compared to the general healthy population. US born participants (n = 97) reported poorer psychological QoL and social relations, more somatic symptoms, and were more likely to be depressed than non-US born participants (n = 89). Higher numbers of somatic symptoms were associated with poorer environmental QoL. Depression was associated with all aspects of QoL; a higher level of depression was related to poorer QoL in all aspects. Our findings show that free clinic patients, especially US born patients, have poor HRQoL. Depression and perceived neighborhood satisfaction are key factors to determine HRQoL among free clinic patients. Mental health services and collaboration with other community organizations may help in improving HRQoL among free clinic patients. Finally, health promotion programs at the community level, not just at the clinic level, would be valuable to improve health of free clinic patients as perceived neighborhood environment is associated with their HRQoL.

Introduction: We hypothesized that serum levels of C-X-C motif chemokine 13 (CXCL13), a B-cell chemokine, would delineate a subset of rheumatoid arthritis (RA) patients characterized by increased humoral immunity., Methods: Serum from patients with established RA (the Dartmouth RA Cohort) was analyzed for CXCL13, rheumatoid factor (RF) levels, anticitrullinated peptide/protein antibody (ACPA) and total immunoglobulin G (IgG); other parameters were obtained by chart review. A confirmatory analysis was performed using samples from the Sherbrooke Early Undifferentiated PolyArthritis (EUPA) Cohort. The Wilcoxon rank-sum test, a t-test and Spearman's correlation analysis were utilized to determine relationships between variables., Results: In both the Dartmouth and Sherbrooke cohorts, CXCL13 levels were selectively increased in seropositive relative to seronegative RA patients (P = 0.0002 and P < 0.0001 for the respective cohorts), with a strong correlation to both immunoglobulin M (IgM) and IgA RF levels (P < 0.0001). There was a weaker relationship to ACPA titers (P = 0.03 and P = 0.006, respectively) and total IgG (P = 0.02 and P = 0.14, respectively). No relationship was seen with regard to age, sex, shared epitope status or inclusion high-sensitivity C-reactive protein (hsCRP) in either cohort or regarding the presence of baseline erosions in the Sherbrooke Cohort, whereas a modest relationship with Disease Activity Score in 28 joints CRP (DAS28-CRP) was seen in the Dartmouth cohort but not the Sherbrooke cohort., Conclusion: Using both established and early RA cohorts, marked elevations of serum CXCL13 levels resided nearly completely within the seropositive population. CXCL13 levels exhibited a strong relationship with RF, whereas the association with clinical parameters (age, sex, DAS28-CRP and erosions) or other serologic markers (ACPA and IgG) was either much weaker or absent. Elevated serum CXCL13 levels may identify a subset of seropositive RA patients whose disease is shaped by or responsive to RF production.

Objective: To evaluate loss of the B cell-specific marker CD19 after the addition of rituximab (RTX) to healthy donor blood and to determine the role of complement-mediated cytotoxicity in these cells., Methods: Whole blood and peripheral blood mononuclear cells (PBMCs) from healthy donors were evaluated for the loss of CD19 in the presence of RTX using flow cytometry. The effect of complement on CD19 loss was examined using serum-free media, C3- and C5-deficient sera, and a C5-blocking antibody. Evidence of B cell death was evaluated by measuring messenger RNA (mRNA) levels as well as by flow cytometry. Transfer of CD19 antigen to monocytes and neutrophils was evaluated by flow cytometry and confocal microscopy., Results: RTX induced a rapid decrease in CD19 count (mean 51%; n = 37) in PBMCs. This reduction occurred in the absence of complement. Despite the decrease in CD19 expression, B cell death did not occur, as evidenced by a lack of change in CD19 or CD20 mRNA levels and a lack of change in CD19 levels determined by intracellular staining and through the use of viability dyes. The CD19 antigen was shown to be transferred to monocytes and neutrophils in an Fc-dependent manner., Conclusion: Our findings indicate that the addition of RTX to healthy donor PBMCs in vitro results in complement-independent loss of CD19 without causing B cell death. CD19 is transferred from B cells to monocytes and neutrophils during shaving of the RTX-CD20 complex in an Fc-dependent manner. These data suggest that monitoring the effect of RTX by measuring the CD19+ cell count may be compromised by this activity., (Copyright © 2012 by the American College of Rheumatology.)

The study of post-transcriptional regulation is constrained by the technical limitations associated with both transient and stable transfection of chimeric reporter plasmids examining the activity of 3'-UTR cis-acting elements. We report the adaptation of a commercially available system that enables consistent stable integration of chimeric reporter cDNA into a single genomic site in which transcription is induced by tetracycline. Using this system, we demonstrate the tight control afforded by this system and its suitability in mapping the regulatory function of defined cis-acting elements in the human TNF 3'-UTR, as well as the distinct effects of serum starvation on transiently transfected and stably integrated chimeric reporter genes.

We report a role for CA repeats in the 3'-untranslated region (3'-UTR) in regulating CD154 expression. Human CD154 is encoded by an unstable mRNA; this instability is conferred in cis by a portion of its 3'-UTR that includes a polypyrimidine-rich region and CA dinucleotide repeat. We demonstrate similar instability activity with the murine CD154 3'-UTR. This instability element mapped solely to a conserved 100-base CU-rich region alone, which we call a CU-rich response element. Surprisingly, the CA dinucleotide-rich region also regulated reporter expression but at the level of translation. This activity was associated with poly(A) tail shortening and regulated by heterogeneous nuclear ribonucleoprotein L levels. We conclude that the CD154 3'-UTR contains dual cis-acting elements, one of which defines a novel function for exonic CA dinucleotide repeats. These findings suggest a mechanism for the association of 3'-UTR CA-rich response element polymorphisms with CD154 overexpression and the subsequent risk of autoimmune disease.

Glucose transporter-1 (GLUT1) mediates uptake of glucose and is up-regulated in some cancers. The amount of this membrane protein is regulated by a post-transcriptional mechanism in which mRNA binding proteins recognize cis-acting elements in the 3'-untranslated (3'UTR) of the mRNA. To identify cis elements in GLUT1 mRNA we introduced 3'UTR sequences into the 3'UTR of the luciferase gene in a reporter construct. A 30 nt adenosine-uridine-rich element ("GLUT1 AURE") inhibited luciferase activity in HEK-293 cells. This inhibitory effect was confirmed by deleting the GLUT1 AURE from a reporter containing the full-length 3'UTR. Deletion of the GLUT1 AURE caused reporter activity to increase. Deletion of a larger fragment ("Bsu" region) containing the GLUT1 AURE increased reporter activity still further, suggesting that there are additional cis elements in the GLUT1 mRNA. The GLUT1 AURE was also active in GBM-T98G glioblastoma cells. Next, we tested the action of a trans-acting factor, hnRNP A2, on GLUT1 gene expression. We show that a cytoplasmic-localizing isoform of hnRNP A2 binds human GLUT1 RNA by gel-shift assay and by UV-crosslinking. Finally, over-expression of the hnRNP A2 isoform inhibited GLUT1 reporter expression in GBM-T98G cells. These results identify the AURE cis element in human GLUT1 mRNA and show that hnRNP A2 acts on GLUT1 mRNA to inhibit expression of GLUT1 in a brain cancer cell line.

The expression of CD154 (CD40 ligand) by activated T lymphocytes plays a central role in humoral and cellular immunity. The fundamental importance of this protein in mounting an immune response has made it an attractive target for immunomodulation. Several studies have demonstrated that CD154 expression is regulated at the level of mRNA turnover in a manner distinct from other cytokine genes. We have purified, sequenced, and characterized the two major proteins that bind the CD154 3' untranslated region (3'UTR) as members of the polypyrimidine tract binding protein (PTB) family. One of these proteins is a previously unreported alternatively spliced PTB isoform, which we call PTB-T. These proteins interact with a polypyrimidine-rich region within the CD154 3'UTR that lacks any known cis-acting instability elements. The polypyrimidine-rich region of the CD154 3'UTR was both necessary and sufficient to mediate changes in reporter gene expression and mRNA accumulation, indicating the presence of a novel cis-acting instability element. The presence of a cis-acting instability element in the polypyrimidine-rich region was confirmed using a tetracycline-responsive reporter gene approach. The function of this cis-acting element appears to be dependent on the relative cytoplasmic levels of PTB and PTB-T. Cotransfection of vectors encoding PTB-T consistently decreased the CD154 3'UTR-dependent luciferase expression. In contrast, transfection of plasmids encoding PTB tended to increase CD154 3'UTR-dependent luciferase expression. Thus, the CD154 3'UTR contains a novel cis-acting element whose function is determined by the binding of PTB and PTB-T. These data identify a specific pathway that regulates CD154 expression that can potentially be selectively targeted for the treatment of autoimmune disease and allograft rejection.

Post-transcriptional pathways provide a major means of regulating eukaryotic gene expression. Reiterations of the AU-rich element (ARE) within the 3'-untranslated region of many cytokine and proto-oncogene mRNAs serve as signals for rapid degradation and translational repression. The identification of this cis-acting stability determinant has fueled the search for ARE-binding proteins (AUBP) that function as trans-acting factors that transduce this function. Previous work identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as a major AUBP capable of binding the ARE of granulocyte-macrophage colony stimulating factor (GM-CSF) RNA in the context of a full-length mRNA. We report here that functional studies failed to indicate a role for hnRNP A1 in ARE-dependent mRNA turnover. In an effort to identify other functionally relevant AUBP, the major GM-CSF ARE-specific binding protein in cells lacking hnRNP A1 was purified from CB3 mouse erythroleukemia cells. Microsequencing identified this protein as the glycolytic enzyme lactate dehydrogenase (LDH) M. RNA binding by LDH was shown to occur in the NAD(+)-binding region (Rossmann fold). Polysome gradient analysis demonstrates that LDH is found in the translationally active fraction. Polysomal localization of LDH was dependent on RNA binding. Moreover, polysomal LDH exists in a complex with AUF1 and hsp-70, which has been implicated previously in the regulation of mRNA turnover. The interaction between LDH and AUF1 is direct as it can be demonstrated in vitro with purified proteins. Collectively these data implicate a role for LDH in the post-transcriptional regulation of gene expression.

Background: This meta-analysis was performed to determine the degree to which improvements in open hernia repair (OHR) in the last decade have altered the relative benefit of laparoscopic hernia repair (LHR)., Methods: Twenty-seven comparative trials including 4,688 randomized patients were evaluated., Results: Within the control OHR, patients with routine mesh repair returned to work earlier than a sutured repair (16.4 versus 27.3 days, P = 0.010). During the study period, the increased use of mesh in OHR (3 of 12 initially versus 9 of 15 subsequent studies) was associated with an earlier return to work (25.9 to 16.8 days, P = 0.017); there was no significant improvement with corresponding LHR., Conclusions: Although LHR was associated with an earlier return to work compared with conventional sutured OHR, more recent mesh OHRs provide equivalent outcomes but at lower costs and potentially less severe complications, supporting an open technique using preperitoneal mesh prostheses as the optimal hernia repair.

The heterogeneous nuclear ribonucleoproteins (hnRNP) associate with pre-mRNA in the nucleus and play an important role in RNA processing and splice site selection. In addition, hnRNP A proteins function in the export of mRNA to the cytoplasm. Although the hnRNP A proteins are predominantly nuclear, hnRNP A1 shuttles rapidly between the nucleus and the cytoplasm. HnRNP A2, whose cytoplasmic overexpression has been identified as an early biomarker of lung cancer, has been less well studied. Cytosolic hnRNP A2 overexpression has also been noted in brain tumors, in which it has been correlated with translational repression of Glucose Transporter-1 expression. We now examine the role of arginine methylation on the nucleocytoplasmic localization of hnRNP A2 in the HEK-293 and NIH-3T3 mammalian cell lines. Treatment of either cell line with the methyltransferase inhibitor adenosine dialdehyde dramatically shifts hnRNP A2 localization from the nuclear to the cytoplasmic compartment, as shown both by immunoblotting and by immunocytochemistry. In vitro radiolabeling with [(3)H]AdoMet of GST-tagged hnRNP A2 RGG mutants, using recombinant protein arginine methyltransferase (PRMT1), shows (i) that hnRNP A2 is a substrate for PRMT1 and (ii) that methylated residues are found only in the RGG domain. Deletion of the RGG domain (R191-G253) of hnRNP A2 results in a cytoplasmic localization phenotype, detected both by immunoblotting and by immunocytochemistry. These studies indicate that the RGG domain of hnRNP A2 contains sequences critical for cellular localization of the protein. The data suggest that hnRNP A2 may contain a novel nuclear localization sequence, regulated by arginine methylation, that lies in the R191-G253 region and may function independently of the M9 transportin-1-binding region in hnRNP A2., (Copyright 2000 Academic Press.)

CD154 (CD40 ligand (CD40L)) has been demonstrated to play an essential role in the development of humoral and cellular immunity through its interaction with CD40. While earlier studies have examined the regulation of CD154 expression by transcriptional and posttranslational pathways, scant data exist on its regulation at a posttranscriptional level. In this report we demonstrate that CD154 mRNA is rapidly turned over in primary culture of activated human T lymphocytes. Moreover, we demonstrate that CD154 mRNA is unstable, but can be stabilized by treatment with either phorbol esters or calcium ionophores. To address this lability of CD154 mRNA, we examined the ability of cytoplasmic proteins to bind to its 3' untranslated region (3'UTR). Two major proteins (p25 and p50) capable of binding the 3'UTR of CD154 were identified. The p25 binding activity was associated with polysomes and appeared to correlate with CD154 mRNA instability. Intriguingly, these proteins did not appear to bind to the AU-rich elements present in the 3'UTR of CD154. Rather, their binding was localized to unique sites between nt 471-811 of the 3'UTR, which lack any classical AU-rich elements. These data suggest that these proteins interact with distinct cis-acting elements that are important in the posttranscriptional regulation of CD154 expression. As such, identifying these proteins will help us understand the signals that are necessary for CD154 expression by activated T cells.

Recent work identified an RNA binding protein whose presence in brain tumors correlated with translational repression of Glut1 expression. RNase T1 mapping demonstrated that this protein bound an AU-rich response element (AURE) in the Glut1 3'UTR. Facilitated by its differential expression in brain tumor cytosols, we identified this Glut1 RNA binding protein as hnRNP A2. Studies further demonstrated that hnRNP A2 was the major Glut1 RNA binding activity in other cell lines. Recombinant hnRNP A2 exhibited equivalent Glut1 RNA binding specificity, quite distinct from the related AURE binding protein hnRNP A1. These data indicate that hnRNP A2 is the Glut1 AURE binding protein whose cytoplasmic expression in gliomas is associated with translational repression and mRNA instability. Using this approach, we also identified the other major Glut1 3'UTR RNA binding activity as hnRNP L. Stimuli (hypoxia and hypoglycemia) which increase Glut1 mRNA stability selectively decreased polysomal levels of hnRNP A2 and L. Immunoprecipitation demonstrated that hnRNP A2 and L exist as a complex in vivo. As a result of these and other studies, we conclude that hnRNP A2 and L associate in vivo and independently bind the 3'UTR of Glut1 mRNA., (Copyright 1999 Academic Press.)

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) shuttles between the cytoplasm and nucleus and plays important roles in RNA metabolism. Whereas nuclear hnRNP A1 has been shown to bind intronic sequences and modulate splicing, cytoplasmic hnRNP A1 is associated with poly(A)+ RNA, indicating different RNA ligand specificity. Previous studies indicated that cytoplasmic hnRNP A1 is capable of high-affinity binding of reiterated AUUUA sequences (ARE) that have been shown to modulate mRNA turnover and translation. Through a combination of two-dimensional gel and proteolysis studies, we establish hnRNP A1 (or structurally related proteins that are post-translationally regulated in an identical manner) as the dominant cytoplasmic protein in human T lymphocytes capable of interacting with the ARE contained within the context of full-length granulocyte-macrophage colony-stimulating factor mRNA. We additionally demonstrate that cytoplasmic hnRNP A1 preferentially binds ARE relative to pre-mRNAs in both cross-linking and mobility shift experiments. RNA polymerase II inhibition increased the binding of ARE (AUBP activity) and poly(U)-Sepharose by cytoplasmic hnRNP A1, while nuclear hnRNP A1 binding was unaffected. Nuclear and cytoplasmic hnRNP A1 could be distinguished by the differential sensitivity of their RNA binding to diamide and N-ethylmaleimide. The increase in AUBP activity of cytoplasmic hnRNP A1 following RNA polymerase II inhibition correlated with serine-threonine dephosphorylation, as determined by inhibitor and metabolic labeling studies. Thus, cytoplasmic and nuclear hnRNP A1 exhibit different RNA binding profiles, perhaps transduced through serine-threonine phosphorylation. These findings are relevant to the specific ability of hnRNP A1 to serve distinct roles in post-transcriptional regulation of gene expression in both the nucleus and cytoplasm.

We characterized modulation of the gamma-aminobutyric acid (GABA)-evoked responses of the diazepam-insensitive alpha 4 beta 2 gamma2 and alpha 6 beta 2 gamma 2 recombinant GABAA receptors. The partial agonist bretazenil potentiated the responses of both receptors with similar dose dependence but with a higher maximal enhancement at the alpha 4 beta 2 gamma 2 receptor. The bretazenil-induced potentiation was reduced by the benzodiazepine antagonist flumazenil. At a high concentration (10 microM), flumazenil was a weak potentiator of the GABA response. The partial agonist imidazenil was inactive. The imidazobenzodiazepine inverse agonist Ro 15-4513, which is known to bind with high affinity to the alpha 6 beta 2 gamma 2 receptor, potentiated the GABA responses of the alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptor subtypes with similar dose dependence over the concentration range of 0.1-10 microM. Methyl-6, 7-dimethoxy-4-ethyl-beta-carboline, a beta-carboline inverse agonist, had a similar potentiating effect when tested at a concentration of 10 microM. The alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptor-mediated currents had equal sensitivities to furosemide and Zn2+ ions, both of which reduced the GABA-evoked responses. The alpha 6 beta 2 gamma 2 receptor but not the alpha 4 beta 2 gamma 2 receptor exhibited a low level of spontaneous activity in the absence of GABA; this resting current could be directly potentiated by Ro 15-4513, methyl-6,7-dimethoxy-4-ethyl-beta-carboline, bretazenil and flumazenil and was blocked by picrotoxin. Thus, although the alpha 4 beta 2 gamma 2 receptors are insensitive to benzodiazepine binding site full agonists, such as diazepam, they can be modulated by certain ligands acting as partial and inverse agonists at diazepam-sensitive receptors and thereby contribute to the respective pharmacological profiles.

We have recently demonstrated by patch clamp experiments that the four isomers of hexachlorocyclohexane (HCH), alpha-, beta-, gamma- and delta-HCH insecticides, modulated the kinetics of the GABAA receptor-chloride channel complex of rat dorsal root ganglion neurons. The present paper reports the differential effects of HCH isomers of the GABA-induced chloride currents in three combinations of alpha, beta and gamma subunits of GABAA receptor expressed in human embryonic kidney cells. When co-applied with GABA, gamma-HCH strongly suppressed the peak amplitude of GABA-induced current, and delta-HCH strongly enhanced it in the alpha 1 beta 2 gamma 2s, alpha 1 beta 2, alpha 6 beta 2 gamma 2s combinations in a dose-dependent manner. There was little or no difference in the dose dependence of the effects between gamma- and delta-HCH in any of the three subunit combinations. However, alpha- and beta-HCH showed differential effects on GABA-induced chloride currents in the three subunit combinations tested. alpha-HCH showed enhancing effects on the peak current in alpha 1 beta 2 gamma 2s, small enhancing effects on alpha 1 beta 2, and biphasic effects on alpha 6 beta 2 gamma 2s subunit combinations. beta-HCH had little or no effect on the peak current in alpha 1 beta 2 gamma 2s and alpha 1 beta 2 combinations, but suppressed currents in the alpha 6 beta2 gamma 2s subunit combination in a dose-dependent manner. The differential actions of HCH isomers may produce variable effects on different regions of the nervous systems and in different species of animals.

Voltage-activated sodium channels and GABAA receptor-chloride channel complex are among the most important target sites of various environmental neurotoxicants. Pyrethroids keep the sodium channels open for prolonged periods of time leading to hyperexcitation of the entire nervous system. In rat cerebellar Purkinje neurons and dorsal root ganglion neurons, only about 1% of sodium channel population needed to be modified by the pyrethroid tetramethrin to increase the depolarizing after-potential to the level of the threshold membrane potential for generation of repetitive after-discharges. This concept of toxicity amplification is applicable to other chemicals that go through a threshold phenomenon to exert their effects. The potency of pyrethroids on neuronal sodium channels increased with lowering the temperature with a Q10 value of 0.2. The selective pyrethroid toxicity between mammals and insects can be quantitatively explained on the basis of the differences in 5 factors, i.e. the intrinsic sodium channel sensitivity, the sodium channel modification due to temperature difference, the reversibility of sodium channel, the detoxication of pyrethroids, and body size. These 5 factors are multiplied to approximately 2000 which is in the same order of magnitude as that of the difference in LD50. Dieldrin had a dual action on the GABAA receptor-chloride channel complex of rat dorsal root ganglion neurons. The initial transient potentiation of GABA-induced currents after application of dieldrin was followed by a suppression. Dieldrin-induced potentiation of current was observed only when the gamma 2 subunit was present in embryonic kidney cells (HEK-293) transfected with GABA receptor subunits. Dieldrin-induced suppression was observed in the presence and absence of the gamma 2 subunit. The dieldrin suppression of GABA-induced currents is deemed directly responsible for hyperactive symptoms of poisoning in animals.

A group of pyrrolopyrimidine derivatives were examined for their interaction with rat recombinant gamma-aminobutyric acid (GABA)A receptors using the whole cell patch clamp and equilibrium binding techniques. In the alpha 1 beta 2 gamma 2 subtype of GABAA receptors expressed in human embryonic kidney cells, a prototype pyrrolopyrimidine, U-89843A (7H-pyrrol[2,3-d]pyrimidine,6,7-methyl-2,4-di- 1-pyrrolidinyl,hydrochloride), dose-dependently enhanced 5 microM GABA-induced Cl- currents with a maximal enhancement of 362 +/- 91%, a half-maximal concentration of 2 +/- 0.4 microM and a slope factor of 1.1 +/- 0.4. The drug also inhibited [35S]t-butylbicyclophosphorothionate binding in rat cerebrocortical membranes with a similar half-maximal inhibitory concentration. The enhancement of Cl- currents by U-89843A was insensitive to Ro 15-1788 (a benzodiazepine antagonist), was also observed in the alpha 3 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 subtypes (no selectivity to different alpha-isoforms unlike many benzodiazepines), but was absent in the receptor subtypes consisting of two subunits (alpha 1 beta 2, alpha 1 gamma 2 and beta 2 gamma 2). It has been known that neurosteroids and barbiturates are uniformly active in both the two subunit receptors, substituted pyrazinones are only active in the alpha 1 beta 2 subtype and loreclezole is active in the subtypes containing beta 2. We propose that U-89843A interacts with an allosteric site on GABAA receptors distinct from the sites for benzodiazepines, barbiturates, neurosteroids, substituted pyrazinones or loreclezole.(ABSTRACT TRUNCATED AT 250 WORDS)

The presentation, diagnosis, and successful management of posttraumatic pseudoaneurysms of the intraparenchymal splenic artery after nonoperative therapy in an adult patient is described. Pseudoaneurysm formation of the intraparenchymal splenic artery is a rare complication of traumatic splenic injury, which is a potential mechanism of delayed splenic rupture and demonstrates the importance of follow-up in the nonoperative therapy of blunt injury to the spleen.

1. U-97775 (tert-butyl 7-chloro-4,5-dihydro-5-[(1-(3,4,5-trimethyl)piperazino)carbonyl]- imidazo[1,5-a])quinoxaline-3-carboxylate) is a novel GABAA receptor ligand of dual functionality and was characterized for its interactions with cloned rat GABAA receptors expressed in human embryonic kidney cells. 2. The drug produced a bell-shaped dose-response profile in the alpha 1 beta 2 gamma 2 receptor subtype as monitored with GABA-induced Cl- currents in the whole cell patch-clamp technique. At low concentrations (< 0.5 microM), U-97775 enhanced the currents with a maximal increase of 120% as normalized to 5 microM GABA response (control). An agonist interaction of U-97775 with the benzodiazepine site is suggested, because Ro 15-1788 (an antagonist at the benzodiazepine site) abolished the current increase and [3H]-flunitrazepam binding was inhibited by U-97775 with a Ki of 1.2 nM. 3. The enhancement of GABA currents progressively disappeared as the U-97775 concentration was raised above 1 microM, and the current amplitude was reduced to 40% below the control at 10 microM U-97775. The current inhibition by U-97775 (10 microM) was not affected by Ro 15-1788. It appears that U-97775 interacts with a second site on GABA receptors, distinct from the benzodiazepine site, to reverse its agonistic activity on the benzodiazepine site and also to inhibit GABA currents. 4. U-97775 at low concentrations reduced and at high concentrations enhanced [35S]-TBPS binding. Ro 15-1788 selectively blocked the effect of U-97775 at low concentrations. Analysis of the binding data in the presence of Ro 15-1788 yielded a single low affinity site with an estimated Kd of 407 nM.5. In other alpha beta upsilon receptor subtypes, U-97775 at low concentrations enhanced Cl- currents in the alpha 3 beta 2 upsilon 2,but not in the alpha 6 beta 2 upsilon 2 subtype. On the other hand, U-97775 at high concentrations reduced Cl- currents in all the receptor subtypes we examined, including those of two subunits, alpha 1 beta 2, beta 2 upsilon 2 and alpha 1 upsilon 2 subtypes.6. Therapeutically, U-97775 could be unique among benzodiazepine ligands because of its ability to limit its own agonistic activity such that, at high doses the appearance of agonistic activity would be delayed until occupancy of its second site wanes. This property should make the total agonistic activity of U-97775 relatively constant over a wide range of drug doses, and may minimize its liability to abuse.

U-93631,[4-dimethyl-3-t-butylcarboxyl-4,5-dihydro (1,5-a) quinoxaline], represents a GABAA receptor ligand of novel chemical structure, and has been shown to induce a rapid, time-dependent decay of GABA-induced whole-cell Cl- currents in recombinant GABAA receptors (Dillon et al., 1993). In this study, we found that the drug competitively inhibited [35S]t-butylbicyclophosphorothionate binding to the picrotoxin site on a cloned GABAA receptor, alpha 1 beta 2 gamma 2, and preempted the action of picrotoxin and [35S]t-butylbicyclophosphorothionate on GABA-induced Cl- currents. We further examined the effect of U-93631 on GABA-induced single channel openings in outside-out patches. U-93631 (5 microM) showed no effect on single channel conductance, the duration of channel open states or the short closed state, but increased the duration of the long closed state and its relative contribution to total closed time. In addition, closings of very long duration (> 500 msec), albeit rare, occurred more frequently in the presence of U-93631. Thus, the probability of single channel openings decreased from 0.12 +/- 0.02 to 0.04 +/- 0.01 in the presence of U-93631 (5 microM). These properties of U-93631 are analogous to those of picrotoxin and [35S]t-butylbicyclophosphorothionate reported in earlier studies with native neurons. We conclude that U-93631 at least shares overlapping binding domains with picrotoxin and [35S]t-butylbicyclophosphorothionate, and the ability to stabilize the GABAA receptor/Cl- channel complex in an inactivated state(s).(ABSTRACT TRUNCATED AT 250 WORDS)

We have recently demonstrated that the cyclodiene insecticide dieldrin modulates the kinetics of the GABAA receptor-chloride channel complex of rat dorsal root ganglion neurons in a complex manner, causing both stimulatory and inhibitory effects. We now report that the differential effects of dieldrin on the GABA-induced chloride current of human embryonic kidney cells expressing three different combinations of alpha, beta and gamma subunits. The EC50 values for GABA induction of current were estimated to be 9.8 microM for the alpha 1 beta 2 gamma 2s combination, 2.0 microM for the alpha 1 beta 2 combination and 3.0 microM for the alpha 6 beta 2 gamma 2s combination. When co-applied with GABA, dieldrin exerted a dual effect, enhancement and suppression, on the GABA-induced chloride currents in the alpha 1 beta 2 gamma 2s and alpha 6 beta 2 gamma 2s combinations. However, only suppression was observed in the alpha 1 beta 2 combination, indicating that the gamma subunit is necessary for dieldrin's enhancing effect. Dieldrin was more efficacious in enhancing the current in the alpha 6 beta 2 gamma 2s combination than in the alpha 1 beta 2 gamma 2s combination, indicating some specific role of alpha subunits in the dieldrin enhancement of current. Dieldrin suppressed the GABA-induced current in a non-competitive manner, with an EC50 value of 2.1 microM for alpha 1 beta 2 gamma 2s, 2.8 microM for alpha 1 beta 2 and 1.0 microM for alpha 6 beta 2 gamma 2s combination. These results indicated that dieldrin suppression did not require specific subunit combinations among the three tested.

Several previous studies implicated alpha 6 and gamma 2L subunits as potential determinants of gamma-aminobutyric acid A (GABAA) receptor channel sensitivity to alcohol modulation. The effects of ethanol and n-octanol were studied on GABA-induced currents in human embryonic kidney cells transfected to express one of three different GABAA receptor channel subunit combinations: alpha 1 beta 2 gamma 2S, alpha 6 beta 2 gamma 2S or alpha 6 beta 2 gamma 2L. No increase in the current amplitude of any subunit combination was observed after the coapplication of GABA and physiological concentrations (10-100 mM) of ethanol. By contrast, the coapplication of GABA and 100 microM octanol increased the current amplitude by 50% to 100% in all three subunit combinations. Octanol produced a shift of the current dose-response curve toward lower concentrations of GABA. Ethanol was effective in increasing the rate of desensitization produced by higher concentrations of GABA in the alpha 6 beta 2 gamma 2S cells but not the alpha 1 beta 2 gamma 2S combination. This ethanol-induced modification of desensitization was not altered by the presence of the protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). These experiments indicate that the presence of alpha 6 or gamma 2L subunits, in itself, does not result in the potentiation of GABA-induced currents by ethanol, as described in some reports. However, the presence of either the alpha 6 or alpha 1 subunit may determine whether the desensitization rate of the GABAA current is affected by the alcohol.

The MLA 144 gibbon T cell line is infected with a type C retrovirus and constitutively expresses interleukin-2 (IL-2) and granulocyte macrophage colony-stimulating factor (GM-CSF). IL-2 mRNA levels are 10-fold more abundant than GM-CSF in these cells. Comparable transcriptional rates for these lymphokines suggested the involvement of post-transcriptional mechanisms in selective IL-2 mRNA accumulation. IL-2 mRNA is exceptionally stable in MLA cells with a t1/2 of more than 8 h. The presence of reiterated AUUUA sequences in the 3'-untranslated region (UTR) has been shown to confer mRNA lability. The provirally altered MLA IL-2 allele encodes an mRNA in which three AUUUA motifs have been deleted. Six major cytoplasmic proteins bound in vitro transcribed RNA probes containing sequences from the 3'-UTR of normal human IL-2 (3'-IL-2), GM-CSF (delta 2R1), and the virally altered MLA IL-2 (3'-IL-2 PV) mRNA. Increased binding of these proteins to 3'-IL-2 PV was observed relative to 3'-IL-2 or delta 2R1. Northwestern blotting demonstrated similar differential ability of a 36- and 43-kDa protein to bind, as well as showed that these proteins colocalized by immunoblotting as hnRNP A1 and C, respectively. These findings suggest a direct correlation between differential binding of cytoplasmic proteins to AU-rich 3'-UTRs in vitro and lymphokine mRNA stability in vivo.

The effects of n-octanol on GABA-induced currents were examined on the alpha 1 beta 2 gamma 2s and alpha 1 beta 2 combinations of GABAA receptor subunits expressed in a human kidney cell line (HEK 293), using the whole-cell variation of the patch clamp technique. The EC50 of the GABA dose-response curve for the alpha 1 beta 2 combination was lower than that for the alpha 1 beta 2 gamma 2s combination. n-Octanol at 100 microM augmented the GABA-induced currents in a dose-dependent manner, decreasing the EC50 of the GABA dose-response curve without affecting the maximal response. The magnitude of n-octanol potentiation was nearly the same in both combinations. In contrast, a benzodiazepine agonist, chlordiazepoxide, augmented the currents of the alpha 1 beta 2 gamma 2s combination only. We conclude that the potentiation of GABAA receptor-mediated currents by a long carbon chain n-alcohol does not require the gamma 2 subunit.

Previous studies with cloned gamma-aminobutyric acid type A receptors expressed in human embryonic kidney cells have indicated that the alpha 1 beta 2 gamma 2 and alpha 1 gamma 2 (but not alpha 1 beta 2) subtypes have benzodiazepine sites. We found in this study that even the beta 2 gamma 2 subtype displays gamma-aminobutyric acid-induced Cl- currents that are potentiated by triazolam (a triazolobenzodiazepine). The maximal efficacy of the drug among the subtypes was highest with the alpha 1 beta 2 gamma 2 subtype, followed by the alpha 1 gamma 2 and beta 2 gamma 2 subtypes. These observations led us to compare the ability of several benzodiazepine site agonists of diverse chemical structures to potentiate Cl- currents with these subtypes. With the alpha 1 gamma 2 subtype, diazepam, alpidem, zolpidem, Cl-218872, zopiclone, U-79098 (an imidazoquinoxaline derivative), and U-90167 (a diimidazoquinazoline derivative) at 5 microM potentiated Cl- currents to essentially similar levels (slightly lower for a few ligands), compared with those with the alpha 1 beta 2 gamma 2 subtype. With the beta 2 gamma 2 subtype, the type 1 ligands zolpidem, alpidem, and Cl-218872 showed no or very low levels of potentiation, whereas less selective ligands such as diazepam, zopiclone, U-78098, and U-90167 displayed levels of Cl- current potentiation comparable to those observed with the subtypes containing the alpha 1 and gamma 2 subunits. These data indicate that, in the presence of gamma 2, beta 2 may substitute for alpha 1 in forming the benzodiazepine site of limited sensitivity to the type 1 ligands. It appears that individual ligands for benzodiazepine sites have their own sets of interacting domains, which are distributed in alpha 1 and gamma 2, and the agonistic activity of type 1 ligands may be more dependent on the alpha 1-specific domains than is that of less selective ligands.

1. In this study, we compared two series of newly discovered ligands for their selectivity to benzodiazepine sites in the alpha 1 beta 2 gamma 2 and the alpha 6 beta 2 gamma 2 subtypes of cloned gamma-aminobutyric acidA (GABAA) receptors, the latter being unique in not interacting with classical benzodiazepines. 2. The prototype compounds, U-85575 (12-chloro-5-(5-cyclopropyl-1',2',4'- oxadiazol-3'-yl)-2,3-dihydro-diimidazo [1,5-a;1,2-c]quinazoline), and U-92330 (5-acetyl-3-(5'-cyclopropyl-1',2',4'-oxadiazole-3'-yl)-7-chloro-4,5-d ihy dro [1,5-a]quinoxaline), appear to share an overlapping recognition site with classical benzodiazepines on the GABAA receptor, because their potentiation of GABA-mediated Cl- currents in both subtypes were sensitive to Ro 15-1788, a classical benzodiazepine antagonist. 3. Minor changes in the ring substituents of the drugs reduced their affinity to the alpha 6 beta 2 gamma 2 subtype more pronouncedly than to the alpha 1 beta 2 gamma 2 subtype. The diimidazoquinazoline containing a 2-methyl group which projected below the plane of the rigid ring showed a markedly lower affinity to the alpha 6 beta 2 gamma 2 subtype as compared to its stereoisomer having the methyl group above the plane of the ring. Also, the dihydroimidazoquinoxalines containing the 5-benzoyl group showed a lower affinity to the alpha 6 beta 2 gamma 2 subtype than the 5-acetyl counterpart. In particular, the 5-benzoyl analogue containing a 6-fluoro group showed no interaction with the alpha 6 beta 2 gamma 2 subtype even at the concentration of 10 microM, probably due to stabilization of the benzoyl group in the out-of-plane region by the steric and electrostatic effects of the 6-fluoro group.4. We propose that the benzodiazepine site of the alpha 6 beta 2 gamma 2 subtype shares overlapping regions with that of the alpha 1 beta 2 gamma 2 subtype, but has a sterically restricted out-of-plane region, which may be also incompatible with the 5-phenyl group of classical benzodiazepines.

We discovered the ability of U-93631 (4-dimethyl-3-t-butylcarboxyl-4,5- dihydro[1,5-a]imidazoquinoxaline) to accelerate decay of gamma-aminobutyric acid (GABA)-induced currents, and we explored its mechanism in human embryonic kidney cells (HEK-293) stably expressing the alpha 1 beta 2 gamma 2 subtype of GABAA receptors. Inward currents (Cl- efflux) induced by 5 microM GABA at the holding potential of -60 mV (under a symmetrical Cl- gradient) decayed with an exponential time course with a mean time constant (tau) of 222 +/- 25 sec, as examined with the whole-cell configuration of the patch-clamp technique. The monoexponential decay was greatly accelerated in the presence of U-93631 at 5 microM, with the mean tau value being 5.2 +/- 0.5 sec. The tau values were dependent on the concentration of U-93631, with an estimated Kd of approximately 2 microM. Outward currents at the holding potential of +60 mV decayed with a similar tau value in the presence of the drug, suggesting the voltage independence of the drug action. The initial amplitude of the GABA (5 microM)-induced Cl- current was not affected by preincubation with U-93631 (5 microM) or GABA (200 nM) alone but was reduced by preincubation with the combination of the two. In the presence of U-93631 at 5 microM, the peak amplitude decreased as a function of GABA concentration, with the half-maximal inhibitory concentration being approximately 100 nm, which is close to the Kd for the high affinity GABA site (85 nM). It appears that the drug interacts with GABA-bound receptors (at least monoliganded) and accelerates receptor desensitization, rather than acting as an open channel blocker. The binding site for U-93631 on GABAA receptors seems not to overlap with GABA, barbiturate, or benzodiazepine sites, because the drug effect persisted in the presence of excess ligands for those sites. With cloned GABAA receptors composed of only alpha 1 beta 2, beta 2 gamma 2, or alpha 1 gamma 2 subunits, U-93631 also accelerated the decay rate. This lack of subtype selectivity raises the possibility that the compound interacts with a region common among the three subunits, probably a novel modulatory site, which can possibly be exploited as a novel therapeutic target.

We discovered substituted pyrazinones as a new class of allosteric modulators of gamma-aminobutyric acid (GABA)A receptors. Prototype pyrazinones, U-92813 [1-(furfuryl)-3,5-dichloro-6-phenylpyrazinone] and U-94863 [1-benzyl-3,5-dichloro-6-(2-chlorophenyl)pyrazinone], potentiated GABA-mediated Cl- currents in cloned GABAA receptors with certain subtype selectivity. The drugs markedly enhanced the GABA response in the alpha 1 beta 2 gamma 2 and alpha 1 beta 2 subtypes but not in the alpha 1 gamma 2 and beta 2 gamma 2 subtypes expressed in human kidney cells. The dose-response profile of U-94863 in the alpha 1 beta 2 subtype was largely indistinguishable from that in the alpha 1 beta 2 gamma 2 subtype, suggesting no critical role for the gamma 2 subunit in potentiation of the GABA response by the pyrazinones. The drugs also potentiated the GABA response in the alpha 3 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 subtypes, indicating their nonselectivity toward the alpha isotypes. With respect to subtype selectivity, the pyrazinones differ not only from ligands for benzodiazepine receptors, which interact only with the subtypes containing alpha beta gamma subunits, but also from barbiturates and neurosteroids, which interact with all the subtypes tested in this study. The unique binding site for U-92813 on GABAA receptors was confirmed by the insensitivity of its action to Ro 15-1788, a classical benzodiazepine antagonist, and by the additive nature of its agonistic activity with that of barbiturates and neurosteroids. With respect to the mechanism of potentiation, the pyrazinones are similar to the other allosteric modulators, in that they potentiate the GABA response more effectively at low GABA concentrations than at high GABA concentrations. We propose that substituted pyrazinones represent a novel class of allosteric modulators of GABAA receptors, with their binding site probably located between the alpha and beta subunits.

We have used a reverse genetics approach to isolate genes encoding two subunits of Drosophila melanogaster RNA polymerase II. RpII18 encodes the 18-kDa subunit and maps cytogenetically to polytene band region 83A. RpII140 encodes the 140-kDa subunit and maps to polytene band region 88A10:B1,2. Focusing on RpII140, we used in situ hybridization to map this gene to a small subinterval defined by the endpoints of a series of deficiencies impinging on the 88A/B region and showed that it does not represent a previously known genetic locus. Two recently defined complementation groups, A5 and Z6, reside in the same subinterval and thus were candidates for the RpII140 locus. Phenotypes of A5 mutants suggested that they affect RNA polymerase II, in that the lethal phase and the interaction with developmental loci such as Ubx resemble those of mutants in the gene for the largest subunit, RpII215. Indeed, we have achieved complete genetic rescue of representative recessive lethal mutations of A5 with a P-element construct containing a 9.1-kb genomic DNA fragment carrying RpII140. Interestingly, the initial construct also rescued lethal alleles in the neighboring complementation group, Z6, revealing that the 9.1-kb insert carries two genes. Deleting coding region sequences of RpII140, however, yielded a transformation vector that failed to rescue A5 alleles but continued to rescue Z6 alleles. These results strongly support the conclusion that the A5 complementation group is equivalent to the genomic RpII140 locus.

A predominant form of the GABAA/benzodiazepine receptor-Cl- channel complex is believed to consist of three different 48-55 kDa subunits (alpha, beta, gamma) with unknown stoichiometry. Plasmids containing the rat GABAA receptor cDNAs coding for alpha 1, beta 2, and gamma 2 were co-transfected, along with a plasmid encoding G418 resistance, into human embryonic kidney cells previously transformed with Adenovirus 5 (HEK-293) [J. Gen. Virol., 36 (1977) 59-72]. Four percent of the G418 resistant colonies were found to express mRNA for all three of the GABAA subunits constitutively. A single cell clone derived from one of the alpha 1 beta 2 gamma 2 expressors has demonstrated stable electrophysiological characteristics over 25 passages. The GABA-activated Cl- current in this cell line is blocked by picrotoxin and bicuculline, and is modulated by a variety of agonist and inverse agonist ligands including diazepam, Ro 154513, zolpidem, and beta-CCE. The cell line has been used successfully over a 12-month period as a screen for novel drugs modulating GABA-mediated polarization of neuronal cells.

Post-transcriptional regulatory mechanisms have been shown to play a major role in gene expression in eukaryotic cells. The presence of a reiterated pentamer (AUUUA) in the 3'-untranslated region (UTR) of mRNAs encoding lymphokines, cytokines, transcription factors, and proto-oncogenes has been shown to be associated with rapid turnover and translation attenuation. Cytoplasmic proteins (70, 50, 43, 36, and 25 kDa) capable of specifically binding to RNAs containing these AU-rich sequences were identified in human peripheral blood T lymphocytes. Levels of the 36-kDa protein were markedly increased following transcriptional, but not translational inhibition, a feature recently reported for hnRNP A1, a protein of comparable mass. Antibodies directed against heterogeneous nuclear ribonucleoproteins (hnRNPs) A1 and C immunoprecipitated 36- and 43-kDa proteins that had bound the AUUUA-rich region contained in the 3'-UTR of granulocyte-macrophage colony-stimulating factor mRNA. Recombinant hnRNP A1 was shown to preferentially bind to RNAs containing AUUUA sequences in a specific manner, and displayed comparable patterns to the 36-kDa AU-specific binding proteins following partial proteolysis. These data identify for the first time hnRNP A1 and C as cytoplasmic proteins in human lymphocytes that are capable of specifically associating with reiterated AUUUA sequences present in the 3'-UTR of labile mRNAs. As such, they may play a role as trans-acting factors in the modulation of cytoplasmic mRNA turnover and translation, in addition to their previously characterized roles as pre-mRNA binding proteins involved in nuclear mRNA processing.

1. U-84935, diimidazo[1,5-a;1',2'-C]quinazoline,5-(5-cyclopropyl-1,2,4-oxid iazol-3yl)- 2,3-dihydro, is a ligand of high affinity for the benzodiazepine site of the GABAA receptor composed of alpha 1 beta 2 gamma 2 subunits. 2. The efficacy of its analogues was measured with their ability to potentiate GABA-mediated Cl- currents in the whole cell configuration of the patch clamp techniques in human kidney cells (A293 cells) expressing the subtype of the GABAA receptor. 3. The analogues displayed various levels of efficacy including agonists, partial agonists and antagonists without marked changes in their affinity for the receptors. 4. The major determinant of their efficacy was the spacial configuration of a methyl substituent of the C2 atom of the rigid and planar diimidazoquinazoline ring: U-90167, containing the methyl substituent projected below the plane of the ring, markedly enhanced the GABA current with a maximal potentiation of 220 +/- 25%, while its stereoisomer, U-90168, marginally increased the GABA response with a maximal potentiation of 45 +/- 10%, to which its methyl group appeared to contribute very little. 5. U-90167 potentiated the GABA response with an EC50 of 8.1 nM and a Hill coefficient of 1.1 and did not alter the reversal potential for the Cl- current. 6. From computational modelling, the sensitive methyl group of U-90167 could be assigned to the general region for the 5-phenyl group of diazepam. The diimidazoquinazoline, because of its rigid and plantar ring structure, may be useful to define further the out-of-plane region responsible for agonistic activity and to pinpoint other areas pivotal to the functionality of benzodiazepine ligands.

The effect of lanthanum ion (La3+) on gamma-aminobutyric acid (GABA)-mediated Cl- currents was examined in the alpha 1 beta 2 or alpha 1 beta 2 gamma 2 subtype of GABAA receptors expressed in a human kidney cell line (A293), using a whole-cell configuration of patch-clamp techniques. La3+ dose-dependently stimulated the Cl- currents in the alpha 1 beta 2 gamma 2 subtype with an EC50 of 21.3 +/- 3.5 microM with a maximal potentiation of 240 +/- 16% as normalized to the GABA response at 5 microM. In the alpha 1 beta 2 subtype, however, the ion marginally potentiated GABA response, a maximal stimulation being less than 70% with an EC50 for La3+ near 200 microM. The stimulation of GABA response by La3+ in the alpha 1 beta 2 gamma 2 subtype was due to a decrease in the half maximal concentration for GABA and was more pronounced at the negative membrane potentials. This selectivity of La3+ toward the subtypes of GABAA receptors contrasts to that of Zn2+ which inhibits the currents in the alpha 1 beta 2, but not in the alpha 1 beta 2 gamma 2 subtype (Neuron, 5: (1990) 781-788). It appears that these polyvalent cations are useful in understanding the molecular basis for the functional diversity and in characterizing the molecular organization of native GABAA receptors.

U-78875 [imidazo[1,5-a]quinoxalin-4(5H)-one, 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-(1-methylethyl)] belongs to a series of imidazoquinoxaline derivatives, recently discovered ligands with high affinity for benzodiazepine receptors. In this study, we have examined the drug and its analogs for their modes of interaction with the receptors, with a particular emphasis on finding molecular determinants for their functional properties. Changes in the substituents on N5 and C6 of the heterocyclic ring produced no major effects on binding characteristics but yielded drugs of widely varying efficacy (antagonist to full agonist), measured as gamma-aminobutyric acid (GABA)-mediated 36Cl- uptake and t-butylbicyclophosphoro[35S]thionate binding in rat cerebrocortical membranes. The relative binding affinity and efficacy of the analogs measured in brain membranes were similar to those in cloned GABAA receptors of the alpha 1 beta 2 gamma 2 (type I) and alpha 3 beta 2 gamma 2 (type II) subtypes. The imidazoquinoxalines showed no marked subtype selectivity. Their Ki value against [3H]flunitrazepam binding for type I was only 2-3 times lower than that for type II, and their rank order for agonistic activity was the same in the two subtypes, measured as GABA-mediated Cl- currents in human kidney cells (A293) expressing the subtypes of GABAA receptors. According to computational modeling of the drugs using both molecular and quantum mechanics, the agonistic activity of the imidazoquinoxaline derivatives depends on the presence of a bulky alkyl substituent at N5 and the deformation of the substituted portion of the otherwise planar ring system induced by a bulky moiety at N5 or C6. With a fixed N5 substituent (isopropyl), the relative efficacy in the brain membranes, as well as in the cloned receptors, appeared to be dependent on the degree of the ring deformation. This out-of-plane portion of the imidazoquinoxalines can be assigned to the general region occupied by the 5-phenyl group of diazepam and other agonistic functional groups of several nonbenzodiazepine ligands. It seems that this region, apparently common to various agonistic ligands, interacts with an agonistic pocket in type I and type II subtypes of the benzodiazepine receptors in the brain. Our results also provide direct support for the view that the agonists and nonagonists share largely overlapping binding regions in the benzodiazepine receptor, which has been proposed earlier from in vivo efficacy measurements of other series of ligands.

Specific mutations in the gene encoding the largest subunit of RNA polymerase II (RpII215) cause a partial transformation of a structure of the third thoracic segment, the capitellum, into the analogous structure of the second thoracic segment, the wing. This mutant phenotype is also caused by genetically reducing the cellular concentration of the transcription factor Ultrabithorax (Ubx). To recover mutations in the 140,000-D second-largest subunit of RNA polymerase II (RpII140) and determine whether any can cause a mutant phenotype similar to Ubx we attempted to identify all recessive-lethal mutable loci in a 340-kilobase deletion including this and other loci. One of the 13 complementation groups in this region encodes RpII140. Three RpII140 alleles cause a transformation of capitellum to wing but unlike RpII215 alleles, only when the concentration of Ubx protein is reduced by mutations in Ubx.

Previous studies have shown that 1,25-dihydroxyvitamin D3 (calcitriol) is a macrophage-derived cytokine and a potent inhibitor of IL-2 and interferon-gamma (IFN-gamma) production and T lymphocyte proliferation. The growth inhibitory effect of calcitriol is only partially reversed by IL-2 addition, suggesting IL-2 independent effects. In this report we characterize the IL-2-independent effects of calcitriol on lymphocyte activation. Calcitriol inhibited cellular transition from early to late G1 (G1A-G1B transition) in both the absence and presence of IL-2. Exogenous IL-2 did not increase either IFN-gamma production or transferrin receptor (TfR) expression in the presence of calcitriol despite increases in cell entry into late G1 and proliferation. Calcitriol treatment reduced TfR expression by activated T lymphocytes independent of their location in the cell cycle, further suggesting its independence from IL-2-mediated events. Combinations of rIL-2 and rIL-4 did not reverse calcitriol-dependent inhibition of proliferation and TfR expression to any greater degree than rIL-2 alone. Northern blot analysis demonstrated the decrease in IFN-gamma and TfR mRNA accumulation with calcitriol treatment was unaffected by exogenous IL-2. In contrast, IL-2R mRNA and protein were increased by IL-2, with superinduction in the presence of calcitriol, demonstrating that the lack of effect on IFN-gamma and TfR was not due to IL-2 insensitivity. Moreover, equivalent numbers of high-affinity IL-2R were expressed by both control and calcitriol-treated T lymphoblasts. Thus, lectin-activated T lymphocyte responsiveness to IL-2, as measured by IL-2R expression and proliferation, can be partly to completely dissociated from IFN-gamma production and TfR expression in the presence of calcitriol. Finally, IL-2-induced proliferation of unstimulated mononuclear cells and purified T lymphocytes was inhibited by calcitriol. These data indicate that local production of calcitriol by activated macrophages is capable of regulating T lymphocyte activation not only through suppression of IL-2 production, but also through additional mechanism(s), that are mediated at a post-IL-2R level.

The inability to perceive and coordinate both internal and external signals that function to regulate cellular growth and proliferation is a hallmark of oncogenic transformation. To examine the effects of the v-mos oncogene on distinct signal transduction pathways, the 6m2 cell line was used, in which expression of the p85gag-mos oncogene, and consequently transformation, are temperature sensitive. Through the analysis of endogenous metallothionein 1 (Mt-1) gene expression in 6m2 cells, p85gag-mos effects on glucocorticoid, cAMP, and heavy-metal induction were examined. While heavy-metal induction of Mt-1 mRNA was found to be unaffected by p85gag-mos, differential effects were exerted upon glucocorticoid and cAMP induction of Mt-1. Glucocorticoid induction of Mt-1 mRNA in p85gag-mos-transformed 6m2 cells was initiated normally but not maintained to the same extent as in nontransformed 6m2 cells. In contrast, cAMP did not induce Mt-1 mRNA in p85gag-mos-transformed 6m2 cells, although a significant induction was noted in nontransformed 6m2 cells. Thus, an oncoprotein interferes with different steps in each particular signal transduction pathway, ultimately causing abnormalities of inducible gene expression.

Expression of the p85gag-mos oncoprotein in temperature sensitive transformed 6m2 cells results in desensitization of glucocorticoid induction of metallothionein-1 mRNA. Indirect immunofluorescence analyses demonstrate that hormone insensitivity in v-mos transformed cells is associated with inefficient nuclear retention of glucocorticoid receptor (GR) protein. Desensitized receptors that accumulate in the cytoplasm of transformed 6m2 cells do not regain the capacity for hormone-dependent nuclear translocation after turnover of the thermo-labile p85gag-mos oncoprotein. Although ligand induced down-regulation of immunoreactive GR protein occurs in transformed 6m2 cells, desensitized receptors appear to retain some capacity to bind hormone in vivo. Thus alterations in the intracellular partitioning of GR protein in v-mos-transformed cells result in the generation of a novel desensitized receptor that is apparently trapped in the cytoplasm and incapable of being reutilized.