Biomedical subjects

D E Afar

Publications and source records attributed to D E Afar.

25 records · Page 2Linked to original sources

PubMed1993

Immunoelectron microscopic localization of monoclonal IgM antibodies in gammopathy associated with peripheral demyelinative neuropathy.

A sural nerve biopsy from a patient with benign monoclonal IgM kappa gammopathy and sensory-motor demyelinative neuropathy, revealed marked loss of myelinated fibers and focal axonal degeneration as well as widespread demyelination and remyelination with onion-skin formation. Almost all myelinated fibers displayed characteristic widening of the myelin lamellae as well as excessive thickness and/or exuberant outfoldings of myelin, reminiscent of that seen in tomaculous neuropathy. Many endoneurial capillaries were lined by fenestrated endothelium, indicating breakdown of a normal blood-nerve barrier. The endoneurium contained large amounts of extracellular proteinaceous material. Immunofluorescence and immunoelectron microscopy performed on the nerve of the patient, demonstrated selective deposition of IgM kappa gammaglobulin, exclusively in the areas of splittings of the myelin lamellae. Schwann cells contained cytoplasmic myelin debris labelled with IgM kappa only. In the indirect immunofluorescence and immunoelectron microscopy, serum of the patient reacted with the whole thickness of compact peripheral myelin of a normal human nerve. There was no immunoreactivity with the central myelin, Schwannoma cells, glial cells, axons or neurons. Demonstration of the selective presence of monoclonal IgM in widened lamellae of myelinated fibers, as well as bound to the internalized myelin debris in Schwann cells and macrophages, indicates a pathogenetic role of monoclonal paraprotein in myelin injury. Demyelination is promoted by development of endothelial fenestrations in the endoneurial capillaries and breakdown of the blood-nerve barrier.

Aged↗

PubMed1992

Tyrosine phosphorylation of biliary glycoprotein, a cell adhesion molecule related to carcinoembryonic antigen.

Biliary-glycoprotein (BGP), a cell adhesion molecule related to carcinoembryonic antigen (CEA), has been shown to exist as several alternatively spliced isoforms. Here we show that BGPa and BGPb are phosphorylated in the chronic myelogenous leukaemia cell line KG-1, which constitutively expresses several BGP isoforms, and Chinese hamster LR-73 cells transfected with the cDNAs encoding BGPa and BGPb. The phosphorylation can be augmented with the protein tyrosine phosphatase inhibitor ammonium vanadate and with TPA (an activator of protein kinase C). Phospho-amino acid analysis of phosphorylated BGPs demonstrated that phosphorylation occurs on serine, threonine and tyrosine residues. Phosphorylation reactions carried out in in vitro membrane preparations from KG-1 cells revealed a close association of BGP proteins with membrane associated protein tyrosine kinases. These observations suggest an association of BGP proteins with signal transduction molecules which is regulated by alternative splicing of the cytoplasmic domain.

Amino Acid Sequence↗

PubMed1992

Multiple cDNAs encoding the esk kinase predict transmembrane and intracellular enzyme isoforms.

A novel protein kinase, the Esk kinase, has been isolated from an embryonal carcinoma (EC) cell line by using an expression cloning strategy. Sequence analysis of two independent cDNA clones (2.97 and 2.85 kb) suggested the presence of two Esk isoforms in EC cells. The esk-1 cDNA sequence predicted an 857-amino-acid protein kinase with a putative membrane-spanning domain, while the esk-2 cDNA predicted an 831-amino-acid kinase which lacked this domain. In adult mouse cells, esk mRNA levels were highest in tissues possessing a high proliferation rate or a sizeable stem cell compartment, suggesting that the Esk kinase may play some role in the control of cell proliferation or differentiation. As anticipated from the screening procedure, bacterial expression of the Esk kinase reacted with antiphosphotyrosine antibodies on immunoblots. Furthermore, in in vitro kinase assays, the Esk kinase was shown to phosphorylate both itself and the exogenous substrate myelin basic protein on serine, threonine, and tyrosine residues, confirming that the Esk kinase is a novel member of the serine/threonine/tyrosine family of protein kinases.

Amino Acid Sequence↗

PubMed1991

TIK, a novel serine/threonine kinase, is recognized by antibodies directed against phosphotyrosine.

We have isolated cDNAs encoding kinases from a murine pre-B cell line by screening a lambda gt11 cDNA expression library with anti-phosphotyrosine antibodies. One cDNA was identified to encode the previously isolated tyrosine kinase c-lyn. Among the remaining clones, we have characterized a cDNA encoding a novel kinase which we have designated TIK. Sequence analysis of this cDNA indicates that the TIK enzyme lacks the features thought to be conserved among protein tyrosine kinases. Although isolated on the basis of its reactivity with the anti-phosphotyrosine antibody, the TIK enzyme was found to have only serine and threonine kinase activity. The amino-terminal portion of the TIK protein contains a cdc2 phosphorylation consensus sequence. Three mRNA transcripts derived from the TIK gene are detected in a variety of adult murine tissues.

Amino Acid Sequence↗

PubMed1991

Cell adhesion properties of myelin-associated glycoprotein in L cell fibroblasts.

Myelin-associated glycoprotein (MAG) is a cell surface molecule expressed by oligodendrocytes and Schwann cells. In order to determine whether MAG expression can confer adhesive properties to cells which normally do not aggregate in suspension, the cDNA encoding the long form of MAG (L-MAG) was introduced into L cell fibroblasts by retroviral infection. Clonal L cell lines expressing MAG were then subjected to a cell aggregation assay. Our results indicate that L-MAG can function as an intercellular adhesion molecule in a heterologous cell system. A critical threshold value of L-MAG expression was required for cell aggregation to occur. The adhesive properties of these cells were specific to MAG, since monoclonal antibodies directed against its extracellular domain inhibited aggregation. Furthermore, the adhesion was found to be calcium- and temperature-independent. Cell sorting experiments demonstrated that L-MAG-expressing cells bind in a heterotypic fashion to parental L cell fibroblasts. These results suggest that L-MAG can function as a heterotypic cell adhesion molecule recognizing a cell surface molecule(s) expressed by L cells.

Animals↗

PubMed1991

STY, a tyrosine-phosphorylating enzyme with sequence homology to serine/threonine kinases.

We have cloned a novel kinase (STY) from an embryonal carcinoma cell line. Sequence analysis of the STY cDNA reveals that it shares sequence homology with serine/threonine-type kinases and yet the bacterial expression product of the STY cDNA appears to have serine-, threonine-, and tyrosine-phosphorylating activities. The predicted STY protein is highly basic and contains a putative nuclear localization signal. During differentiation, two new mRNAs were detected in addition to the embryonic transcript.

Amino Acid Sequence↗

PubMed1990

Differential phosphorylation of myelin-associated glycoprotein isoforms in cell culture.

The alternative splicing of myelin-associated glycoprotein (MAG) mRNA generates two isoforms that harbor distinct potential phosphorylation sites in their cytoplasmic tails. Here we characterize the in vivo phosphorylation of MAG isoforms in NIH 3T3 cells transfected with the cDNAs encoding the two isoforms of MAG. Our results demonstrate that the longer isoform, L-MAG, is phosphorylated constitutively mainly on serine, but also on threonine and tyrosine residues. This phosphorylation is subject to change by 12-O-tetradecanoylphorbol 13-acetate (TPA) and ammonium vanadate, but not by dibutyryl-cyclic AMP. The shorter isoform, S-MAG, is constitutively phosphorylated only on serine residues. While TPA and dibutyryl-cyclic AMP have no detectable effect, ammonium vanadate induces tyrosine and threonine phosphorylation in S-MAG. 32P labeling of v-src-transformed NIH 3T3 cells that express L-MAG also show that L-MAG is likely to be an in vivo substrate for pp60v-src tyrosine kinase activity. These results demonstrate that both MAG isoforms are phosphorylated in a heterologous cell system and that this phosphorylation is subject to pharmacological manipulation.

Animals↗