Biomedical subjects

L J Thomas

Publications and source records attributed to L J Thomas.

At least 37 records · Page 2Linked to original sources

PubMed1991

Functional analysis of T-cell mutants defective in the biosynthesis of glycosylphosphatidylinositol anchor. Relative importance of glycosylphosphatidylinositol anchor versus N-linked glycosylation in T-cell activation.

The glycosylphosphatidylinositol (GPI) anchor, potentially capable of generating a number of second messengers, such as diacylglycerol, phosphatidic acid, and inositol phosphate glycan, has been postulated to be involved in signal transduction in various cell types, including T-cells. We have identified a panel of T-cell hybridoma mutants that are defective at various steps of GPI anchor biosynthesis. Since they were derived from a functional T-T hybridoma, we were able to determine the precise role of the GPI anchor in T-cell activation. Two mutants were chosen for this analysis. The first mutant is defective at the first step of GPI anchor biosynthesis, i.e. in the transfer of N-acetylglucosamine to a phosphatidylinositol acceptor. Thus, it cannot form any GPI precursors or GPI-like compounds. Interestingly, this mutant can be activated by antigen, superantigen, and concanavalin A in a manner comparable to the wild-type hybridoma. These data strongly suggest that the GPI anchor, its precursor, or its potential cleavage product, inositol phosphate glycan, is not required for the early phase of T-cell activation. The second mutant is able to synthesize the first two GPI precursors, but is not able to add mannose residues to them due to a deficiency in dolichol-phosphate-mannose (Dol-P-Man) biosynthesis. Unexpectedly, all of the Dol-P-Man mutants are defective in activation by antigen, suprantigen, and concanavalin A despite normal T-cell receptor expression. Here, we show that the activation defect was due to a pleiotropic glycosylation abnormality because Dol-P-Man is required for both GPI anchor and N-linked oligosaccharide biosynthesis. When the yeast Dol-P-Man synthase gene was stably transfected into the mutants, full expression of surface GPI-anchored proteins was restored. However, N-linked glycosylation was either partially or completely corrected in different transfectants. Reconstitution of activation defects correlates well with the status of N-linked glycosylation, but not with the expression of GPI-anchored proteins. These results thus reveal an unexpected role of N-linked glycosylation in T-cell activation.

Antigens, CD↗

PubMed1991

Identification of defects in glycosylphosphatidylinositol anchor biosynthesis in the Thy-1 expression mutants.

A number of eukaryotic proteins are anchored to the membrane by glycosylphosphatidylinositol (GPI), of which the core structure is conserved from protozoan to mammalian cells. Here, we used a panel of thymoma mutants, which synthesize Thy-1 but cannot express it on the cell surface, to study the GPI biosynthetic pathway in mammalian cells. These mutants have been assigned into six complementation classes (A, B, C, E, F, H) by the technique of somatic cell hybridization. Using a combination of metabolic labeling and chemical/enzymatic tests, the biosynthetic defects were mapped to four different steps. Class A, C, and H mutants cannot transfer N-acetylglucosamine (GlcNAc) to a phosphatidylinositol acceptor, suggesting that the first step of GPI synthesis is regulated by at least three genes. The Class E mutant does not synthesize dolichol-phosphate-mannose, the donor for the first mannose residue transferred to the GPI core, and thus cannot form any mannose-containing GPI precursors. Class B and F mutants are defective in the addition of the third mannose residue or ethanolamine phosphate, respectively, to the elongating GPI core. Our findings have implications for the biosynthesis and attachment of the mammalian GPI anchor.

Autoradiography↗

PubMed1990

Correction of a defect in mammalian GPI anchor biosynthesis by a transfected yeast gene.

Glycosylphosphatidylinositol (GPI) serves as a membrane anchor for a large number of eukaryotic proteins. A genetic approach was used to investigate the biosynthesis of GPI anchor precursors in mammalian cells. T cell hybridoma mutants that cannot synthesize dolichol-phosphate-mannose (Dol-P-Man) also do not express on their surface GPI-anchored proteins such as Thy-1 and Ly-6A. These mutants cannot form mannose-containing GPI precursors. Transfection with the yeast Dol-P-Man synthase gene rescues the synthesis of both Dol-P-Man and mannose-containing GPI precursors, as well as the surface expression of Thy-1 and Ly-6A, suggesting that Dol-P-Man is the donor of at least one mannose residue in the GPI core.

Animals↗

PubMed1990

The relation of diabetic control to in vivo pH of soft tissue abscesses.

It has been shown that induced soft tissue abscesses have a lower intra-abscess pH in the uncontrolled diabetic host than in the nondiabetic control. These differences were felt to be secondary to alterations in white cell metabolism. The current study compares the intra-abscess pH in three groups of mice: (I) nondiabetic, (II) untreated diabetic, and (III) insulin-treated diabetic. Diabetes was induced with streptozotocin in male white mice. The bacteria used to induce the abscesses were a combination of B. fragilis and Enterococcus. The blood glucose values of groups I, II, and III were 189 mg% (+/- 20.3), 256 mg% (+/- 121.9), and 712.8 mg% (+/- 169.7), respectively. None of the animals were ketotic, and peritoneal pH (reflecting systemic pH) showed no significant differences between groups. There were no significant differences in colony counts between any groups. The intra-abscess pH values of groups I, II, and III were 6.97 (+/- 0.26), 6.85 (+/- 0.41), and 6.08 (+/- 0.70). The differences in intra-abscess pH and blood glucose levels were all significantly different from each other when all three groups were compared. The insulin-treated mice tended to return to normality but had the widest spread of values. Since a decrease in intra-abscess pH has been felt to be a reflection of white cell activity, our studies may be the first to demonstrate an in vivo effect of insulin on white cell activity.

Abscess↗

PubMed1990

Spatial normalization of one-dimensional electrophoretic gel images.

A strategy for using processed, digitized images of one-dimensional electrophoretic gels to facilitate the analysis of large sets of overlapping clones is described. The images are acquired from fluorescently stained gels or from transilluminated gel photographs using a cooled, solid-state charge-coupled device camera. By employing sets of bands in the size-standard lanes as reference points, all the gel images are spatially normalized to a common reference template. After normalization, lane images from different gels can be compared as though the gels had been electrophoresed under identical, uniform-field conditions. Applications of this procedure to the analysis of a large set of overlapping lambda clones from chromosome VII of Saccharomyces cerevisiae and to the estimation of fragment sizes are illustrated.

Analog-Digital Conversion↗

PubMed1989

Time-dependent cleavage of a high-mannose form of Ii to p25 in an intracellular compartment.

The cleavage of a high-mannose form of Ii to p25 was demonstrated in an intracellular compartment of B cells. Subcellular fractions of 72 hr-activated B cells, separated by Percoll density gradient centrifugation, were immunoprecipitated with anti-class II or anti-Ii serum and characterized for 5'-nucleotidase, acid phosphatase, and radiolabeled transferrin. The cleavage of p25 from Ii as a C-terminal fragment occurred from 20 to 60 min after synthesis in an intracellular compartment which was intermediate in density between lysosomal and plasma membrane fractions and coincided with the lighter to two internalized transferrin compartments. Chloroquine or monensin treatments, at maximal nontoxic doses, which block Golgi and lysosomal functions, did not seem to alter the cleavage of Ii to p25. p25 molecules were reduced to about 10,500 daltons by treatment with endoglycosidases F or H. We conclude that p25 was generated from a high mannose form of Ii in the endoplasmic reticulum or cis-Golgi. This finding could either implicate that site for class II MHC desetope charging with foreign peptides or reflect a mechanism for degradation of "excess" Ii molecules.

Chemical Phenomena↗

PubMed1989

In vivo pH of induced soft-tissue abscesses in diabetic and nondiabetic mice.

Infections in the diabetic host have been shown to persist longer than those in the nondiabetic host. To investigate whether intra-abscess milieu might be a contributing factor to this persistence, the in vivo intra-abscess pH was measured in induced soft-tissue abscesses in diabetic and nondiabetic mice. Two models (female genetically obese insulin-resistant and male streptozocin-induced diabetic mice) were used with appropriate controls. The bacteria injected to produce the soft-tissue abscesses were Bacteroides fragilis and Enterococcus (B + E), Staphylococcus epidermidis and Enterococcus (S + E), and S. aureus (SA). Intra-abscess pH measured on day 3 was consistently and significantly lower in all diabetic mice compared with their controls. In the diabetic mice, the pH of an abscess induced with B + E, S + E, and SA was 6.28 (n = 17), 6.79 (n = 10), and 6.52 (n = 10), respectively; the pH in the controls was 7.21 (n = 20), 7.30 (n = 10), and 7.17 (n = 10), respectively. Differences in all groups between diabetic and nondiabetic mice were significant. The blood glucose values of the diabetic mice averaged 722 mg/dl, and in the nondiabetic mice were 210 mg/dl. No animals were ketotic. There were no significant differences in total colony counts between any groups. In conclusion, there is a significantly lower pH in the abscess of the diabetic host compared with the nondiabetic host that is not related to the numbers or types of causative bacteria.

Abscess↗

PubMed1988

Proteolytic cleavage of Ii to p25.

The 25,000-Da protein that is seen in immunoprecipitates with antibodies to class II MHC molecules or to Ii was shown to be a C-terminal fragment of Ii. [35S]Methionine pulse-chase labeling of polyclonally activated B lymphocytes or lymphoblastoid cell lines demonstrated maximal appearance of p25 in Percoll-separated endosomal fractions at 20- to 40-min chase times (studies in progress). This finding was consistent with the view that proteolysis of Ii to p25 and its release might catalyze the binding of digested foreign peptides to class II molecules and/or govern release of such charged complexes to traffic to the cell surface. We examined the structural relationship of p25 to Ii and the basis for cleavage of a relatively restricted site just external to the transmembranal segment. [35S]Methionine-labeled Ii and associated molecules were immunoprecipitated with a mAb to native Ii and then denatured, resolubilized, and subjected to a second immunoprecipitation with various antibodies. Two antisera to C-terminal peptides of Ii (183 to 193 and 192 to 211), but not antibodies to an N-terminal peptide (12 to 28), did immunoprecipitate p25. The three antibodies to C-terminal and N-terminal peptides all immunoprecipitated denatured Ii proteins. The mAb to Ii immunoprecipitated [35S]methionine-labeled p25 but not [35S]cysteine-labeled p25. This finding was consistent with loss of a portion of Ii containing the only cysteine in Ii, Cys28. Digestion of class II MHC Ag-Ii complexes with various proteases yielded proteins migrating at and near p25 in two-dimensional electrophoretic gels. Upon increasing the duration of protease digestion, the 25,000-Da fragments were relatively resistant to further digestion. This observation was consistent with the presence of secondary structures (domains) leaving a relatively protease-sensitive (Ig hinge-like) region in Ii near its insertion into the membrane.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1987

An hypothesis on the binding of an amphipathic, alpha helical sequence in Ii to the desetope of class II antigens.

When we investigated the hypothesis that amphipathic alpha helical peptides digested from foreign antigen bind to class II major histocompatability complex (MHC) molecules' binding site (desetope) for foreign antigen to be presented to T cell receptors, we found such an extended amphipathic helix in Ii. This amphipathic helix was hypothesized to bind Ii to class II MHC antigens until release in endosomes containing digested foreign antigen. Then these amphipathic Ii polypeptides might polymerize so as not to compete with foreign antigen for binding to class II MHC molecules. Various structural models were consistent with these views and led to the suggestion of specific forms of polymeric interaction.

Animals↗

PubMed1987

Data acquisition system for maximum-likelihood analysis of electron microscopic autoradiographs.

EMAMAP is a program for the data acquisition phase of maximum-likelihood analysis of electron microscope autoradiographs. This program is written in C and has been implemented on a Masscomp MC-500 which supports a graphics processor and a digitizing tablet. The image analysis is automated at a low level: the program operator outlines the edges of the structures of interest using the digitizing tablet, while contiguous regions formed by closed contours are automatically filled by the software. The resulting image is compressed for efficient storage by a quadtree encoding technique for which data compression ratios of greater than 25:1 have been achieved. In practical terms, this implies that the data from a typical experiment of 50 autoradiographs could be stored on a single floppy disk. The system is currently in use for acquiring actual biological experimental data.

Autoradiography↗

PubMed1987

Hydrophobic strip-of-helix algorithm for selection of T cell-presented peptides.

In extension of the hypothesis that an amphipathic alpha helix of Ii (Phe146-Val164) bound to the foreign antigen-presenting site (desetope) of class II MHC molecules through hydrophobic amino acid residues (Phe146, Leu150, Leu153, Met157, Ile160, Val164) which were present in an axial strip along one side of the Ii helix, we developed an algorithm to search for T cell-presented peptides showing a similar hydrophobic strip-of-helix. Such peptides might bind to the class II MHC molecule site which was complementary to the Ii hydrophobic strip-of-helix. The strip-of-helix hydrophobicity index was the mean hydrophobicity (from Kyte-Doolittle values) of sets of amino acids in axial strips down sides of helices for 3-6 turns, at positions, n, n + 4, N + 7, n + 11, n + 14, and n + 18. Peptides correlating well with T cell responsiveness had: (1) 12-19 amino acids (3-5 cycles or 4-6 turns of an alpha helix), (2) a strip with highly hydrophobic residues, (3) adjacent, moderately hydrophilic strips, and (4) no prolines. The degree of hydrophilicity of the remainder of a putative antigenic helix above a threshold value did not count in this index. That is, the magnitude of amphipathicity was not judged to be the principal selecting factor for T cell-presented peptides. This simple algorithm to quantitate strip-of-helix hydrophobicity in a putative amphipathic alpha helix, allowing otherwise generally hydrophilic residues, predicted 10 of 12 T cell-presented peptides in seven well-studied proteins. The derivation and application of this algorithm were analyzed.

Algorithms↗

PubMed1987

Quantitative ultrasonic tissue characterization with real-time integrated backscatter imaging in normal human subjects and in patients with dilated cardiomyopathy.

We have shown previously that the physical properties of myocardium in dogs can be characterized with quantitative ultrasonic integrated backscatter and that interrogation of the tissue with ultrasound can delineate cardiac cycle-dependent changes in ultrasonic backscatter in normal tissue that disappear with ischemia and reappear with reperfusion if functional integrity is restorable. To determine whether this approach can be applied to man, we implemented an automatic gain compensation and continuous data acquisition system to characterize myocardium with quantitative ultrasonic backscatter and to detect cardiac cycle-dependent changes in real time. We developed a two-dimensional echocardiographic system with quantitative integrated backscatter imaging capabilities for use in human subjects that can automatically differentiate ultrasonic signals from blood as opposed to those obtained from tissue and adjust the slope of the gain compensation appropriately. Real-time images were formed from a continuous signal proportional to the logarithm of the integrated backscatter along each A-line. In our initial investigation, 15 normal volunteers (ages 17 to 40 years, heart rates 44 to 88 beats/min) and five patients with dilated cardiomyopathy (ages 22 to 52, heart rates 82 to 120 beats/min) were studied with conventional parasternal long-axis echocardiographic views. Diastolic-to-systolic variation of integrated backscatter in the interventricular septum and left ventricular posterior wall was seen in each of the normal subjects averaging 4.6 +/- 1.4 dB (SD) and 5.3 +/- 1.5 dB (n = 127 sites), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1987

Hyperexpressed hairy leukemic cell Ii might bind to the antigen-presenting site of class II MHC molecules.

The p35 protein which is hyperexpressed on hairy leukemic cells was determined to be Ii, the electrophoretically invariant glycoprotein that is associated with class II major histocompatibility complex (Ia) antigens from the time of their synthesis. The principal function of class II MHC antigens is to present to T cell receptors those digested foreign antigenic peptides that probably fold as amphipathic alpha-helices and adsorb to a hydrophobic surface (desetope) on Ia. By a novel strip-of-helix hydrophobicity algorithm we found that the sequence Leu-142 to His-170 in Ii formed a five-cycle, amphipathic, alpha-helix, the highest scoring one among a series of proteins commonly used as experimental antigens. This finding led to the hypothesis that this sequence in Ii bound to the antigen-binding site (desetope) of Ia until release and self-aggregation in the endosome in order that digested foreign peptides could then bind to Ia. Abundant expression of Ii in leukemic cells might be associated with an altered capacity of those cells to present foreign or leukemic antigens to the host's immune system.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1986

Sudden death and its relation to QT-interval prolongation after acute myocardial infarction: two-year follow-up.

Risk of sudden death was assessed in 533 patients who survived 10 days after acute myocardial infarction (AMI) and were followed for up to 24 months (mean 18) in the Multicenter Investigation of the Limitation of Infarct Size. Analysis of clinical and laboratory variables measured before hospital discharge revealed that the QT interval, either corrected (QTc) or uncorrected for heart rate, did not contribute significantly to prediction of subsequent sudden death or total mortality. In this population, frequent ventricular premature complexes (more than 10 per hour) on ambulatory electrocardiographic monitoring and left ventricular (LV) dysfunction (radionuclide LV ejection fraction of 0.40 or less) identify patients at high risk of sudden death. In patients with these adverse clinical findings, the QTc was 0.468 +/- 0.044 second among those who died suddenly and 0.446 +/- 0.032 second in survivors, and was not statistically significant as an additional predictor of sudden death. Consideration of the use of type I antiarrhythmic agents, digoxin, presence of U waves and correction for intraventricular conduction delay did not alter these findings. Although QT-interval prolongation occurs in some patients after acute myocardial infarction, reduced LV ejection fraction and frequent ventricular premature complexes are the most important factors for predicting subsequent sudden death in this patient population.

Adrenergic beta-Antagonists↗

PubMed1986

Abrupt withdrawal of beta-blockade therapy in patients with myocardial infarction: effects on infarct size, left ventricular function, and hospital course.

The effects of abrupt withdrawal or continuation of beta-blockade therapy during acute myocardial infarction were evaluated in 326 patients participating in the Multicenter Investigation of the Limitation of Infarct Size (MILIS). Thirty-nine patients previously receiving a beta-blocker and randomly selected for withdrawal of beta-blockers and placebo treatment during infarction (group 1) were compared with 272 patients previously untreated with beta-blockers who were also randomly assigned to placebo therapy (group 2). There were no significant differences between the two groups in MB creatine kinase isoenzyme (15.8 +/- 10.9 vs 18.2 +/- 14.4 g-eq/m2, respectively) estimates of infarct size, radionuclide-determined left ventricular ejection fractions within 18 hr of infarction (0.44 +/- 0.15 vs 0.47 +/- 0.16) or 10 days later (0.42 +/- 0.14 vs 0.47 +/- 0.16), creatine kinase-determined incidence of infarct extension (13% vs 6%), congestive heart failure (43% vs 37%), nonfatal ventricular fibrillation (5% vs 7%), or in-hospital mortality (13% vs 9%). Patients in group 1 had more recurrent ischemic chest pain (p = .002) within the first 24 hr after infarction, but not thereafter. However, this did not appear to be related to a rebound increase in systolic blood pressure, heart rate, or double product. In a separate analysis, 20 propranolol-eligible group 1 patients randomly selected for withdrawal of beta-blockade (group 3) were compared with 15 patients randomly selected for continuation of prior beta-blockade therapy (group 4). This comparison yielded similar results. These data indicate that the beta-blockade withdrawal phenomenon is not a major clinical problem in patients with acute myocardial infarction. beta-Blockade therapy can be discontinued abruptly during acute myocardial infarction if clinically indicated.

Aged↗

PubMed1986

Sensitive detection of the effects of reperfusion on myocardium by ultrasonic tissue characterization with integrated backscatter.

We have shown recently that tissue characterization of myocardium with ultrasound reflects changes associated with contractile function throughout the cardiac cycle. To determine whether ultrasonic tissue characterization can sensitively detect the impact of ischemic injury and reperfusion on contractile properties of the heart, we studied the time course of change of backscatter after 5, 20, and 60 min of coronary occlusion followed by reperfusion in 15 dogs. The time-averaged integrated backscatter (IB) and the amplitude and phase of cyclic variation of IB (phase relative to the left ventricular pressure waveform) were measured. A novel ultrasonic index of acute injury was identified, the phase-weighted amplitude of cyclic variation, and calculated by weighting the amplitude of cyclic variation of IB with respect to the phase. We hypothesized that backscatter variables would change dramatically after occlusion and that their restitution after reperfusion would sensitively reflect the extent and time course of reversibility of ischemic injury. After coronary occlusion, segmental wall thickening decreased from approximately 55% to 5% regardless of the duration of ischemia. Changes in backscatter associated with this decrease included an increase in time-averaged IB of approximately 5 dB, a 5 dB decrease in cyclic variation, an 80 degree phase shift, and a 7 dB decrease in phase-weighted amplitude. Wall thickening after reperfusion immediately after the 5, 20, or 60 min occlusions recovered to 45%, 27%, and 12% of baseline values, respectively. Within 3 hr it recovered to 53%, 44%, and 22%. Time-averaged IB recovered initially by 89%, 61%, and 44% (all p less than .05) and continued to recover subsequently although more slowly. Ultimate recovery was virtually complete. In contrast to the rapid recovery of time-averaged IB, phase-weighted amplitude recovered initially to only 72%, 41%, and -7% of baseline (all p less than .05) and manifested slower and incomplete recovery when ischemia had been present for 20 or 60 min. After reperfusion, the time course of both cyclic variation and phase were reflected by changes in the phase-weighted amplitude. The backscatter variables assessed appear to sensitively delineate the duration, time course of recovery, and reversibility of ischemic injury in response to reperfusion. The results suggest that early recovery of time-averaged IB corresponds in part to the restoration of tissue ultrastructural integrity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1985

The dependence of myocardial ultrasonic integrated backscatter on contractile performance.

We have recently shown that the cardiac cycle-dependent variation in myocardial ultrasonic integrated backscatter is blunted with regional ischemia in dogs. To determine if global and intramural regional myocardial contractile performance can be quantified by integrated backscatter, we analyzed ultrasonic responses after induction of increased and decreased contractility in five dogs. A recently developed analog data-acquisition system for measuring integrated backscatter in real time was used to sample radiofrequency signals gated from subepicardial or subendocardial regions. Base-line recordings of integrated backscatter, left ventricular pressure, left ventricular dP/dt, and wall thickness were made at 12 left ventricular sites for both intramural regions. Contractility was modified subsequently by either paired pacing or propranolol to produce significantly elevated or depressed values for maximum left ventricular dP/dt compared with baseline (1083 +/- 289 to 3001 +/- 570 mm Hg/sec; p less than .01 for all). The amplitude of the cyclic variation of integrated backscatter was 50% greater (arithmetically) in subendocardial than in subepicardial regions for all treatments (7.6 +/- 0.3 vs 6.0 +/- 0.5 dB, p less than .001). The maximum rate of change in integrated backscatter waveforms during isovolumetric contraction was faster with paired pacing and slower with propranolol than at baseline for all regions (56 +/- 6 to 74 +/- 6 to 82 +/- 5 dB/sec, p less than .005). The maximum rate of change in integrated backscatter also was greater in subendocardial than subepicardial regions (p less than .001). Thus, both regional and global differences in myocardial contractile performance are manifest quantitatively in integrated backscatter waveforms. We propose that the physiologic determinants of these differences may depend on regional and global variations in myofibril elastic characteristics.

Animals↗

PubMed1985

A relationship between ultrasonic integrated backscatter and myocardial contractile function.

We have shown previously that the physiologic, mechanical cardiac cycle is associated with a parallel, cardiac cycle-dependent variation of integrated backscatter (IB). However, the mechanisms responsible are not known. The mathematical and physiological considerations explored in the present study suggest that the relationship between backscatter and myocardial contractile function reflects cyclic alterations in myofibrillar elastic parameters, with the juxtaposition of intracellular and extracellular elastic elements that have different intrinsic acoustic impedances providing an appropriately sized scattering interface at the cellular level. Cardiac cycle-dependent changes in the degree of local acoustic impedance mismatch therefore may elicit concomitant changes in backscatter. Because acoustic impedance is determined partly by elastic modulus, changes in local elastic moduli resulting from the non-Hookian behavior of myocardial elastic elements exposed to stretch may alter the extent of impedance mismatch. When cardiac cell mechanical behavior is represented by a three-component Maxwell-type model of muscle mechanics, the systolic decrease in IB that we have observed experimentally is predicted. Our prior observations of regional intramural differences in IB and the dependence of IB on global contractile function are accounted for as well. When the model is tested experimentally by assessing its ability to predict the regional and global behavior of backscatter in response to passive left ventricular distention, good concordance is observed.

Animals↗