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Biomedical subjects

L J Thomas

Publications and source records attributed to L J Thomas.

At least 19 recordsLinked to original sources
PubMed1992

Biosynthesis of phosphatidylinositol-glycan (PI-G)-anchored membrane proteins in cell-free systems: PI-G is an obligatory cosubstrate for COOH-terminal processing of nascent proteins.

It is generally recognized that nascent proteins destined to be processed to a phosphatidylinositol-glycan (PI-G)-anchored membrane form contain a hydrophobic signal peptide at both their NH2 and COOH termini. In previous studies we showed that rough microsomal membranes (RM) prepared from CHO cells can carry out COOH-terminal processing. We have now investigated RM prepared from many additional cell types, including frog oocytes, B cells, and T cells, and found that all are competent with respect to COOH-terminal processing. Exceptions were certain mutant T cells that had been shown to be defective at various steps of PI-G anchor biosynthesis [Sugiyama, E., De Gasperi, R., Urakaze, M., Chang, H.-M., Thomas, L. J., Hyman, R., Warren, C. D. & Yeh, E. T. H. (1991) J. Biol. Chem. 266, 12119-12122]. In one such defective mutant, COOH-terminal processing activity of RM could be restored either by transfecting the intact cells with the gene for the deficient step in PI-G synthesis or by adding PI-G extracts to the RM in vitro. Cleavage of the COOH-terminal signal peptide in the RM is therefore dependent on the presence of intact PI-G incorporated into the mature protein.

Acyltransferases↗
PubMed1992

A personal computer based implementation of the maximum-likelihood method of analysis of electron microscope autoradiographs.

The maximum-likelihood (ML) method for the quantitative analysis of electron-microscopic autoradiographs has been shown to be substantially superior to the conventional crossfire (CF) method. It can generate reliable and accurate tracer concentration estimates with far fewer micrographs and produce valid estimates even at counts low enough to preclude the use of the crossfire method while eliminating the need for special ad hoc treatment of narrow membranous structures as well as the secondary verification of the tracer concentration estimates. Despite these significant advantages, the large computational requirements of the ML method has to date hampered its widespread use. In this paper, we present a new line-integration method that allows us to reduce the computational requirements of the ML method to a point where it becomes feasible to implement it on a small computer system of the type typically available to a laboratory user of EM autoradiography. We present the complete line-integration method for the particular case of EM autoradiography with tritium, and show how it can be adapted to other isotopes. We have constructed a software package that implements the complete maximum-likelihood method on the IBM PC class of machines using our line-integration method. Features of this software package which are of particular importance to the research community are device independence, which makes it usable with a large variety of currently available laboratory equipment, and easy portability of the software and data between different computer systems.

Algorithms↗
PubMed1992

Differential expression of glycosylphosphatidylinositol-anchored proteins in a murine T cell hybridoma mutant producing limiting amounts of the glycolipid core. Implications for paroxysmal nocturnal hemoglobinuria.

A T cell hybridoma mutant, which expressed a markedly reduced level of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface, was characterized. The surface expression level of Thy-1 was approximately 17% of the wild-type level, whereas the surface expression of Ly-6A was approximately 2.4% of the wild-type level. We show here that these cells synthesized limiting amounts of the GPI core and that the underlying defect in these cells was an inability to synthesize dolichyl phosphate mannose (Dol-P-Man) at the normal level. The defect in Ly-6A expression could be partially corrected by tunicamycin, which blocked the biosynthesis of N-linked oligosaccharide precursors and shunted Dol-P-Man to the GPI pathway. Full restoration of Thy-1 and Ly-6A expression, however, required the stable transfection of a yeast Dol-P-Man synthase gene into the mutants. These results revealed that when the GPI core is limiting, there is a differential transfer of the available GPI core to proteins that contain GPI-anchor attachment sequences. Our findings also have implications for the elucidation of the defects in paroxysmal nocturnal hemoglobinuria.

Animals↗
PubMed1992

Regularized linear method for reconstruction of three-dimensional microscopic objects from optical sections.

The inverse problem involving the determination of a three-dimensional biological structure from images obtained by means of optical-sectioning microscopy is ill posed. Although the linear least-squares solution can be obtained rapidly by inverse filtering, we show here that it is unstable because of the inversion of small eigenvalues of the microscope's point-spread-function operator. We have regularized the problem by application of the linear-precision-gauge formalism of Joyce and Root [J. Opt. Soc. Am. A 1, 149 (1984)]. In our method the solution is regularized by being constrained to lie in a subspace spanned by the eigenvectors corresponding to a selected number of large eigenvalues. The trade-off between the variance and the regularization error determines the number of eigenvalues inverted in the estimation. The resulting linear method is a one-step algorithm that yields, in a few seconds, solutions that are optimal in the mean-square sense when the correct number of eigenvalues are inverted. Results from sensitivity studies show that the proposed method is robust to noise and to underestimation of the width of the point-spread function. The method proposed here is particularly useful for applications in which processing speed is critical, such as studies of living specimens and time-lapse analyses. For these applications existing iterative methods are impractical without expensive and/or specially designed hardware.

Computer Simulation↗
PubMed1992

A graphical user interface for quantitative imaging and analysis of electrophoretic gels and autoradiograms.

DNA/GUI (DNA Graphical User Interface) is an interactive software system for rapid and efficient analysis of images of the types used in genome mapping, such as autoradiograms and electrophoretic gels. Images are digitized using a commercially available charge-coupled-device (CCD) camera system and analyzed on a graphics workstation using a menu-driven user interface. DNA/GUI features automatic lane and band detection, simultaneous display of multiple images and a unique spatial-normalization algorithm. Images and their associated data are archived and easily available for later recall. Preliminary results indicate that DNA/GUI is a useful tool in the analysis and comparison of images used in a variety of applications such as genetic-linkage analysis and DNA restriction mapping. The interactive display software is based on the X Window System and is therefore readily portable to a variety of graphics workstations.

Autoradiography↗
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

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↗