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A Pallant

Publications and source records attributed to A Pallant.

7 recordsLinked to original sources
PubMed2000

Determination of plasma methoxyamines.

The determination of plasma methoxyamines is the most informative parameter for the diagnosis of pheochromocytoma. Very good sensitivity and specificity are necessary for this. The measurement of deconjugated (free plus sulfate-conjugated) metanephrines is more useful. Concentrations are 10-fold higher than free metanephrine concentrations. The methodology generally uses a column purification and high performance liquid chromatography with electrochemical detection. We have tested six commercial columns used for the purification step. We chose a rapid and reliable method with a mixture of strong cation-exchange and strong anion-exchange groups bonded onto a silica column. This protocol has been validated with samples of plasma from normal subjects, healthy elderly people, renal failure patients and patients with pheochromocytoma.

Chromatography, High Pressure Liquid↗
PubMed1992

CD43 is expressed normally on Wiskott-Aldrich-derived lymphocytes.

The Wiskott-Aldrich syndrome (WAS) is an X-linked disease characterized by eczema, thrombocytopenia, and profound immunodeficiency in affected males. While the etiology of the syndrome is currently unknown, abnormalities of CD43 have been described as a biochemical marker of the disease. Several investigators have demonstrated alterations in the expression of the CD43 surface antigen on WAS hematopoietic cells, noting either absence, decreased levels or changes in the characteristic molecular weight of the protein on the lymphocytes of affected patients. Biochemical studies have further indicated that glycosylating activity of specific enzymes which may post-translationally modify CD43 is altered in both T cells and Epstein-Barr-virus (EBV)-transformed B cells in WAS patients when compared to unaffected controls. Here we present data on cells derived from two males with a clinical diagnosis of WAS. Analysis of genomic DNA from the mothers of each of these patients (obligate carriers) showed a nonrandom X-chromosome inactivation pattern of nucleated blood cells, confirming the diagnosis of the X-linked syndrome. CD43 was characterized on peripheral blood lymphocytes and long-term EBV-transformed B cell lines, both to further analyze the molecular defects of WAS, as well as to attempt to generate a reproducible method for disease detection. Surprisingly, surface expression, molecular weight and two-dimensional gel analysis failed to demonstrated any reproducible differences in the CD43 expression, whether from disease or normal lymphocytes. Such results suggest possible heterogeneity of this syndrome.

Antigens, CD↗
PubMed1990

CD43 (leukosialin, sialophorin, large sialoglycoprotein) can be expressed in both normal and Wiskott-Aldrich fibroblasts via transfection of a leukosialin cDNA.

Human leukosialin is among the most abundant sialoglycoproteins found on the surface of cells of the lympho-hematopoietic system. Leukosialin, also known as sialophorin, is involved in T cell proliferation, and its molecular isoform changes upon cellular activation. We show that human leukosialin is identical to the antigens described by the monoclonal antibodies (mAb) G10-2, G19-1 (CD43) and B1B6 (large sialoglycoprotein). This identity was suggested by immunoblot analysis of transformed cell lysates. Further, fibroblasts transfected with the human leukosialin cDNA gain reactivity to these mAb, showing conclusively that molecules recognized by these mAb are determined by the same cDNA. Expression of the leukosialin gene is readily detected on the surface of transfected human and mouse fibroblasts. Immunoblot analysis of the transfectants indicates that processing of the human protein occurs in both species. Alterations of leukosialin expression have been reported in patients with the Wiskott-Aldrich Syndrome (WAS), an X-chromosome-linked immunodeficiency disease. While essentially all of the transfected tumor and primary fibroblasts from normal individuals express the transfected gene on the cell surface, only half of the transfected Wiskott-Aldrich fibroblasts express CD43. Nonetheless, the antigenic pattern by immunoblot analysis of both normal and WAS-transfected fibroblasts appears identical. These results indicate that WAS-derived cells can express leukosialin and that the product of WAS X-chromosome mutation may not be expressed in fibroblasts.

Animals↗
PubMed1989

Characterization of cDNAs encoding human leukosialin and localization of the leukosialin gene to chromosome 16.

We describe the isolation and characterization of cDNA clones encoding human leukosialin, a major sialoglycoprotein of human leukocytes. Leukosialin is very closely related or identical to the sialophorin molecule, which is involved in T-cell proliferation and whose expression is altered in Wiskott-Aldrich syndrome (WAS), an X chromosome-linked immunodeficiency disease. Using a rabbit anti-serum to leukosialin, a cDNA clone was isolated from a lambda gt11 cDNA library constructed from human peripheral blood cells. This lambda gt11 clone was used to isolate longer cDNA clones that correspond to the entire coding sequence of leukosialin. DNA sequence analysis reveals three domains in the predicted mature protein. The extracellular domain is enriched for Ser, Thr, and Pro and contains four contiguous 18-amino acid repeats. The transmembrane and intracellular domains of the human leukosialin molecule are highly homologous to the rat W3/13 molecule. RNA gel blot analysis reveals two polyadenylylated species of 2.3 and 8 kilobases. Southern blot analysis suggests that human leukosialin is a single-copy gene. Analysis of monochromosomal cell hybrids indicates that the leukosialin gene is not X chromosome linked and in situ hybridization shows leukosialin is located on chromosome 16. These findings demonstrate that the primary mutation in WAS is not a defect in the structural gene for leukosialin.

Amino Acid Sequence↗
PubMed1986

Free kappa light chains in multiple sclerosis spinal fluid.

Based on prior reports of free light chains of immunoglobulin G (IgG) in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS), we quantitated free kappa and lambda chains and whole IgG concentrations using sensitive and specific radioimmunoassays (RIAs). The RIA for free kappa chains had a sensitivity of 0.25 micrograms/ml and was capable of specifically measuring free kappa chains in whole CSF or serum even in the presence of a 4-log excess of whole IgG. By RIA, free kappa chains were detected in CSF samples from 33 (84%) of 39 MS patients but in only 1 (2.4%) of 42 controls. The control patients included 10 with noninfectious inflammatory diseases and 9 with central nervous system infections. The concentration of free kappa chains in the CSF of the MS patients was 1.40 +/- 1.21 micrograms/ml. Free kappa chains were concentrated in the CSF 71- to 120-fold relative to reference proteins. In contrast, increased levels of free lambda chains or of whole IgG were nonspecific; abnormalities were seen in controls with infections or inflammatory diseases as often as in MS patients. These studies suggest that the measurement of free kappa light chains may have important diagnostic usefulness, since the specificity of the finding for MS appears to be high.

Communicable Diseases↗
PubMed1985

Amine transport into chromaffin ghosts. Kinetic measurements of net uptake of biologically and pharmacologically relevant amines using an on-line amperometric technique.

The kinetic parameters for net transport of dopamine, epinephrine, norepinephrine, 5-hydroxytryptamine, S alpha-methyldopamine, R alpha-methyldopamine, and 1R,2S alpha-methylnorepinephrine into highly purified bovine chromaffin ghosts were determined using an on-line amperometric technique. Chromaffin ghosts devoid of endogenous amines were formed from lysis of chromaffin granules under hypotonic conditions, extensive washing of the scattered membranes, followed by resuspension in iso-osmotic media and overnight dialysis. When chromaffin ghosts formed so as to generate and maintain a large delta pH were suspended in 185 mM KCl, 10 mM Hepes at pH 7.0, 37 degrees C, the addition of MgATP resulted in rapid acidification of the intravesicular space, which was maintained at pH 6.0 (+/- 0.1) for over 30 min. Kinetic net amine transport was subsequently measured with a glassy carbon electrode. The initial rates of uptake were found to follow Michaelis-Menten kinetics. Computer based statistical analysis of the data using distribution-free procedures yielded Km (and V) values as follows: in microM (nmol X mg protein-1 X min-1) dopamine, 16.2 (14.0); R-norepinephrine, 32.5 (12.9); R-epinephrine, 35.1 (15.2); 5-hydroxytryptamine, 4.7 (5.1); S alpha-methyldopamine, 17.7 (11.2); R alpha-methyldopamine, 44.2 (9.9); 1R,2S alpha-methylnorepinephrine, 76.5 (12.5). The physiologic and pharmacologic implications of these kinetic parameters are discussed.

Adrenal Medulla↗