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Biomedical subjects

P Lock

Publications and source records attributed to P Lock.

16 recordsLinked to original sources
PubMed1998

A new method for isolating tyrosine kinase substrates used to identify fish, an SH3 and PX domain-containing protein, and Src substrate.

We describe a method for identifying tyrosine kinase substrates using anti-phosphotyrosine antibodies to screen tyrosine-phosphorylated cDNA expression libraries. Several potential Src substrates were identified including Fish, which has five SH3 domains and a recently discovered phox homology (PX) domain. Fish is tyrosine-phosphorylated in Src-transformed fibroblasts (suggesting that it is a target of Src in vivo) and in normal cells following treatment with several growth factors. Treatment of cells with cytochalasin D also resulted in rapid tyrosine phosphorylation of Fish, concomitant with activation of Src. These data suggest that Fish is involved in signalling by tyrosine kinases, and imply a specialized role in the actin cytoskeleton.

3T3 Cells↗
PubMed1994

Cross-species receptor binding characteristics of human and mouse leukemia inhibitory factor suggest a complex binding interaction.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine whose activities appear to be mediated through a single heterodimeric receptor complex. Human LIF (hLIF) can bind to and activate mouse LIF (mLIF) receptors but mLIF is unable to bind to hLIF receptors. Cross-species competition of mLIF and hLIF for binding to the mLIF receptor was found to be dependent on which ligand was used as the radioactive tracer (Layton, M. J., Cross, B. A., Metcalf, D., Ward, L. D., Simpson, R. J., and Nicola, N. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8616-8620), and this phenomenon was investigated in the present study. We found that hLIF bound to the low affinity mLIF receptor with a 100-500-fold higher primary affinity and lower kinetic dissociation rate than mLIF, but both ligands displayed a single rate of ligand dissociation. In contrast, the binding of hLIF to low and high affinity hLIF receptors revealed two classes of binding site. The observed tracer-dependent phenomena suggested that both mLIF and hLIF interfere with the binding of each other to the mLIF receptor. A model is presented in which hLIF binds to two sites on mLIF and hLIF receptors, one of which interferes with the common site for mLIF. This model may reconcile some of the observed complexities of LIF/LIF receptor interactions.

Animals↗
PubMed1994

CD4-mediated enhancement or inhibition of T cell activation does not require the CD4:p56lck association.

CD4 is the coreceptor molecule expressed on the surface of T cells specific for or restricted by class II molecules of the major histocompatibility complex (MHC). Its expression on T cells is required for an optimal response to antigen (Ag). Three mechanisms have been invoked for the involvement of CD4 in T cell activation. First, it was shown that CD4 binds to MHC class II molecules on antigen presenting cells (APCs) thereby favoring an adhesion between effector cells and APCs. Association of CD4 to the T cell receptor and to the tyrosine kinase p56lck have also been shown to be critically involved in the positive function of CD4. Here, we demonstrate that the interaction of CD4 with p56lck is not required to enhance the response of two CD4-dependent, Ag-specific T cell hybridomas. Mutant forms of CD4 (TCD4), which lose association to p56lck, were expressed in these T cells and were shown to enhance the Ag-specific response as efficiently as the wild-type CD4. Moreover both CD4-dependent and independent T cell responses were inhibited by CD4-specific mAbs even when CD4 was not associated with p56lck. These results indicate that mechanisms distinct from sequestration of p56lck and/or negative signaling operate in these inhibitions. Results demonstrating enhancement of TCR-mediated signaling by the coaggregation of TCD4 mutant to the TCR further confirm that the association of p56lck to CD4 is not absolutely required for the regulatory functions of CD4. Our results suggest that the mechanisms implicated in the enhancement of T cell stimulation via CD4 depend solely on the extracellular and transmembrane domains of CD4.

Animals↗
PubMed1994

Histidine-367 of the human common beta chain of the receptor is critical for high-affinity binding of human granulocyte-macrophage colony-stimulating factor.

High-affinity receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3, and interleukin 5 consist of ligand-specific alpha chains (low-affinity subunits) and a common beta chain (beta c) that converts each complex to a high-affinity form. Although beta c alone has no detectable cytokine-binding activity, amino acid substitutions for Glu-21 of human GM-CSF significantly reduce high-affinity but not low-affinity binding, implying that beta c interacts directly with GM-CSF during formation of the high-affinity receptor but only in the presence of the alpha chain. A potential GM-CSF-binding determinant was identified in the second hemopoietin domain of beta c, and the role of individual residues within this region was investigated by determining the ability of mutated beta c chains to confer high-affinity binding when coexpressed with the alpha subunit of the GM-CSF receptor in COS cells. Substitutions involving Met-363, Arg-364, Tyr-365, and Glu-366 did not affect high-affinity binding. However, substitution of His-367 by lysine or glutamine abolished high-affinity binding, suggesting that this residue may form an important part of the high-affinity GM-CSF-binding determinant. Consistent with the loss of high-affinity binding, higher concentrations of human GM-CSF were required to stimulate proliferation of CTLL-2 cell lines transfected with cDNAs for GM-CSF receptor alpha chain and His-367 beta c mutant than those expressing GM-CSF receptor alpha subunit and beta c wild type.

Amino Acid Sequence↗
PubMed1994

Reliability of the Spanish version of the Nottingham Health Profile in patients with stable end-stage renal disease.

OBJECTIVE: Since reproducibility of results is a basic prerequisite of health status measures for its use in prospective and evaluative studies, the reliability of the Spanish version of the Nottingham Health Profile (NHP), a multi-dimensional perceived health status measure, was assessed in a sample of stable end-stage renal disease (ESRD) patients. METHODS: The NHP was administered on two occasions four weeks apart to a group of hospital hemodialysis program patients who were clinically stable according to their physicians. Correlations of scores and agreement of first and second administrations were assessed together with internal consistency. Afterwards, analyses were repeated taking into account the time (before, during or after the dialysis) and the method of administration (self vs interviewer), and the interviewer. RESULTS: Spearman correlation coefficients (rs) between responses to the first and to the second administration were > 0.6 for all of the six dimensions of the NHP (range = 0.69-0.85) and in every sub-group analyzed (P < 0.01). Agreement percent (AP) between items was > 0.4 (0.48-0.65). Internal consistency was 0.91 for the whole profile and > 0.5 (0.58-0.86) when analyzed by individual dimensions. Reliability did not vary significantly with the time nor the method of administration (self or interviewer). CONCLUSIONS: Overall, results suggest that the Spanish version of NHP is sufficiently reliable to be used in ESRD patients. While a higher reliability would have been achieved by a shorter retest period, the study provides a realistic approximation to the reliability of the questionnaire in actual research and clinical applications.

Female↗
PubMed1994

Receptor insertion into factor-dependent murine cell lines to develop specific bioassays for murine G-CSF and M-CSF and human GM-CSF.

cDNAs encoding the receptors for murine G-CSF, M-CSF or human GM-CSF were inserted into the murine hemopoietic continuous cell lines Ba/F3 or FDC-P1 and sublines selected that were then responsive to proliferative stimulation by these growth factors. When used in microwell assays the Ba/F3 G-CSF receptor-expressing cell line was able to detect 100 pg G-CSF per ml, the Ba/F3 M-CSF receptor-expressing cell line 100-400 pg M-CSF per ml and the FDC-P1 line expressing the alpha- and beta-chains of the human GM-CSF receptor detected 5-10 pg/ml of GM-CSF in test material. These cell lines appear satisfactory for use as selective bioassays for these colony stimulating factors in material potentially containing a mixture of growth factors.

Animals↗
PubMed1993

Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF-R), while mouse LIF (mLIF) binds only to mouse LIF-R. Moreover, hLIF binds with higher affinity to the mLIF-R than does mLIF. In order to define the regions of the hLIF molecule responsible for species-specific interaction with the hLIF-R and for the unusual high-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mapped to the same region of the hLIF molecule. The predominant contribution was from residues in the loop linking the third and fourth helices, with lesser contributions from residues in the third helix and the loop connecting the second and third helices in the predicted three-dimensional structure. Since all chimeras retained full biological activity and receptor-binding activity on mouse cells, and there was little variation in the specific biological activity of the purified proteins, it can be concluded that the overall secondary and tertiary structures of each chimera were intact. This observation also implied that the primary binding sites on mLIF and hLIF for the mLIF-R were unaltered by inter-species domain swapping. Consequently, the site on the hLIF molecule that confers species-specific binding to the hLIF-R and higher affinity binding to the mLIF-R, must constitute an additional interaction site to that used by both mLIF and hLIF to bind to the mLIF-R. These studies define a maximum of 15 amino acid differences between hLIF and mLIF that are responsible for the different properties of these proteins.

Amino Acid Sequence↗
PubMed1992

Lipopolysaccharide- and interferon-gamma-induced expression of hck and lyn tyrosine kinases in murine bone marrow-derived macrophages.

We have examined the role of tyrosine phosphorylation during the course of macrophage activation. Initial experiments indicated that vanadate, a known phosphotyrosine phosphatase inhibitor, enhanced the phorbol 12-myristate 13-acetate (PMA)-triggered respiratory burst and potentiated the priming effects of bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), suggesting that tyrosine phosphorylation may be important in these end cell functions. As src-related kinases have been implicated in the activation of cells of other haemopoietic lineages, we examined the relationship between the activity of two such kinases, hck and lyn, and priming of the respiratory burst. We found that the level of hck and lyn is increased following exposure of bone marrow-derived macrophages (BMM) to LPS or IFN-gamma. The induction of both of these kinases follows similar kinetics with maximal activity occurring at 24-48 h. Interestingly, the kinetics of induction of hck and lyn kinase activity in BMM demonstrated a close temporal relationship with the priming effects of LPS and IFN-gamma on the macrophage respiratory burst. Collectively, these observations raise the possibility that modulation of expression of hck and lyn is involved in the regulation of the respiratory burst.

Animals↗
PubMed1991

Alternatively spliced murine lyn mRNAs encode distinct proteins.

Two lyn proteins of 56 and 53 kDa have been observed in immunoprecipitates from a variety of murine and human cell lines and tissues. We report the cloning and nucleotide sequence of two distinct murine lyn cDNAs isolated from an FDC-P1 cDNA library. One of the cDNAs, designated lyn11, encodes a protein of 56 kDa which shares 96% similarity with human lyn. The other cDNA, designated lyn12, encodes a protein of 53 kDa. The proteins differ in the presence or absence of a 21-amino-acid sequence located 24 amino acids C terminal of the translational initiation codon. Using RNase protection analysis, we have identified mRNAs corresponding to both cDNAs in murine cell lines and tissues. Sequence analysis of murine genomic clones suggests that the distinct mRNAs are alternatively spliced transcripts derived from a single gene. Expression of both cDNAs in COS cells leads to the production of lyn proteins with the same molecular weight as the two forms of lyn proteins immunoprecipitated from extracts of FDC-P1 cells and mouse spleen. Subcellular fractionation studies and Western immunoblotting analysis suggest that both isoforms of lyn are membrane associated. The association of both lyn isoforms with the membrane fraction supports the notion that lyn, like other src-related kinases, may interact with the intracellular domain of cell surface receptors.

Amino Acid Sequence↗
PubMed1991

Two isoforms of murine hck, generated by utilization of alternative translational initiation codons, exhibit different patterns of subcellular localization.

Mammalian hck, a member of the src family of tyrosine kinases, is expressed predominantly in cells of the myeloid and B-lymphoid lineages. Using mutational analysis, we have investigated the molecular basis of two immunoreactive forms of murine hck of 56 and 59 kDa found in numerous hemopoietic cell types. Our results indicate that translation of murine p59hck initiates from a CTG codon located 21 codons 5' of an ATG that is utilized to generate p56hck. We provide evidence that two human hck isoforms are generated by the same mechanism. Subcellular fractionation studies reveal that while p59hck and p56hck are associated with membranes of various murine B-lymphoid and myeloid cell lines, p59hck alone is also located in the cytosol. In contrast to membrane-associated p59hck, which is metabolically labeled with [3H]myristic acid and exhibits amphiphilic properties in Triton X-114 detergent, cytosolic p59hck is hydrophilic, suggesting that it is not acylated. Possible mechanisms are proposed to account for these observations.

Amino Acid Sequence↗
PubMed1990

Cloning and characterization of the rat alpha-inhibin gene.

The gene for the rat glycoprotein hormone alpha-inhibin has been cloned and characterized. The entire gene was found to be contained within a 5.5 kilobase EcoRI fragment. It is composed of two exons separated by a 1.5 kb intron. Primer extension and S1 nuclease analysis showed that the major transcription initiation site in the ovary was 77 bp from the start of translation. The promoter region of the gene did not contain a conventional TATA box but instead a number of GA rich repeated sequences were found to be present. Other potential regulatory elements found included a repeating purine-pyrimidine tract (TG)28, cAMP and phorbol ester response elements and a putative glucocorticoid response element. Southern blot analysis of rat genomic DNA indicated that there is a single gene for alpha-inhibin in the rat.

Amino Acid Sequence↗
PubMed1990

Functional analysis and nucleotide sequence of the promoter region of the murine hck gene.

The structure and function of the promoter region and exon 1 of the murine hck gene have been characterized in detail. RNase protection analysis has established that hck transcripts initiate from heterogeneous start sites located within the hck gene. Fusion gene constructs containing hck 5'-flanking sequences and the bacterial Neor gene have been introduced into the hematopoietic cell lines FDC-P1 and WEHI-265 by using a self-inactivating retroviral vector. The transcriptional start sites of the fusion gene are essentially identical to those of the endogenous hck gene. Analysis of infected WEHI-265 cell lines treated with bacterial lipopolysaccharide (LPS) reveals a 3- to 5-fold elevation in the levels of endogenous hck mRNA and a 1.4- to 2.6-fold increase in the level of Neor fusion gene transcripts, indicating that hck 5'-flanking sequences are capable of conferring LPS responsiveness on the Neor gene. The 5'-flanking region of the hck gene contains sequences similar to an element which is thought to be involved in the LPS responsiveness of the class II major histocompatibility gene A alpha k. A subset of these sequences are also found in the 5'-flanking regions of other LPS-responsive genes. Moreover, this motif is related to the consensus binding sequence of NF-kappa B, a transcription factor which is known to be regulated by LPS.

Animals↗
PubMed1980

Triage and the patient with renal failure.

The call for 'triage' as a specific policy for the selection of patients presenting with chronic renal failure, in the light of increasingly limited resources prompted us to question nephrologist on their bases for selection. We discovered no absolute criteria for rejection, but a consensus of opinion against those with additional and complicating factors to their renal disease such as age, hepatitis carriers and mental illness-a bias seen throughout the National Health Service. In this paper we discuss the validity of such criteria, the implications of the currently pragmatic and often covert practice of selection, and in this potentially finite area of demand we question the rationale for the limitation of resources.

Cost-Benefit Analysis↗