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M Boothby

Publications and source records attributed to M Boothby.

At least 37 records · Page 2Linked to original sources
PubMed1997

HMG-I(Y) phosphorylation status as a nuclear target regulated through insulin receptor substrate-1 and the I4R motif of the interleukin-4 receptor.

Interleukin (IL)-4 is a cytokine that regulates both the growth and differentiation of hematopoietic cells. Its ligand binding specificity and important signal transduction mechanisms are conferred by the IL-4 receptor alpha chain (IL-4Ralpha). The I4R is a tyrosine-containing motif within IL-4Ralpha that is critical for proliferative responses to IL-4. Although the I4R also contributes to gene regulation, nuclear targets directly regulated by this motif have not been described. It is shown here that the tyrosine at position 497 in the I4R is critical for regulation of the phosphorylation status of a set of nuclear proteins that includes HMG-I(Y), small non-histone chromosomal proteins involved in the control of gene expression in hematopoietic cell lines. Moreover, IL-4 is unable to induce HMG-I(Y) phosphorylation in insulin receptor substrate-1-deficient cells, and the inhibitor wortmannin completely blocks IL-4 regulation of HMG-I(Y) phosphorylation status but not activation of an IL-4 Stat protein. Taken together, these data indicate that HMG-I(Y) is a nuclear target whose phosphorylation status is regulated through the I4R motif via insulin receptor substrate proteins, independent of activation of the Stat pathway.

Androstadienes↗
PubMed1997

Von Willebrand factor and risk of ischemic stroke.

We carried out a case-control study to determine whether von Willebrand factor (vWF) antigen (and factor VII and tissue plasminogen activator [tPA] antigens) are associated with ischemic stroke. Ninety-five patients with transient ischemic attack or minor ischemic stroke recruited from the Oxfordshire Community Stroke Project and one neurology clinic were compared with 236 controls, group-matched for age and sex, from the same general practitioners as the incident cases. In crude analyses, concentrations of vWF antigen were significantly higher in cases than in controls (p = 0.004). The age- and sex-adjusted odds ratios from lowest (referent) to highest quartile of vWF antigen were 1.00, 1.15, 2.34, and 2.36 (trend test, p = 0.006). Factor VII antigen and tPA antigen were not significantly different between cases and controls. Although adjustment for other potential risk factors abolished the statistical significance of the association between vWF and disease, this was largely due to the influence of history of ischemic heart disease. We conclude that vWF is a potent and independent risk factor for transient ischemic attack, minor ischemic stroke, and, by extrapolation, ischemic stroke in general. The data also suggest that vWF may be a risk factor for both ischemic stroke and ischemic heart disease. We found no evidence to implicate factor VII and tPA as risk factors for ischemic stroke.

Adult↗
PubMed1996

Rapid commitment to negative selection of a thymocyte line independent of CD4 co-engagement.

Biochemical and genetic investigations of thymocyte negative selection will be facilitated by the availability of cultured cell lines representing immature thymocytes, but to date thymocyte lines have demonstrated resistance to Ag receptor-mediated death. It is shown here that a CD4+CD8+ TCR alpha beta+ cell line with a TCR of known Ag and class II MHC specificity (cytochrome c with I-Ek) undergoes Ag dose-dependent apoptotic death in a manner consistent with negative selection of normal thymocytes. In contrast to superantigen-induced selection, apoptosis in this model is unaffected by blocking co-engagement of CD4 and the TCR. At high Ag doses, a significant fraction of thymocytes becomes committed to apoptosis within 2 h of contact with antigen-pulsed APCs, while increased interaction times increase the probability of death. Longer contact periods are required at lower Ag doses. These kinetics of commitment to apoptosis suggest that, in normal negative selection, a thymocyte clone may be deleted after any of several encounters with MHC-bearing cells during progress through the thymus. They also suggest why negative selection predominates when a specific peptide is competent both to delete and to select positively.

Animals↗
PubMed1996

Independent pathways for de-repression of the mouse Ig heavy chain germ-line epsilon promoter: an IL-4 NAF/NF-IL-4 site as a context-dependent negative element.

The activation of germ-line promoters in the Ig heavy chain loci is regulated by cytokines as part of the regulation of B cell commitment to production of new antibody isotypes. Activation of the germ-line promoter of the epsilon heavy chain locus (Gepsilon) and production of IgE are induced by IL-4 and each is virtually undetectable in the absence of IL-4 or the homologous cytokine IL-13. Basal expression of the Gepsilon promoter is repressed by the non-histone chromosomal protein HMG-I(Y), which also contributes to promoter inducibility, and IL-4 stimulates phosphorylation of the C-terminus of HMG-I(Y) through a rapamycin-sensitive pathway. IL-4 treatment of mouse B cells also induces a Gepsilon DNA binding activity with the properties of IL-4 NAF, which is rapidly induced and requires phosphotyrosine for DNA binding activity. This protein binds to a different site from HMG-I(Y), but the IL-4 NAF/NF-IL-4 binding site also is a negative element more active in repression of basal transcription of the Gepsilon promoter. This site acts as a negative element when transferred to the thymidine kinase promoter, but does not confer inducibility. In contrast to HMG-I(Y), IL-4 NAF/NF-IL-4 activation is refractory to rapamycin but sensitive to genistein. These findings indicate that two independent signal transduction pathways diverge from the IL-4 receptor and suggest that normal expression of Gepsilon RNA or IgE is low in part because the germ-line promoter is kept in a state of repression which requires de-repression through several cooperative pathways. These pathways target conserved nucleotide sequence motifs whose precise function depends on the promoter context in which they are situated.

Animals↗
PubMed1995

Interleukin 4-inducible phosphorylation of HMG-I(Y) is inhibited by rapamycin.

The non-histone chromosomal protein HMG-I(Y) participates in repression of transcription directed by a promoter which confers interleukin 4 (IL-4)-inducible activation in transfected B cell lines. Metabolic labeling, phosphoamino acid analyses, and in vitro phosphorylation studies demonstrate that IL-4 induces serine phosphorylation of HMG-I(Y) in B lymphocytes. Phosphopeptide mapping shows that the predominant site of phosphorylation contains a casein kinase II consensus motif. The immunosuppressive agent rapamycin has been shown preferentially to inhibit IgE production by IL-4-treated human B cells. It is shown here that rapamycin inhibits both activation of the human germ line epsilon promoter by IL-4 and IL-4-inducible phosphorylation of HMG-I(Y). These findings demonstrate a rapamycin-sensitive pathway that transduces signals from the IL-4 receptor to nuclear factors that regulate inducible transcription. The affinity of normal nuclear HMG-I(Y) for DNA is increased by dephosphorylation in vitro, whereas in vitro kinase reactions using casein kinase II decrease recombinant HMG-I(Y) binding to DNA. These data further suggest a novel mechanism in which phosphorylation triggered by IL-4 or other cytokines could regulate the effects of HMG-I(Y) on gene transcription.

Amino Acid Sequence↗
PubMed1995

The non-histone chromosomal protein HMG-I(Y) contributes to repression of the immunoglobulin heavy chain germ-line epsilon RNA promoter.

The rate of germ-line RNA transcription correlates with the rate of immunoglobulin heavy chain isotype switching. A promoter element for the transcription of RNA from the germ-line mouse immunoglobulin epsilon heavy chain constant region gene is induced by interleukin(IL)-4 and lipopolysaccharide, and is bound at its transcription initiation sites by an IL-4-inducible nuclear protein, NF-BRE. To examine the function of the binding site for this IL-4-inducible complex, substitution mutations were introduced in the promoter. These binding site mutations increased promoter activity and decreased binding of NF-BRE. To investigate the paradox of an IL-4-inducible protein binding to a repressor site in an IL-4-inducible promoter, we determined that the non-histone chromosomal protein HMG-I(Y) binds at the transcription initiation sites of the germ-line epsilon promoter. Assays with antisera against HMG-I(Y) revealed monomeric HMG-I(Y) in nuclear extracts. Cotransfection of an expression construct directing the synthesis of anti-sense HMG-I(Y) RNA also increased promoter activity, consistent with a repressor function of HMG-I(Y). Thus, the data are most consistent with a model in which HMG-I(Y) participates in repression of promoter activity. The effects of IL-4 may include derepression at this site.

Animals↗
PubMed1993

Distinct IL-4 response mechanisms of the MHC gene A alpha in different mouse B cell lines.

Interleukin-4 (IL-4) is a multipotent cytokine which stimulates proliferation of B and T lymphocytes, induces B lymphocyte expression of major histocompatibility complex (MHC) class II molecules and Fc epsilon R II (CD23) molecules, and promotes immunoglobulin class switching to IgE and IgG1. The mechanisms by which IL-4 induces these changes are unclear. To study the basis for heterogeneity in induction of class II MHC proteins observed in splenic B cells, three mouse B cell lines were treated with IL-4, and the response of MHC class II A alpha mRNA was analyzed. Each of the three cell lines responded with a distinctive profile. In one line, 70Z/3, A alpha mRNA was induced greater than 10 fold by 65 hr of IL-4 stimulation. Additional studies showed that A alpha mRNA was stabilized by IL-4 treatment of 70Z/3 cells, and that changes in gene transcription accounted for little of the increase in mRNA levels. A second line, WEHI.231, was shown to increase A alpha mRNA levels 4 fold after 48 hr of IL-4 treatment. In contrast to 70Z/3, when A alpha mRNA stability in the IL-4 treated WEHI.231 cells was compared to untreated cells, no difference was observed, IL-4 treatment induced A alpha transcription. The third cell line, M12.4.1, expressed high basal levels of A alpha, and these levels increased only slightly following IL-4 stimulation. The small increase correlated with a comparable transcriptional response. These data shown that the nature of the A alpha gene response to IL-4 differs among B cell lines. This heterogeneity of response is consistent with responses in total splenic B cells, and with the existence of functionally distinct subpopulations of B cells.

Animals↗
PubMed1993

Lack of evidence for abnormal fibrinolysis in chronic low back pain.

It has been reported that plasma fibrinolytic activity is abnormal in some patients with chronic low back pain. In an attempt to confirm this finding we studied 22 patients with chronic mechanical low back pain and compared them with 18 healthy controls who denied symptoms of back pain. Factors known to interfere with plasma fibrinolysis such as age, weight, seasonal and diurnal variation, exercise, smoking and drugs were controlled as far as possible. Plasma fibrinogen was significantly higher (2.8 versus 2.3 g/l, P < 0.005) in patients than in controls, but there were no significant differences in the median plasma concentrations of euglobulin clot lysis time, fibrin plate lysis area, plasminogen, alpha-2-antiplasmin, tissue plasminogen activator activity, and antigen, tissue plasminogen activator inhibition and plasminogen activator inhibitor-1 antigen level. The results fail to confirm abnormalities of plasma fibrinolytic activity in a group of unselected cases of chronic low back pain.

Adult↗
PubMed1992

IL-4 induces IL-2 receptor p75 beta-chain gene expression and IL-2-dependent proliferation in mouse T lymphocytes.

T and B cells exhibit complex responses to the combination of IL-2 and IL-4, each of which can act as a growth or differentiation factor for lymphocytes under certain circumstances. To characterize better the mechanism by which these cytokines interact, mRNA levels of the signal-transducing p75 beta-chain of the IL-2R were analyzed. These studies show that IL-4 increases expression of the IL-2R beta-chain in mouse splenic B and T cells, and the response of B cells was potentiated by concurrent cross-linking of surface Ig. Kinetic analysis of the IL-2R beta response showed a slow onset but maintenance of peak levels of expression between 10 and 24 h. These data indicate that the pathways involved in the lymphocyte response to IL-4 differ for IL-2R and IL-4R, and that the induction of IL-4R precedes the increase in IL-2R. The effect of IL-4 on IL-2R beta mRNA levels was mediated in part by an increase in the rate of gene transcription, and was associated with increased IL-2 binding in the absence of any change in IL-2R alpha levels. In addition, IL-4 increased the level of IL-2R beta expression in thymocytes. Proliferation assays demonstrated that pretreatment of splenic T cells with IL-4 led to a substantial increase in IL-2-dependent proliferation. These results are consistent with a mechanism by which IL-4 can prime T cells and certain thymocytes for responsiveness to IL-2 by increasing IL-2R p75 chain gene expression, independent of general T cell activation.

Animals↗
PubMed1991

Role of lipopolysaccharide and IL-4 in control of transcription of the class II A alpha gene.

The class II (Ia) MHC molecules are cell surface proteins that regulate the activation of T cells. B lymphocyte expression of class II molecules has been shown to be influenced by a number of external stimuli. It has been previously demonstrated that treatment of these cells with IL-4 leads to an increase in class II gene transcription at 18 h as well as to an increase in steady state class II mRNA. It has also been previously demonstrated that LPS treatment of splenic B cells from athymic mice results in a decrease in steady state mRNA encoding the A alpha class II protein. This decrease persists for at least 18 h. Nuclear run-on transcription assays now demonstrate that although steady state mRNA levels for A alpha are decreased by LPS treatment of athymic mouse lymphocytes, LPS does not decrease A alpha gene transcription, but rather modestly activates transcription of this class II gene. LPS and IL-4 have been demonstrated to be synergistic stimuli for a number of genes. Costimulation of splenic lymphocytes from athymic mice with IL-4 plus LPS leads to activation of transcription, but the increase in transcription is no more than that seen with IL-4 stimulation alone. However, in costimulated lymphocytes, steady state A alpha-encoding mRNA levels are intermediate between the increased levels seen with IL-4 stimulation and the decreased levels seen with LPS stimulation. Therefore, LPS and IL-4 act nonsynergistically in class II gene transcription and the effects of LPS in decreasing steady state mRNA are most likely posttranscriptional. An IL-4-inducible and an LPS-inducible DNA-binding protein have been previously identified in splenic lymphocytes from athymic mice. Both nuclear binding proteins form complexes with the same DNA fragments from a control region of the A alpha gene. These nucleoprotein complexes comigrate under nondenaturing conditions and display identical patterns of binding with a panel of oligonucleotide competitors. Oligonucleotides representing protein binding sites of the IL-4 and LPS-induced DNA-binding proteins cross-compete for protein binding. Therefore, the binding proteins induced by LPS and IL-4 are likely related, and may function at different efficiencies as activators of A alpha gene transcription.

Animals↗
PubMed1991

Identification of a conserved lipopolysaccharide-plus-interleukin-4-responsive element located at the promoter of germ line epsilon transcripts.

Treatment of splenic B lymphocytes and certain B-lineage cell lines with the mitogen lipopolysaccharide (LPS) and the lymphokine interleukin-4 (IL-4) induces expression of germ line immunoglobulin C epsilon transcripts and class switching to the C epsilon gene. We show that LPS-plus-IL-4 induction of germ line epsilon transcripts (termed I epsilon transcripts) occurs at the transcriptional level in an Abelson murine leukemia virus-transformed pre-B-cell line. A 1.1-kb region of DNA surrounding the I epsilon promoter endows inducible transcription to a heterologous reporter gene stably transfected into these cells; such inducible expression depends on combined treatment with LPS and IL-4. Analyses of constructs transiently introduced into a B-cell lymphoma line demonstrated that LPS-plus-IL-4-inducible expression can be conferred by a 179-bp segment of DNA spanning the I epsilon transcriptional initiation site. Mutational analyses demonstrated that this expression depended on DNA sequences within a conserved region directly upstream from the I epsilon transcriptional initiation region. One nuclear protein that is constitutively expressed in normal B cells binds to the downstream end of the conserved sequence; its binding specificity correlates with the functional effect of several mutations. Two additional proteins, which are induced by IL-4 treatment of splenic B cells, bind to the transcription initiation sites of I epsilon. These proteins are indistinguishable in binding assays from proteins previously shown to bind an enhancer region of the class II major histocompatibility complex gene A alpha.

Abelson murine leukemia virus↗
PubMed1990

The effect of modified electroconvulsive therapy on vasopressin release and haemostasis in man.

Plasma concentrations of plasminogen activator activity (PAA) and factor VIII are partly controlled by circulating adrenaline and vasopressin. Acute rises in PAA and factor VIII occur during electroconvulsive therapy (ECT). To investigate the relationships between vasopressin (aVP), adrenaline and changes in PAA and factor VIII during ECT, 8 female and 2 male patients, median age 57 years (range 39-75) undergoing modified ECT had venous blood samples taken before and at 2 min, 15 min, 60 min and 24 h after cessation of seizure activity. AVP rose from 0.5 before ECT to 35.5 pg/ml at 2 min (P less than 0.005) and fell thereafter. PAA (10(6)/ECLT2) increased from 22 to 69 units (P less than 0.005) over the same time and fell to 13 units at 24 h (P less than 0.02). Tissue plasminogen activator activity (tPA) rose from 162 before to 1447 mIU/ml at 2 min. (P less than 0.005) and its inhibition activity fell from 8 to 3.75 IU/ml (P less than 0.005) over the same time and rose to 10.4 IU/ml after 24 h (P less than 0.02). There were no changes in adrenaline, noradrenaline, factor VIIIc, vWF or fibrinopeptides A and B beta 15-42. AVP correlated with tPA (rs = 0.64, P = 0.0022) and PAA (rs = 0.61, P = 0.004). These results support the hypothesis that aVP has a role in the regulation of fibrinolytic activity mediated by an increase in tPA. The absence of a factor VIII response may indicate that adrenaline is more important in the regulation of factor VIIIc and vWF.

Adult↗
PubMed1990

Effect of warfarin on plasma concentrations of vitamin K dependent coagulation factors in patients with stable control and monitored compliance.

There is a discrepancy in the results of reported studies of levels of vitamin K dependent coagulation factors in patients on warfarin therapy. This may have arisen partly because of the problem of assuring compliance with therapy in outpatients. The plasma concentrations of the vitamin K dependent clotting factors II, VII, IX and X were studied in 23 outpatients whose adherence to prescribed warfarin therapy was determined using a pharmacological indicator of compliance. In these patients, who were shown to have consistently good compliance and stable anticoagulant control over a period of 3-6 months, the activities in plasma of the four coagulation factors were not equally suppressed. Factor IX levels were significantly greater than those of factor VII (P less than 0.0001) which in turn were significantly greater than the levels of factor II (P less than 0.0001) or factor X (P less than 0.0001). There was no significant difference between the levels of factors II and X which were depressed to a similar extent. The proportion of variability of the International Normalized Ratio (INR) explained by linear regression was 51-77% and a model was derived to predict the INR from the mean of the levels of the four clotting factors. The concentrations of the coagulation factors II, VII, IX and X are likely to be highly dependent on the degree of compliance with warfarin therapy which should be taken into account when investigating the behaviour of these factors.

Aged↗
PubMed1990

Therapeutic defibrinogenation by ancrod: effect on limb blood flow in peripheral vascular disease.

Reduction of blood viscosity by venesection may improve peripheral blood flow but similar haemorheological changes have not been conclusively demonstrated following reduction of plasma viscosity by defibrinogenation. Ancrod is a defibrinogenating enzyme from the venom of the Malayan pit viper. We studied six male patients with severe intermittent claudication before, during and after treatment with ancrod for 48 h. Ancrod was given by initial infusion of 2 U/kg over 6 h and four subsequent bolus i.v. injections of 2 U/kg. Skin blood flow in feet and toes were measured repeatedly by laser Doppler velocimetry and calf blood flow by electronic plethysmography both before and after 2 min arterial occlusion. Plasma fibrinogen [median (range)] fell from 2.3(1.4-3.9) to 0.1(0.1-0.3) g/l and plasma viscosity from 1.81(1.61-1.90) to 1.49(1.45-1.72) cp. Dorsal foot and toe skin blood (resting or post-ischaemic) flows were not changed significantly by ancrod. Resting calf blood flow fell significantly after 49 h treatment with ancrod (P less than 0.04). Brachial and ankle blood pressures remained unchanged and haematocrit was not changed. Thus, treatment with ancrod for 48 h did not improve peripheral blood flow in patients with peripheral vascular disease. The fall observed in calf blood flow could be related to increased circulating fibrin/fibrinogen high-molecular-weight complexes and degradation products.

Adult↗
PubMed1989

Differences in DNA sequence specificity among MHC class II X box binding proteins.

The class II (Ia) MHC Ag are integral membrane proteins whose expression is limited to specific cell types. A pair of consensus sequences, X and Y, is found upstream from all class II genes and deletion of each of these sequences eliminates expression of transfected genes. Cells that express Ia demonstrate a coordinate response to lymphokines and other stimuli. These conserved sequences might, therefore, play a role in tissue specificity or lymphokine inducibility of Ia gene expression. The X box sequence of the murine class II A alpha gene diverges much more substantially from the X consensus than does the Y box motif of this gene. We demonstrate that this X box motif is nonetheless recognized by sequence-specific DNA-binding proteins, as is the more closely conserved Y box. Gel retardation assays and DNase I footprints were compared for a panel of Ia+ and Ia- cells as well as for cells stimulated with the Ia-inducing lymphokines IL-4 and IFN-gamma. The level, retardation pattern and region of DNA contact were comparable in all instances. Thus the availability of active DNA-binding X and Y box factors cannot alone account for the regulation of A alpha expression. To test whether the same set of proteins binds all class II MHC conserved motifs, oligonucleotide probe binding and cross-competition experiments with X box sequences from A alpha, E alpha, and E beta genes were performed. These studies demonstrated A alpha, E alpha, and E beta DNA-protein complexes with unique mobilities and specificities. In addition, all three X box oligonucleotide probes generated one faint complex with an affinity profile of E beta greater than E alpha much greater than A alpha. These three complexes comigrated and thus may represent a communal binding protein. The data are most consistent with the conclusion that multiple proteins bind class II MHC X boxes. For A alpha, the predominant complexes represent different specificities from the predominant E alpha and E beta X box binding proteins.

Animals↗