Search PubMed⌕ Search

Biomedical subjects

D J Rawlings

Publications and source records attributed to D J Rawlings.

At least 37 records · Page 2Linked to original sources
PubMed1998

Phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3)/Tec kinase-dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals.

Tec family non-receptor tyrosine kinases have been implicated in signal transduction events initiated by cell surface receptors from a broad range of cell types, including an essential role in B-cell development. A unique feature of several Tec members among known tyrosine kinases is the presence of an N-terminal pleckstrin homology (PH) domain. We directly demonstrate that phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) interacting with the PH domain acts as an upstream activation signal for Tec kinases, resulting in Tec kinase-dependent phospholipase Cgamma (PLCgamma) tyrosine phosphorylation and inositol trisphosphate production. In addition, we show that this pathway is blocked when an SH2-containing inositol phosphatase (SHIP)-dependent inhibitory receptor is engaged. Together, our results suggest a general mechanism whereby PtdIns-3,4,5-P3 regulates receptor-dependent calcium signals through the function of Tec kinases.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation.

Bruton's tyrosine kinase (Btk) is essential for B-lineage development and represents an emerging family of non-receptor tyrosine kinases implicated in signal transduction events initiated by a range of cell surface receptors. Increased dosage of Btk in normal B cells resulted in a striking enhancement of extracellular calcium influx following B-cell antigen receptor (BCR) cross-linking. Ectopic expression of Btk, or related Btk/Tec family kinases, restored deficient extracellular Ca2+ influx in a series of novel Btk-deficient human B-cell lines. Btk and phospholipase Cgamma (PLCgamma) co-expression resulted in tyrosine phosphorylation of PLCgamma and required the same Btk domains as those for Btk-dependent calcium influx. Receptor-dependent Btk activation led to enhanced peak inositol trisphosphate (IP3) generation and depletion of thapsigargin (Tg)-sensitive intracellular calcium stores. These results suggest that Btk maintains increased intracellular calcium levels by controlling a Tg-sensitive, IP3-gated calcium store(s) that regulates store-operated calcium entry. Overexpression of dominant-negative Syk dramatically reduced the initial phase calcium response, demonstrating that Btk/Tec and Syk family kinases may exert distinct effects on calcium signaling. Finally, co-cross-linking of the BCR and the inhibitory receptor, FcgammaRIIb1, completely abrogated Btk-dependent IP3 production and calcium store depletion. Together, these data demonstrate that Btk functions at a critical crossroads in the events controlling calcium signaling by regulating peak IP3 levels and calcium store depletion.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

Comparison of ultrasound measurements at the heel between adults with mental retardation and control subjects.

Little is known about the prevalence of metabolic bone disease among adults with mental retardation (now known as learning disability), although they may be at increased risk of fractures. Broadband-ultrasound attenuation (BUA) and velocity-of-sound (VOS) measurements were performed on the left heel of 170 patients in a large hospital for adults with mental retardation. For 108 of these patients, age- and gender-matched control subjects were recruited from the local community, who also underwent BUA and VOS measurements. The mean age of matched pairs of patients and control subjects was 54 (range 32-83) years for men and 53 (range 27-82) years for women. Mean +/- SEM BUA for male patients was 52 +/- 4 dB/MHz and 89 +/- 2 for control subjects, whereas for female patients it was 34 +/- 3 dB/MHz and 68 +/- 2 for control subjects. VOS was 1603 +/- 7 m/sec for male patients and 1649 +/- 5 for control subjects, and 1573 +/- 7 m/sec for female patients and 1623 +/- 5 for control subjects. All differences between patients and control subjects were significant (p < 0.005). Dual-energy X-ray absorptiometry bone mineral density (BMD) measurements were also performed in seven patients with BUA less than 50 dB/MHz, four of whom were found to have a lumbar spine or femoral neck BMD more than 2.5 SD below the mean value for young adults. This study shows that patients with mental retardation have a marked reduction in BUA and VOS measurements at the heel, compared with age-matched control subjects. There is a need to identify the major causes of low bone mass in this group, as there may be potentially avoidable risk factors for osteoporosis, such as vitamin D deficiency and hypogonadism.

Absorptiometry, Photon↗
PubMed1997

Phosphorylation of two regulatory tyrosine residues in the activation of Bruton's tyrosine kinase via alternative receptors.

Mutation of Bruton's tyrosine kinase (Btk) impairs B cell maturation and function and results in a clinical phenotype of X-linked agammaglobulinemia. Activation of Btk correlates with an increase in the phosphorylation of two regulatory Btk tyrosine residues. Y551 (site 1) within the Src homology type 1 (SH1) domain is transphosphorylated by the Src family tyrosine kinases. Y223 (site 2) is an autophosphorylation site within the Btk SH3 domain. Polyclonal, phosphopeptide-specific antibodies were developed to evaluate the phosphorylation of Btk sites 1 and 2. Crosslinking of the B cell antigen receptor (BCR) or the mast cell Fcepsilon receptor, or interleukin 5 receptor stimulation each induced rapid phosphorylation at Btk sites 1 and 2 in a tightly coupled manner. Btk molecules were singly and doubly tyrosine-phosphorylated. Phosphorylated Btk comprised only a small fraction (</=5%) of the total pool of Btk molecules in the BCR-activated B cells. Increased dosage of Lyn in B cells augmented BCR-induced phosphorylation at both sites. Kinetic analysis supports a sequential activation mechanism in which individual Btk molecules undergo serial transphosphorylation (site 1) then autophosphorylation (site 2), followed by successive dephosphorylation of site 1 then site 2. The phosphorylation of conserved tyrosine residues within structurally related Tec family kinases is likely to regulate their activation.

3T3 Cells↗
PubMed1997

Constitutive membrane association potentiates activation of Bruton tyrosine kinase.

Mutations in the nonreceptor tyrosine kinase Btk result in the B cell immunodeficiencies X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. Genetic and biochemical evidence implicates Btk as a key component of several B cell signaling pathways. Activation of Btk by a point mutation (E41K) within the PH domain (Btk*) results in fibroblast transformation and is correlated with increased membrane localization of Btk. When wild type Btk is activated by coexpression with Lyn, the tyrosine phosphorylated pool of Btk is highly enriched in the membrane fraction. To determine whether membrane association is sufficient to activate Btk, we targeted Btk to the plasma membrane using a series of fusion proteins including GagBtk, CD16Btk and CD4Btk. Constitutive membrane association greatly enhanced the ability of Btk to transform Rat2 fibroblasts in the presence of high levels of Src activity. All membrane targeted forms of Btk were highly tyrosine phosphorylated. Transformation required membrane localization, Btk kinase activity, transphosphorylation by Src family kinases, and an intact SH2 domain but not the PH or SH3 domains. These data suggest that membrane localization is a critical early step in Btk activation.

3T3 Cells↗
PubMed1997

Effect of intermittent cyclical disodium etidronate therapy on bone mineral density in men with vertebral fractures.

OBJECTIVES: to investigate the effects of oral intermittent cyclical etidronate therapy on bone mineral density (BMD) in men with idiopathic vertebral osteoporosis. DESIGN: consecutive case series. SETTING: regional specialist clinic for metabolic bone disease. SUBJECTS: 42 men aged 35-81 (median 60.5) with established vertebral crush fractures and back pain, in whom secondary causes of osteoporosis had been excluded. INTERVENTION: repeated cycles of treatment with oral disodium etidronate 400 mg daily for 14 days followed by oral calcium 500 mg as citrate daily for 76 days. OUTCOME MEASURES: BMD measurement of the lumbar spine and femoral neck by dual energy x-ray absorptiometry at 6-12-month intervals; bone biochemistry (serum calcium, phosphate, alkaline phosphatase and urine calcium/creatinine and hydroxyproline/creatinine ratios) at 6-month intervals. RESULTS: all 42 men have been treated for more than 18 months, and 35 of them for more than 24 months. Median follow-up for the group as a whole is 31 months (range 18-45). The treatment was well tolerated. BMD at the lumbar spine increased by a mean of 0.024 g/cm2 per year of follow-up (95% confidence interval 0.017-0.032 g/cm2). This is equivalent to an average annual rate of change of 3.2% of baseline values. There was a small, non-significant rise in mean BMD at the hip equivalent to 0.7% of baseline values per year. Serum alkaline phosphatase tended to fall in the first 6 months of treatment, returning to baseline values at 2 years. Serum calcium and phosphate were unchanged and no decrease in urinary calcium/creatinine ratio or hydroxyproline/creatinine ratio was seen. CONCLUSIONS: intermittent cyclical etidronate therapy increased lumbar spine BMD over a 2-year period in an unselected group of men with osteoporotic vertebral fractures. This treatment warrants further evaluation in a randomized controlled trial.

Administration, Oral↗
PubMed1997

Differentiation of human CD34+CD38- cord blood stem cells into B cell progenitors in vitro.

Umbilical cord blood CD34+CD38- cells are a rare, quiescent, primitive progenitor subpopulation lacking expression of lymphoid and myeloid lineage specific antigens. Although myeloid, erythroid, and megakaryocytic differentiation from these cells has been described, B lineage differentiation has not been demonstrated previously. We report here that highly enriched human B cell progenitors can be consistently generated from CD34+CD38- cord blood cells using long-term culture on the murine stromal line, S17, in the absence of added cytokines. After 6-8 weeks, cell numbers increased up to 160-fold, and cultures contained > 80-90% CD10+CD19+ B progenitors. Consistent with previous reports describing delayed myeloid cell differentiation from CD34+CD38- cells, production of B cell progenitors from CD34+CD38- cord blood cells was delayed 2-4 weeks relative to cultures initiated with either CD34+CD38bright or CD34+CD38dim progenitors. Addition of Flt3 ligand to long-term cultures resulted in a 2-3-fold greater increase in the number of CD19+ cells generated after 4-8 weeks. The selective outgrowth of B cell progenitors using this culture model will be useful for a range of in vitro studies using primitive hematopoietic progenitors.

ADP-ribosyl Cyclase↗
PubMed1997

Preliminary evidence for cyclosporin A as an alternative in the treatment of recalcitrant juvenile rheumatoid arthritis and juvenile dermatomyositis.

OBJECTIVE: To evaluate the safety and efficacy of cyclosporin A (CyA) with and without methotrexate (MTX) in refractory juvenile rheumatoid arthritis (JRA) and juvenile dermatomyositis (JDMS). METHODS: Twenty-two patients (17 with JRA, 5 with JDMS) with refractory disease were studied retrospectively. All received CyA at a mean dose of 3.2 mg/kg/day over a mean period of 16 mo (range 6-42). All other medications except nonsteroidal antiinflammatory drugs, prednisone, and hydroxychloroquine were discontinued. In addition, 16/22 patients received concomitant MTX. RESULTS: Improvements in laboratory variables, joint counts, joint swelling, and morning stiffness were observed in most of the children with JRA. Muscle strength increased and muscle enzyme levels decreased in the patients with JDMS. CyA treatment permitted prednisone to be discontinued in 5/20 and reduced by greater than 50% in 10/20 patients. There was no evidence of hepatic or bone marrow toxicity or lymphoproliferative disease. Serum creatinine increased in 13/22 patients, but the actual values all remained within normal limits. CONCLUSION: CyA may be an effective agent in the treatment of refractory JRA and JDMS and concomitant MTX seems to be well tolerated. These preliminary data also suggest that combined CyA/MTX therapy may be associated with further improvement in clinical outcome.

Adolescent↗
PubMed1996

Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases.

Bruton's tyrosine kinase (BTK) is pivotal in B cell activation and development through its participation in the signaling pathways of multiple hematopoietic receptors. The mechanisms controlling BTK activation were studied here by examination of the biochemical consequences of an interaction between BTK and SRC family kinases. This interaction of BTK with SRC kinases transphosphorylated BTK on tyrosine at residue 551, which led to BTK activation. BTK then autophosphorylated at a second site. The same two sites were phosphorylated upon B cell antigen receptor cross-linking. The activated BTK was predominantly membrane-associated, which suggests that BTK integrates distinct receptor signals resulting in SRC kinase activation and BTK membrane targeting.

3T3 Cells↗
PubMed1996

Regulation of Btk function by a major autophosphorylation site within the SH3 domain.

Bruton's tyrosine kinase (Btk) plays a crucial role in B cell development. Overexpression of Btk with a Src family kinase increases tyrosine phosphorylation and catalytic activity of Btk. This occurs by transphosphorylation at Y551 in the Btk catalytic domain and the enhancement of Btk autophosphorylation at a second site. A gain-of-function mutant called Btk* containing E41 to K change within the pleckstrin homology domain induces fibroblast transformation. Btk* enhances the transphosphorylation of Y551 by endogenous Src family tyrosine kinases and autophosphorylation at the second site. We mapped the major Btk autophosphorylation site to Y223 within the SH3 domain. Mutation of Y223 to F blocks Btk autophosphorylation and dramatically potentiates the transforming activity of Btk* in fibroblasts. The location of Y223 in a potential ligand-binding pocket suggests that autophosphorylation regulates SH3-mediated signaling by Btk.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1996

Uncertainties in dosemeter intercomparison techniques.

The results of 180 intercomparisons of field dosemeters with an NPL secondary-standard exposure meter (type 2560, with a type 2561 chamber) have been analyzed in order to study variations in readings when ionization chambers are interchanged during the comparison process. It is suggested that the percentage standard error of the mean of the combined series of chamber intercomparisons be used to set a target uncertainty (0.3%), action level (0.5%) and limit of acceptability (1.0%). The target uncertainty is consistent with IAEA estimates of uncertainty for this part of the calibration chain. It is shown that if the calibration procedure is repeated, the percentage difference between the geometric means calculated from each calibration (i.e. a single interchange of chambers) may be as high as +/- 1%. This reinforces the need for at least one repetition of the intercomparison procedure.

Biophysical Phenomena↗
PubMed1996

Assessment of effective dose to staff in brachytherapy.

The aim of this paper is to investigate the problem of monitoring effective dose to hospital staff who are involved in the treatment of tumors using sealed sources placed inside the body (brachytherapy). In addition, the use of an unsealed source to treat the thyroid was also considered. Radiation distributions produced by both sealed sources commonly used in brachytherapy (192I, 137Cs, 226Ra) and an unsealed source used in the treatment of the thyroid (131I) were used to irradiate a Rando phantom. The brachytherapy treatments of esophageal and gynecological carcinoma were simulated. The Rando phantom was loaded with lithium fluoride thermoluminescent dosimeters at positions corresponding to a number of radiosensitive organs. Film badges and electronic personal dosimeters were attached to the Rando phantom at various anatomical sites. The Rando phantom was positioned adjacent to the patient at an angle of 90 degrees to the longitudinal axis of the patient. Irradiations were performed with and without a portable lead screen used on the radiotherapy wards. Effective dose was estimated for each simulated radiotherapy treatment and compared with the personal monitor readings. The data were used as a basis for the provision of advice on the wearing of the film badge dosimeters and the design of portable lead screens. The data also permitted a comparison between the two types of dosimeter when used for personal monitoring in brachytherapy.

Brachytherapy↗
PubMed1996

Regulation of Btk by Src family tyrosine kinases.

Loss of function of Bruton's tyrosine kinase (Btk) results in X-linked immunodeficiencies characterized by a broad spectrum of signaling defects, including those dependent on Src family kinase-linked cell surface receptors. A gain-of-function mutant, Btk*, induces the growth of fibroblasts in soft agar and relieves the interleukin-5 dependence of a pre-B-cell line. To genetically define Btk signaling pathways, we used a strategy to either activate or inactivate Src family kinases in fibroblasts that express Btk*. The transformation potential of Btk* was dramatically increased by coexpression with a partly activated c-Src mutant (E-378 --> G). This synergy was further potentiated by deletion of the Btk Src homology 3 domain. Downregulation of Src family kinases by the C-terminal Src kinase (Csk) suppressed Btk* activation and biological potency. In contrast, kinase-inactive Csk (K-222 --> R), which functioned as a dominant negative molecule, synergized with Btk* in biological transformation. Activation of Btk* correlated with increased phosphotyrosine on transphosphorylation and autophosphorylation sites. These findings suggest that the Src and Btk kinase families form specific signaling units in tissues in which both are expressed.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1995

Long-term culture system for selective growth of human B-cell progenitors.

We describe a simple reproducible system for enrichment and long-term culture of human B-cell progenitors. Enriched CD34+ cord blood mononuclear cells are seeded onto a murine stromal cell line to establish a biphasic culture system. These cultures are characterized by transient growth of myeloid cells followed by outgrowth of cells highly enriched for early B-cell progenitors. Cultures consisting of > 90% early B-lineage cells [expressing CD10, CD19, CD38, and CD45 but lacking CD20, CD22, CD23, and surface IgM] are maintained for > 12 weeks without growth factor addition. Cells remain predominantly germ line at the immunoglobulin locus and express only low levels of cytoplasmic mu chain, terminal deoxynucleotidyltransferase, and recombination-activating gene 1 product. They are unresponsive to the pre-B-cell growth factors interleukin 7 or stem cell factor, or both, suggesting that growth support is provided by a cross-reactive murine stromal cell factor. Cultured B-cell progenitors are generated in large numbers ( > 10(8) cells from a typical cord blood specimen) suitable for use in biochemical analysis and gene-transfer studies. This system should be useful for study of normal and abnormal early human B-lymphopoiesis.

Antigens, CD↗
PubMed1995

The Btk subfamily of cytoplasmic tyrosine kinases: structure, regulation and function.

The genetic defect associated with two closely related primary immunodeficiencies was recently identified as a deficiency of function of a new cytoplasmic tyrosine kinase, Bruton's tyrosine kinase (Btk). Btk and related genes expressed primarily in hematopoietic cells (Itk, Tec, Drsrc28C and Txk) comprise a new subfamily of cytoplasmic tyrosine kinases. These proteins share significant structural and sequence homology including an amino-terminal pleckstrin homology (PH) domain not present in other cytoplasmic tyrosine kinase subfamilies. This domain plays an essential role in regulation and function of the Btk subfamily proteins. Genetic evidence supports a critical role for Btk in B-lineage development. Additional studies demonstrate activation of these proteins in multiple hematopoietic signaling pathways including the B cell antigen receptor, several cytokine receptors, and a potential novel role in heterotrimeric G protein associated receptor signaling.

Agammaglobulinaemia Tyrosine Kinase↗