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M F Wilkinson

Publications and source records attributed to M F Wilkinson.

At least 55 records · Page 3Linked to original sources
PubMed1993

T cell receptor-beta mRNA splicing: regulation of unusual splicing intermediates.

The expression of functional T cell receptor-beta (TCR-beta) transcripts requires the activation of programmed DNA rearrangement events. It is not clear whether other mechanisms dictate TCR-beta mRNA levels during thymic ontogeny. We examined the potential role of RNA splicing as a regulatory mechanism. As a model system, we used an immature T cell clone, SL12.4, that transcribes a fully rearranged TCR-beta gene but essentially lacks mature 1.3-kb TCR-beta transcripts in the cytoplasm. Abundant TCR-beta splicing intermediates accumulate in the nucleus of this cell clone. These splicing intermediates result from inefficient or inhibited excision of four of the five TCR-beta introns; the only intron that is efficiently spliced is the most 5' intron, IVSL. The focal point for the regulation appears to be IVS1C beta 1 and IVS2C beta 1, since unusual splicing intermediates that have cleaved the 5' splice site but not the 3' splice site of these two introns accumulate in vivo. The block in 3' splice site cleavage is of interest since sequence analysis reveals that these two introns possess canonical splice sites. A repressional mechanism involving a labile repressor protein may be responsible for the inhibition of RNA splicing since treatment of SL12.4 cells with the protein synthesis inhibitor cycloheximide reversibly induces a rapid and dramatic accumulation of fully spliced TCR-beta transcripts in the cytoplasm, concomitant with a decline in TCR-beta pre-mRNAs in the nucleus. This inducible system may be useful for future studies analyzing the underlying molecular mechanisms that regulate RNA splicing.

Animals↗
PubMed1993

Vasopressin release within the ventral septal area of the rat brain during drug-induced antipyresis.

The techniques of push-pull perfusion and radioimmunoassay were used to determine concentrations of arginine vasopressin (AVP) in extracellular fluid derived from the ventral septal area (VSA) of the rat brain following antipyresis elicited by acetaminophen or indomethacin in conscious and unrestrained rats. Reduction of bacterial lipopolysaccharide (LPS)-induced fever by intraperitoneal indomethacin resulted in significant increases in AVP levels in VSA perfusion fluid (P < 0.05). In contrast, antipyresis after acetaminophen treatment was without significant effect on AVP output from VSA nerve terminals. In control animals (non-pyrogen treated), body temperature rose in apparent response to the perfusion procedure. Despite this elevation in core temperature, subsequent treatment with acetaminophen or indomethacin did not result in significant changes in AVP release from VSA perfusates. We conclude that AVP release into VSA extracellular fluids following intraperitoneal indomethacin is dependent upon the neuronal sequelae inherent to pyrogen-evoked fever and not nonspecific rises in body temperature. These results support the hypothesis that endogenous AVP, acting within the VSA, participates in the neuronal mechanisms mediating indomethacin-induced antipyresis.

Acetaminophen↗
PubMed1992

A spliced intron accumulates as a lariat in the nucleus of T cells.

The vast majority of mammalian genes are interrupted by non-coding segments of DNA termed introns. Introns are spliced out of RNA transcripts as lariat structures, and then are typically debranched and rapidly degraded. Here, we described an unusual spliced intron from the constant region of the T cell receptor-beta (TCR-beta) locus that is relatively stable in mammalian cells. This intron, IVS1C beta 1, accumulates as a set of lariat RNA structures with different length tails in the nucleus of T cells. The accumulation of this spliced intron is developmentally regulated during murine thymocyte ontogeny. The property of stability appears to be evolutionarily conserved since the human version of this intron also accumulates in T cells. The stability is selective since other spliced TCR-beta introns do not detectably accumulate in T cells. The unusual stability of this intron does not depend on T cell specific factors since non-T cells transfected with TCR-beta gene constructs also accumulate spliced IVS1C beta 1. The discovery of a mammalian intron that accumulates as a lariat in vivo provides an opportunity to elucidate mechanisms that regulate intron debranching, stability, and nuclear localization.

Animals↗
PubMed1992

TcR-alpha mRNA accumulation does not dictate cell surface TcR/CD3 expression.

The TcR-alpha chain is the last subunit of the TcR/CD3 complex to be expressed during thymic ontogeny. Since the presence of all subunits are required for efficient expression of this complex on the cell surface, this has lead to the hypothesis that the TcR-alpha chain is the limiting subunit which controls cell surface TcR/CD3 expression during thymocyte differentiation. We have examined this issue using a T-lymphoma cell clone, RS4.2, which has a CD4- CD8- Thyl+ J11d+ IL2R+ phenotype, identical with immature thymocytes which have the capacity to generate all major T cell subsets. The RS4.2 cell clone accumulates abundant amounts of TcR-beta, CD3-gamma, -delta, -epsilon and -zeta transcripts, but expresses trace levels of TcR-alpha transcripts and cell surface TcR/CD3. TcR-alpha mRNA levels can be dramatically augmented in RS4.2 cells by three distinct mechanisms: in response to treatment with either phorbol myristate acetate (PMA), calcium ionophore (A23187), or cycloheximide (CHX). However, the expression of TcR/CD3 on the cell surface fails to be increased by any of these agents. In fact, PMA induces a rapid down-regulation of cell surface TcR/CD3 expression. In contrast, these agents trigger an increase in cell surface IL2R expression which coincides with augmented IL2R-alpha mRNA levels. Thus, in RS4.2 cells, IL2R surface expression is controlled by transcript levels, while TcR/CD3 surface expression is regulated by post-transcriptional events, independent of TcR-alpha mRNA accumulation.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1992

Central arginine vasopressin and endogenous antipyresis.

Arginine vasopressin (AVP) is a centrally synthesized nonapeptide that exerts classical endocrine effects as well as a host of centrally mediated actions. A strong case can be argued in support of a neurotransmitter-neuromodulator role for AVP. Acting within the central nervous system (CNS), AVP has been demonstrated to be involved in the modulation of febrile body temperature. Because AVP acts to reduce pyrogen-induced fevers, but not normal body temperature, its actions are deemed to be antipyretic. However, to demonstrate an endogenous antipyretic function, AVP must be shown to be active during conditions where fever is naturally suppressed. This review will focus on five such conditions where the absence of pyrogen-induced fever can be linked to the endogenous activity of AVP within the brain. In the neonatal rat pup, the use of specific antagonists to the AVP receptor has revealed a role for CNS AVP in the absence of fever following peripheral injections of bacterial endotoxin. These results may help to explain a similar lack of fever in other newborn species. In parturient animals a reduced or absent febrile response has been linked to the increased presence of AVP within the septal area of the brain. The combined use of AVP receptor antagonism as well as immunohistochemistry has shown enhanced AVP activity within the ventral septal area of the rat and guinea pig brain during tolerance to intravenous pyrogens. These results suggest that the mechanism of fever suppression following repeated systemic injections of bacterial pyrogen includes centrally acting AVP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

CD3-gamma, -delta, -epsilon, -zeta, T-cell receptor-alpha and -beta transcripts are independently regulated during thymocyte ontogeny and T-cell activation.

CD3 proteins transduce signals delivered from the T-cell receptor (TcR) for antigen. The genes that encode the individual CD3 subunits are asynchronously regulated in tumour cell lines in vitro. In this report, we examined the expression of individual CD3 and TcR genes during normal murine thymocyte ontogeny in vivo. We show that CD3-gamma, -delta, -epsilon, and zeta transcripts are all expressed on Day 14 post-coitum (p.c.), along with IL-2R alpha and Thy-1 mRNA, and prior to the expression of functional TcR-alpha, -beta, CD4 and CD8 transcripts. Individual CD3 subunits display unique patterns of increased gene expression as ontogeny proceeds. Unique regulation of these T-cell transcripts is also observed in splenic cells activated with the T-cell mitogen concanavalin A (Con A). CD3-delta, -zeta, TcR-alpha and -beta mRNA expression increases, whilst CD3-gamma and -epsilon mRNA levels decrease after mitogenic activation. The potential implications of this regulation on the composition and expression of the TcR/CD3 complex during T-cell ontogeny and activation is discussed.

Animals↗
PubMed1991

The expression of several T cell-specific and novel genes is repressed by trans-acting factors in immature T lymphoma clones.

Cell surface proteins encoded by members of the immunoglobulin supergene family are sequentially expressed during T cell ontogeny. The molecular mechanisms responsible for the regulation of these surface molecules are not well understood. To investigate this issue, we used a series of well characterized T lymphoma cell clones with phenotypes characteristic of distinct stages of early thymocyte maturation. Somatic cell hybrids formed from these cell lines were employed to detect the presence of negative regulatory molecules. The expression of CD4 and CD8 was strongly repressed in hybrids formed between a CD4+ CD8+ lymphoma clone and "immature" CD4- CD8- lymphoma clones. Individual subunits of the T cell receptor (TCR)/CD3 complex displayed independent regulation in unique patterns in hybrid cells. Hybrids formed by fusing CD3+ and CD3- cells completely repressed CD3-delta mRNA expression while CD3-gamma, -epsilon, and -zeta transcripts were moderately inhibited or codominantly regulated. Similar to CD3-delta, interleukin 2R-alpha(IL-2R-alpha), and TCR-beta mRNA accumulation was trans-negatively regulated. Transcription rate measurements demonstrated that the inhibition of CD4, CD8, CD3-gamma, CD3-epsilon, TCR-beta, and IL-2R-alpha mRNA accumulation in hybrid cells was exerted, at least in part, at the transcriptional level. To test whether repressional regulation is a general feature of T cells, we examined the regulation of six novel genes which were selected solely on the basis of their differential expression between two of the cell lines used in this study. Five of the six novel gene transcripts were repressed in the somatic cell hybrids. Thus, inhibitor factors appear to play a general role in controlling T cell gene expression. The model system presented here may be useful for the identification and characterization of repressor molecules responsible for the regulation of genes expressed during T cell ontogeny.

Animals↗
PubMed1991

The oncofetal gene Pem encodes a homeodomain and is regulated in primordial and pre-muscle stem cells.

The oncofetal gene, Pem, is expressed in a stage specific manner during murine ontogeny. The carboxy terminal portion of the predicted Pem protein has significant similarity to homeodomains of the Drosophila prd family. The Pem gene is expressed in undifferentiated embryonal stem (ES) and embryonal carcinoma (EC) cell lines. Pem mRNA is induced 35-fold in ES cells differentiated in the absence of retinoic acid. Pem mRNA is increased in EC cells differentiated towards parietal or visceral endoderm, consistent with the abundant Pem expression in embryonic yolk sac. In 10T mesenchymal stem cells committed to muscle cell differentiation, Pem mRNA expression is dramatically increased. The elevation in Pem expression preceded the induction of the muscle master regulatory gene, myoD. We conclude that the Pem gene encodes a candidate transcription factor which is developmentally regulated.

Amino Acid Sequence↗
PubMed1990

A novel oncofetal gene is expressed in a stage-specific manner in murine embryonic development.

A novel cDNA clone obtained from a murine T-lymphoma library hybridizes to transcripts expressed in placenta and embryos (Pem) in a stage-specific manner. The Pem cDNA sequence predicts an intracellular hydrophilic protein with no significant sequence similarity to other DNA or protein sequences. Pem transcripts are abundant in 7- and 8-day mouse embryos, but decrease precipitously thereafter. On Day 9 they become abundant in placenta and yolk sac, persisting there until parturition. Although Pem transcripts are present in immortalized and tumorigenic cell lines from several different cell lineages, they are not detectable in any of 15 adult tissues tested. The expression of Pem during fetal development and its presence in immortalized and neoplastic cell lines is consistent with the properties expected of an "oncofetal" gene.

Amino Acid Sequence↗
PubMed1990

Activated T cells express a novel gene on chromosome 8 that is closely related to the murine ecotropic retroviral receptor.

A novel cDNA clone (20.5) which is differentially expressed between two closely related T-lymphoma cell clones was isolated by subtraction-enriched differential screening. SL12.4 cells, from which the cDNA was isolated, have characteristics of thymocytes at an intermediate stage in development. A sister cell clone derived from the same tumor, SL12.3, does not express this mRNA, has a distinct phenotype, and expresses fewer genes required for mature T-cell function. The cDNA sequence predicts a highly hydrophobic protein (approximately 49.5 kilodaltons) which contains seven putative membrane spanning domains. The gene was expressed on concanavalin A-activated T lymphocytes and was designated Tea (T-cell early activation gene). The Tea gene mapped to chromosome 8 and appeared to be conserved among mammalian and avian species. The Tea gene is distinct from, but bears extensive amino acid and DNA sequence similarity with, the murine ecotropic retroviral receptor which is encoded by the Rec-1 gene. Neither gene product displayed significant homology with other known transmembrane-spanning proteins. Thus, the Tea and Rec-1 genes establish a new family encoding multiple membrane-spanning proteins.

Amino Acid Sequence↗
PubMed1990

Centrally acting vasopressin contributes to endotoxin tolerance.

Repeated daily intravenous injections of bacterial endotoxin induce a refractory state to their usual pyrogenic effects. The neuropeptide arginine vasopressin (AVP) has been implicated in natural fever suppression and may be involved in the process of pyrogenic tolerance to intravenous endotoxin. This study was conducted to test this hypothesis. Tolerance was induced by two successive daily intravenous injections of Escherichia coli endotoxin (50 micrograms/kg) into conscious unrestrained rats. This tolerance was maintained, unaltered, after a third or fourth subsequent injection. However, bilateral administration of an AVP V1-receptor antagonist (0.43-4.3 nmol) into the ventral septal area (VSA) of the rat brain markedly enhanced the thermoregulatory response to a third or fourth endotoxin challenge compared with saline controls. The effect of the V1 antagonist was dose related. In contrast, an AVP V2 antagonist (0.43 nmol) bilaterally injected into the VSA did not affect the tolerant reaction to endotoxin. Furthermore, neither saline nor the V1 antagonist significantly affected core temperature when administered within the VSA without subsequent endotoxin. These results are consistent with the hypothesis that AVP acts as an endogenous antipyretic within the VSA during fever. Moreover, the data suggest a possible role for centrally acting vasopressin during pyrogenic tolerance to E. coli endotoxin.

Animals↗
PubMed1990

Central vasopressin V1-blockade prevents salicylate but not acetaminophen antipyresis.

Recent evidence has suggested that the endogenous antipyretic arginine vasopressin (AVP) may participate in drug-induced antipyresis. This study sought to further those investigations by comparing the effects of two other antipyretic drugs, sodium salicylate and acetaminophen, administered intraperitoneally, during AVP V1-receptor blockade within the ventral septal area (VSA) of the rat brain. During endotoxin-evoked fever, V1-receptor blockade within the VSA of the conscious unrestrained rat significantly antagonized the antipyretic effects of salicylate. The effects of the V1-antagonist on salicylate-induced antipyresis were dose related. In contrast, the antipyresis elicited by acetaminophen was unaffected by VSA V1-antagonist pretreatment. Neither saline nor the V1-antagonist microinjected into the VSA of febrile or nonfebrile rats had any significant effects on the normal progression of endotoxin fever or normal core temperature, respectively. These data suggest that the mechanism of action of salicylate-induced antipyresis includes activation of AVP V1-type receptors within the VSA, as has been shown for indomethacin. However, the lack of effect of the V1-antagonist on antipyresis induced by acetaminophen indicates that not all antipyretic drugs act through the same mechanism in the brain.

Acetaminophen↗
PubMed1990

Isolation of novel complementary DNA clones from T lymphoma cells: one encodes a putative multiple membrane-spanning protein.

Five novel complementary DNA (cDNA) clones which are differentially expressed between two closely related T lymphoma cell clones were isolated using subtraction-enriched differential screening. SL12.4 cells, from which the cDNAs were isolated, have characteristics of thymocytes at an intermediate stage in development and cause prominent extranodal ovarian tumors in syngeneic animals. A sister cell clone, SL12.3, derived from the same tumor, has a distinct phenotype and causes more aggressive, diffuse lymphomas. Four of the five novel genes are expressed in normal thymus, activated spleen cells, or gut-associated lymphoid tissue. The DNA sequence and predicted protein sequence are presented for one of the novel cDNA clones. This novel cDNA clone detects mRNA in normal thymus, gut-associated lymphoid tissue, and ovarian tissue. The predicted protein has four putative transmembrane-spanning regions. The expression of the transcript is repressed in somatic cell hybrids formed from SL12.4 cells fused with three different T lymphoma cell lines which lack detectable mRNA complementary to the novel cDNA clone. This trans-negative regulation suggests that the expression of the gene is regulated by repressional mechanisms.

Animals↗
PubMed1989

Antipyretic doses of centrally administered vasopressin reach physiologically meaningful concentrations in the brain of the rat as evaluated by microdialysis.

It was important to determine whether vasopressin (AVP) injected intracerebroventricularly (i.c.v.) in the rat reached the site of action within the ventral septal area (VSA) in sufficient concentrations to account for its physiological effects. Microdialysis was used to evaluate this hypothesis. The exchange rate across the dialysis tubing was determined in vitro to be 0.40%. After placement of the microdialysis cannula in the VSA of the rat the recovery of i.c.v. injected labelled or cold AVP was 0.23 and 0.20%, respectively. Maximum concentrations of AVP in the extracellular fluid of the VSA was determined to be 10.7 nM after 10 ng i.c.v. and hence extrapolated to be 1.07 nM after 1 ng i.c.v. or 2.65 nM after 2.5 ng i.c.v. between which lies the threshold dose of AVP for its antipyretic effects. This can be compared with a reported Kd for these receptors of 1.06 nM as determined by receptor binding assay.

Animals↗
PubMed1989

The CD3 delta gene encodes multiple transcripts regulated by transcriptional and post-transcriptional mechanisms.

CD3 is a multi-subunit complex of proteins noncovalently associated with the T cell receptor (TcR) for antigen. Considerable evidence indicates a role for CD3 molecules in the transduction of activation signals in T cells. The murine CD3 delta gene encodes a 0.7-kb transcript present in mature T cells. Here we report the characterization of several additional CD3 delta transcripts; two nuclear transcripts, 4-4.5 kb in size, and two predominamtly cytoplasmic transcripts of 1.5 kb and 2.5 kb. Both T lymphoma cell lines and normal thymocytes express the 1.5-kb and 2.5-kb CD3 delta transcripts. These cytoplasmic transcripts have long 3'-untranslated sequences which extend beyond the polyadenylation site of the predominant 0.7-kb transcript. The protein synthesis inhibitor cycloheximide (CHX) increases the expression of all three cytoplasmic CD3 delta transcripts, indicating that their level of expression may be regulated by a labile inhibitor protein(s). The CHX elicited increase in CD3 delta mRNA appears to result from post-transcriptional events since the rate of CD3 delta gene transcription remains constant. In contrast to CHX, the calcium ionophore A23187 increases the rate of CD3 delta gene transcription and, like CHX, also increases the level of cellular CD3 delta mRNA. The immunosuppressive agent cyclosporin A inhibits A23187-mediated stimulation of transcription, but has no effect on the CHX-mediated induction of CD3 delta mRNA. We conclude that both transcriptional and post-transcriptional mechanisms can regulate the amount of all three cytoplasmic CD3 delta transcripts.

Animals↗
PubMed1989

Central vasopressin V1-receptors mediate indomethacin-induced antipyresis in rats.

Central microinjection or infusion of an arginine vasopressin (AVP) V1-receptor antagonist within the brain of the conscious, unrestrained, and febrile rat inhibited or abolished the antipyretic effects of peripherally administered indomethacin (Indo). The degree of Indo-induced antipyresis was determined by 2-h thermal indexes (degree C.h) calculated from the time of Indo injection. Microinjection of saline or V1-receptor antagonist within the ventral septal area (VSA) of the rat brain immediately followed by intraperitoneal Indo evoked antipyretic responses of -1.63 +/- 0.17 and -0.24 +/- 0.09 degrees C.h, respectively (P less than 0.01). Infusion of the VSA with saline or V1-receptor antagonist before and after Indo resulted in thermal indexes of -1.35 +/- 0.16 and 0.13 +/- 0.30 degree C.h, respectively (P less than 0.01). Central microinjection of a V2-receptor antagonist did not significantly effect Indo-induced antipyresis compared with paired saline controls. Neither saline nor the V1-receptor antagonist affected nonfebrile body temperature when microinjected into the VSA. These data indicate the importance of AVP V1-receptors within the VSA in mediating the potent fever-reducing properties of the antipyretic drug Indo. Furthermore, these data call into question whether prostaglandin synthesis inhibition is a sufficient explanation of drug-induced antipyresis.

Animals↗
PubMed1988

Complex regulation of the T cell receptor alpha gene: three different modes of triggering induction.

The T cell receptor (TcR) for antigen is composed of variable alpha and beta subunits in noncovalent association with the invariant T3 multimer. TcR/T3 transcripts accumulate in a specific sequence during T cell development; TcR alpha transcripts are the last in the series to accumulate. To explore the regulation of TcR alpha gene expression, we investigated a T lymphoma cell clone which constitutively expresses TcR beta, T3 delta and T3 epsilon mRNA, but essentially lacks detectable TcR alpha mRNA. The cell clone can be induced to accumulate substantial amounts of TcR alpha mRNA in response to phorbol myristate acetate (PMA) or calcium ionophore A23187. Two different protein synthesis inhibitors also induce TcR alpha mRNA. The following evidence indicates that PMA, A23187 and cycloheximide induce TcR alpha by different mechanisms: (a) treatment with a combination of two agents induces greater than additive amounts of TcR alpha mRNA than that induced by a single agent; (b) the immunosuppressant cyclosporin A specifically inhibits A23187-mediated TcR alpha mRNA induction, whereas it fails to inhibit PMA or cycloheximide-mediated induction; and (c) both A23187 and PMA increase the rate of TcR alpha gene transcription, while cycloheximide influences TcR alpha mRNA accumulation by post-transcriptional mechanisms.

Animals↗