Biomedical subjects
Publications and source records attributed to M L Thomas.
CD45 is a leukocyte transmembrane protein tyrosine phosphatase that functions in lymphocyte activation by increasing the kinase activity of Src family members. In contrast, an intracellular protein tyrosine phosphatase that contains Src homology 2 domains, SHP, is required to inactivate receptors. Recent studies have examined the mechanisms of interaction and regulation of these enzymes.
A method for vapor-dosing of charcoal sorbent tubes (CST) that does not require the expense and effort of a test chamber was used to test the desorption efficiency (DE) of seven solvent vapors, representing six classes of solvents as follows: aromatic hydrocarbons (m-xylene); ether/alcohol (2-ethoxyethanol); vinyl monomers (styrene monomer, vinyl acetate); aliphatic hydrocarbons (n-hexane); aliphatic esters (n-butyl acetate); and aliphatic acrylic monomers (methyl methacrylate). The quantities of the solvents used in these experiments would represent eight-hour exposures to concentrations of approximately 0.2 to 10 ppm. The vapor-dosing experimental system consisted of a loaded filter cassette connected directly to a CST. Vapor was generated by injecting liquid solvent onto the glass fiber filter and drawing air through the system. The solvent was desorbed from the filter and charcoal for analysis. Vapor desorption efficiency was determined from the fraction of the injected solvent evaporated from the filter and the amount recovered from the charcoal. The measured DEs were similar to those reported for liquid dosed charcoal. Vapor dosing of sorbent tubes is more representative of samples collected for industrial hygiene exposure assessment. The system is simple to use and applicable for vapor dosing of any sorbent tube.
BACKGROUND: This study was designed to determine whether the genes for both parathyroid hormone-related peptide (PTHrP) and its receptor were expressed in close proximity to one another in various regions of the gut and whether they both were evident in two intestinal epithelial cell lines. The findings would test the idea that PTHrP acts as an autocrine or paracrine factor in the gut. EXPERIMENTAL DESIGN: Reverse transcription/PCR and Northern analysis were used to detect mRNA for PTHrP and its receptor in various regions of the normal rat gut, in epithelial cell populations isolated along the villus tip-crypt axis in rat jejunum, and in a rat and a human gastrointestinal epithelial cell line (IEC-6 and LoVo, respectively). Antisera to PTHrP also were used to detect the peptide in rat gastrointestinal tissues or in growth medium from cultured cells. RESULTS: Both PTHrP and PTHrP receptor mRNA were found in all regions of the rat gut, in all cell populations isolated from rat jejunum, and in the rat and human cell lines. Immunoreactive PTHrP in tissue sections was observed in rat jejunal epithelial cells all along the villus but not in crypt cells, and immunoreactive peptide was found in growth medium from the cultured intestinal epithelial cells. CONCLUSIONS: The results show that genes for PTHrP and its receptor are expressed widely in gut epithelia. Expression of the two genes in the same regions of the gut and in the same cell line implies a local autocrine/paracrine action of the peptide. Expression of the peptide in villus epithelium but not in crypt cells suggests a role in differentiating gastrointestinal epithelial cells.
We have investigated the pattern of PTPase transcript expression during in vitro differentiation of mouse embryonal carcinoma (F9) cells. While the transcripts of most PTPases were unchanged or undetected during embryonal differentiation induced by retinoic acid, several PTPase transcripts exhibited distinct patterns of induction. Mutant cells defective in differentiation did not display the induction of some of these PTPase transcripts. Interestingly, three out of the four PTPase transcripts induced were the same PTPase transcripts induced during in vitro erythroid differentiation of mouse erythroleukemia (MEL) cells [(1994) J. Biol. Chem. 269, 4709-4712] [corrected]. The possible role played by specific PTPases in cell differentiation is discussed.
We reported previously that most of the phosphotyrosine-containing cellular proteins were quickly dephosphorylated at the very early stage of erythroid differentiation of mouse erythroleukemia (MEL) cells. These and other experimental results implicated a specific protein tyrosine phosphatase(s) (PTPase(s)) involved in the commitment of the erythroid differentiation. We have investigated the pattern of transcripts of PTPases during MEL cell differentiation and found that while the transcripts of most PTPases were unchanged or undetected in the cells, transcripts for two PTPases (PTP beta 2 and RIP) exhibited distinct patterns of induction at a very early stage of differentiation. Some of the mutant cells defective in differentiation did not show the induction of these PTPase transcripts. We discuss the possible role played by the PTPases in the commitment of MEL cell differentiation.
The IEC-6 cell line was derived from newborn rat small intestinal crypts and maintains characteristics of nontransformed crypt epithelial cells. We have reported that alkaline phosphatase (ALP) activity, a differentiation marker for intestinal epithelial cells, is regulated in IEC-6 cells by 1,25-dihydroxycholecalciferol (1,25(OH)2D3). Using reverse transcription polymerase chain reaction, we determined that the ALP in IEC-6 cells is the liver/bone/kidney ALP (LALP) isoenzyme and not the mature enterocyte form. Levamisole, a specific blocker for LALP, blocked the ALP activity of IEC-6 cells. Kinetic studies showed that 1,25(OH)2D3 increases Vm values of ALP in IEC-6 cells while Km values remain the same. Northern analysis of LALP mRNA levels in IEC-6 cells indicated that 10(-7) M 1,25(OH)2D3 acts by increasing alkaline phosphatase activity at the gene expression level.
In vivo and epidemiological data suggest a mitogenic role for estrogens (E) in colon cancer. The presence of estrogen receptor (ER) and ER mRNA in colonic epithelium and colon cancer cells, make it necessary to explore the possible direct effects of E on colon cancer growth. In this study, a 15-mer oligodoxynucleotide (oligo) antisense to the region of the translation start codon of estrogen receptor mRNA inhibited ER expression in a mouse colon cancer cell line (MC-26), as determined by receptor binding assay. Antisense oligo also decreased ER mRNA levels in MC-26 cells. The growth-stimulatory effect of E was abolished by antisense oligo treatment, demonstrating that the ER is directly involved in the regulation of colon cancer cell growth.
The expression of transmembrane protein tyrosine phosphatase CD45 is required for antigen-induced activation of T and B lymphocytes. During the past year, advances have been made in our understanding of CD45 regulation and its role in regulating Src-family members. The importance of CD45 in thymocyte maturation has been demonstrated through the generation of CD45-knockout mice.
CD45, the leukocyte-common antigen, is a transmembrane protein tyrosine phosphatase uniquely expressed by cells of hematopoietic origin. We have developed CD4+ and CD8+ T cell clones that are deficient in the expression of CD45 and have previously shown that these cells fail to proliferate in response to antigen or cross-linked CD3. These studies have now been extended to show that stimulation with anti-Thy-1, a mitogenic signal for the CD4+CD45+ and CD8+CD45+ T cells, fails to induce proliferation in the CD45- T cells. Examination of the CD8+CD45- T cells correlates anti-Thy-1 unresponsiveness with a failure to increase in tyrosine phosphorylation. Furthermore, stimulation of CD8+CD45+ T cells with anti-Thy-1 results in an increase in p56lck activity but not in CD8+CD45- T cells. In contrast to the results with anti-Thy-1, both the CD4+CD45- and CD8+CD45- T cells respond to treatment with lectin mitogens, concanavalin A or phytohemagglutinin. Lectin-induced proliferation was inhibited by the addition of cyclosporin A. Treatment of CD45- T cells with PMA and ionomycin also results in proliferation indicating that activation of protein kinase C in conjunction with an increase in intracellular calcium rescues the defect caused by CD45 deficiency. The data suggest that CD45 is required for the activation of tyrosine kinase activity immediate or prior to transmembrane signaling.
PEP is an intracellular protein tyrosine phosphatase expressed primarily by cells of hematopoietic origin that can be divided structurally into a catalytic domain and a large carboxy-terminal domain. The carboxy-terminal domain is enriched in proline, glutamic acid, serine, and threonine residues (PEST sequences) and contains a nonperfect tandem repeat sequence enriched in proline residues and a carboxy terminus enriched in basic amino acids. Here we show that PEP is diffusely expressed in lymphoid tissues, consistent with expression by many different cell types. Analysis of the PEP protein identifies a nuclear localization sequence within the extreme carboxy terminus. Transfer of 18 amino acids from the carboxy terminus of PEP to beta-galactosidase conferred nuclear localization, indicating that this sequence was sufficient for nuclear localization. Proteins enriched in PEST sequences are often rapidly degraded. However, pulse-chase analysis indicates that PEP has a half-life of greater than 5 h.
CD45 is one of the most abundant leukocyte cell surface glycoproteins and is expressed exclusively upon cells of the hematopoietic system. Different isoforms of CD45 are generated by alternative splicing and are expressed in cell type-specific patterns on functional subpopulations of lymphocytes. In a major advance, CD45 was identified as one of the first members of a novel class of enzymes, the protein tyrosine phosphatases (PTPs). This serendipitous discovery linked CD45 to the process of reversible protein tyrosine phosphorylation, a key regulatory mechanism for controlling the growth and division of eukaryotic cells, and provided the impetus for most of the studies described in this review. CD45 is now established as a critical component of the signal transduction machinery of lymphocytes. In particular, evidence from genetic experiments indicates that CD45 plays a pivotal role in antigen-stimulated proliferation of T lymphocytes and in thymic development. Two members of the Src-family of protein tyrosine kinases (PTKs), the p56lck and p59fyn proteins, have been implicated as physiological substrates of CD45, providing an important clue to how the action of this leukocyte-specific PTP might influence signaling by the T cell antigen receptor. Structure-function analysis of CD45 and other PTPs has identified structural features of PTP catalytic domains required for enzymatic activity. However, despite intensive efforts, little is known about how the activity of CD45 is regulated. The external domain of CD45 does not appear to be absolutely required for signal transduction by the T cell receptor, and there is currently no evidence that ligand binding modulates CD45 activity. Analysis of CD45 isoform expression has revealed a hitherto unrecognized plasticity in isoform usage by T cells and other leukocytes, adding to the regulatory complexity of isoform expression.
Weight training (WT) as a countermeasure to +Gz intolerance seems to be an accepted modality, but lacks convincing support in the research literature. This study examines factors associated with WT in an operational environment as a countermeasure to +Gz intolerance. Fourteen naval aviators were assigned to a WT group (n = 7) and a non-weight-trained control (NWT) group (n = 7). All subjects were tested before and after a 10-week training period for aerobic capacity (VO2max), dynamic muscular strength (Cybex II), body fat, gradual onset rate (GOR) for +Gz at a rate of +1 G.15 s-1, and finally a simulated air combat maneuver (SACM) consisting of 15 s at +4.5 Gz and +7 Gz alternating challenges to exhaustion. The results indicate the following: 1) no significant differences in any of the centrifuge variables measured between WT and NWT groups while both groups showed a time or learning effect in SACM from 175 +/- 109 to 239 +/- 110 s (grouped data df = 16, t = 2.97, p < 0.09); 2) no improvement in SACM time for subjects achieving a 300-s SACM pretest value (n = 4); and 3) for SACM times below 300 s, the WT subjects showed a greater improvement than NWT subjects (n = 11, F = 9.12, p < 0.02). The data suggest that although some subjects showed considerable strength gains, WT in a naval aviation operational setting may not provide a universal desired effect.(ABSTRACT TRUNCATED AT 250 WORDS)
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The transmembrane tyrosine phosphatase CD45 is expressed in multiple isoforms on all nucleated hematopoietic cells, resulting from alternative splicing of variable exons. We generated mice with a mutation in the variable CD45 exon 6, using homologous recombination. In mice homozygous for the CD45-exon6 mutation, B cells and most T cells did not express CD45. Development of B cells appeared normal, although Ig mu-induced proliferation was completely abrogated. Thymocyte maturation was blocked at the transitional stage from immature CD4+CD8+ to mature CD4+ or CD8+ cells, and only a few T cells could be detected in peripheral lymphoid organs. Clonal deletion of superantigen-reactive T cells still occurred. Cytotoxic T cell responses to lymphocytic choriomeningitis virus were absent in CD45-exon6-/- mice. These data imply that CD45 is differentially required for the development and function of B and T lymphocytes.
Mice homozygous for the recessive allelic mutation motheaten (me) or viable motheaten (mev) on chromosome 6 develop severe defects in hematopoiesis. In this paper we present the findings that the me and mev mutations are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene. High resolution mapping localized me to an area tightly linked to Hcph on chromosome 6. Abnormalities of the Hcph protein product were demonstrated by Western blot analysis and by activity assays in both me/me and mev/mev mice. Molecular analysis of the Hcph cDNA identified abnormal transcripts in both mutants. DNA sequence analyses of cDNA and genomic clones revealed that both the me and mev mutations are point mutations that result in aberrant splicing of the Hcph transcript. These findings provide the first available animal models for a specific protein-tyrosine phosphatase deficiency, thus facilitating determination of the precise role of this signaling molecule in hematopoiesis.
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Stimulation of tyrosine phosphorylation is an early and important event in antigen-induced T-cell activation. T-cell clones deficient in expression of CD45, a transmembrane protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), are impaired in their ability to respond to either antigen or T-cell receptor cross-linking. Analysis of the CD45-deficient CD8+ T-cell clone L3M-93 demonstrates that the Src family members p56lck and p59fyn show increased immunoreactivity with anti-phosphotyrosine antibody and exhibit decreased kinase activity. The site of increased tyrosine phosphorylation in Src family members was identified by comparison of cyanogen bromide peptide maps. Phosphorylation of the C-terminal phosphopeptide, containing the negative regulatory site of tyrosine phosphorylation, from the CD45-deficient cells was increased 8-fold for p56lck and 2-fold for p59fyn. These data suggest that CD45 dephosphorylates the negative regulatory site of multiple Src family members in the cytotoxic T-lymphocyte clone L3 and show a correlation between the ability to respond efficiently to antigen and the dephosphorylation of Src family members by CD45.
Venous reflux was assessed by duplex scanning and descending phlebography in 98 limbs of 52 patients. There was moderately good agreement for deep vein reflux elicited by a standardised Valsalva manoeuvre, classified by a modified Kistner grading (0 to IV). The same grade was found in 58 limbs, with a difference of one grade in 27 limbs, and more than one grade in 13. Descending phlebography underestimated reflux in the lower leg compared with duplex scanning. The presence of reflux found on duplex scan correlated with the half-volume refilling time on a foot-volume plethysmogram with a below-knee tourniquet. There was no correlation between descending phlebography and the half-refilling time. Ascending phlebography showed post-thrombotic changes in 23 limbs. The duplex scan confirmed severe reflux (grades III and IV) in 14 of these 23 limbs, and demonstrated isolated reflux below normal valves in 4 other limbs. Severe reflux was seen on the descending phlebogram in only 10 of the 23 limbs with post-thrombotic changes. Duplex scanning may be a better method for detecting reflux than descending phlebography and is more acceptable to the patient. Ascending phlebography remains essential to display anatomical abnormalities found in some postthrombotic limbs that are not associated with reflux.