Search PubMed⌕ Search

Biomedical subjects

L A Lasky

Publications and source records attributed to L A Lasky.

At least 37 records · Page 2Linked to original sources
PubMed1997

PSTPIP: a tyrosine phosphorylated cleavage furrow-associated protein that is a substrate for a PEST tyrosine phosphatase.

We have investigated proteins which interact with the PEST-type protein tyrosine phosphatase, PTP hematopoietic stem cell fraction (HSCF), using the yeast two-hybrid system. This resulted in the identification of proline, serine, threonine phosphatase interacting protein (PSTPIP), a novel member of the actin- associated protein family that is homologous to Schizosaccharomyces pombe CDC15p, a phosphorylated protein involved with the assembly of the actin ring in the cytokinetic cleavage furrow. The binding of PTP HSCF to PSTPIP was induced by a novel interaction between the putative coiled-coil region of PSTPIP and the COOH-terminal, proline-rich region of the phosphatase. PSTPIP is tyrosine phosphorylated both endogenously and in v-Src transfected COS cells, and cotransfection of dominant-negative PTP HSCF results in hyperphosphorylation of PSTPIP. This dominant-negative effect is dependent upon the inclusion of the COOH-terminal, proline-rich PSTPIP-binding region of the phosphatase. Confocal microscopy analysis of endogenous PSTPIP revealed colocalization with the cortical actin cytoskeleton, lamellipodia, and actin-rich cytokinetic cleavage furrow. Overexpression of PSTPIP in 3T3 cells resulted in the formation of extended filopodia, consistent with a role for this protein in actin reorganization. Finally, overexpression of mammalian PSTPIP in exponentially growing S. pombe results in a dominant-negative inhibition of cytokinesis. PSTPIP is therefore a novel actin-associated protein, potentially involved with cytokinesis, whose tyrosine phosphorylation is regulated by PTP HSCF.

3T3 Cells↗
PubMed1997

A novel protein-tyrosine phosphatase related to the homotypically adhering kappa and mu receptors.

Here we describe a novel member of the receptor-like protein-tyrosine phosphatases (PTPs) termed PTP lambda, which is homologous to the homotypically adherent PTPs kappa and mu. Murine PTP lambda contains MAM, IgG, fibronectin type III, and dual phosphatase domains. As has been demonstrated for PTPs kappa and mu, PTP lambda mediates homotypic adhesion in vitro, and PTP lambda is associated with beta catenin in kidney epithelial cells. The extracellular domain of PTP lambda is proteolytically processed in cell culture as well as in vivo. Northern blot analysis reveals that PTP lambda is expressed throughout embryonic development and is predominately found in adult brain, lung, and kidney. In situ hybridization to 15.5-day old rat embryos reveals that PTP lambda is expressed in a variety of embryonic neuronal sites as well as in the esophagus, lung bronchiolar epithelium, kidney glomerular epithelium, olfactory epithelium, and various cartilagenous sites. Analysis of neonatal brain demonstrates expression in cells of the hippocampus, cortex, and the substantia nigra. Finally, immunohistochemical analysis reveals expression of this PTP on specific neurons of the spinal cord as well as on isolated cortical neurons.

Amino Acid Sequence↗
PubMed1996

Hematopoiesis: wandering progenitor cells.

The generation of mice that are chimeric for expression of alpha 4 integrin has revealed a critical role for this adhesion molecule, specifically in postnatal lymphopoiesis.

Animals↗
PubMed1996

Characterization of a novel member of the macrophage mannose receptor type C lectin family.

The recognition of a diversity of carbohydrates by the various calcium dependent (type C) lectin family members has been shown to be critical for a variety of processes ranging from cell adhesion to antigen presentation. Examination of the expressed sequence tag (EST) data base for novel type C lectins using E-selectin as a probe resulted in the identification of a distantly related short polypeptide sequence containing many of the conserved residues found in these carbohydrate-binding proteins. Cloning of the full-length murine cDNA containing this region revealed that this protein is a novel member of the family that includes the macrophage mannose, the phospholipase A2, and the DEC 205 receptors, with a cysteine-rich domain, a fibronectin type 2 domain, eight type C lectin domains, a transmembrane domain, and a short cytoplasmic carboxyl terminus. Genomic Southern analysis suggests that this is a conserved protein, and examination of a human homologue revealed a high degree of sequence homology with the murine form. Northern blot analysis revealed expression of a large transcript in a number of different human and murine tissues and tumor cells and an alternatively spliced smaller transcript with a divergent 5' sequence was expressed specifically in the human fetal liver. Analysis of the genomic structure revealed that the gene encoding this lectin was interrupted by a large number of introns, and the intron structure was similar to the macrophage mannose receptor gene. Finally, in situ hybridization analysis demonstrated that the transcript encoding this lectin was found in a number of highly endothelialized sites as well as in chondrocytes in cartilaginous regions of the embryo.

Amino Acid Sequence↗
PubMed1996

A novel protein tyrosine phosphatase expressed in lin(lo)CD34(hi)Sca(hi) hematopoietic progenitor cells.

Stem cells are capable of extensive self-renewal in the absence of differentiation. The maintenance of this undifferentiated state occurs despite the fact that this cell is exposed to a milieu that is rich in a variety of growth and differentiation factors. A unifying feature of such hematopoietic factors is that they mediate their effects through the phosphorylation of tyrosine residues by various cellular kinases. Therefore, one mechanism that might inhibit such differentiation signals in the self-renewing stem cell is the dephosphorylation of tyrosine residues by protein tyrosine phosphatases (PTPs). We have thus investigated the types of tyrosine phosphatases expressed by murine embryonic lin(lo)CD34hiSca(hi) hematopoietic progenitor cells by using a consensus polymerase chain reaction (PCR) approach. Although many known tyrosine phosphatases were detected using this method, a novel PTP related to the previously described PTP PEST type enzymes, murine PTP PEP and murine/human PTP PEST, was also observed. Cloning of the full-length cDNA encoding this enzyme showed that it was indeed a novel new member of this family, with an amino terminal tyrosine phosphatase domain followed by a region rich in serine, threonine, and proline. The carboxy terminus of this novel PTP contained a short sequence that was homologous to a region of the murine PTP PEP that was involved with nuclear localization. Bacterial expression of the phosphatase domain showed that this enzyme could efficiently dephosphorylate tyrosines in vitro. Analysis of the expression of the novel nuclear PTP by quantitative PCR showed that the transcript disappeared as the lin(lo)CD34hiSca(hi) cells differentiated in the presence of interleukin-1, interleukin-3, erythropoietin, and granulocyte-macrophage colony-stimulating factor. In agreement with its potential role in the hematopoietic progenitor cell, this novel PTP was expressed at a barely detectable level in a very limited subset of adult tissues. However, analysis of several murine hematopoietic progenitor cell lines, but not of a differentiated T-cell line, showed a high level of expression of the novel PTP. These data suggest that this novel phosphatase may play a critical role in the maintenance of the undifferentiated state of the hematopoietic stem cell.

Amino Acid Sequence↗
PubMed1996

Hematopoietic defects in mice lacking the sialomucin CD34.

Although the pluripotent hematopoietic stem cell can only be definitively identified by its ability to reconstitute the various mature blood lineages, a diversity of cell surface antigens have also been specifically recognized on this subset of hematopoietic progenitors. One such stem cell-associated antigen is the sialomucin CD34, a highly O-glycosylated cell surface glycoprotein that has also been shown to be expressed on all vascular endothelial cells throughout murine embryogenesis as well as in the adult. The functional significance of CD34 expression on hematopoietic progenitor cells and developing blood vessels is unknown. To analyze the involvement of CD34 in hematopoiesis, we have produced both embryonic stem (ES) cells and mice that are null for the expression of this mucin. Analysis of yolk saclike hematopoietic development in embryoid bodies derived from CD34-null ES cells showed a significant delay in both erythroid and myeloid differentiation that could be reversed by transfection of the mutant ES cells with CD34 constructs expressing either a complete or truncated cytoplasmic domain. Measurements of colony-forming activity of hematopoietic progenitor cells derived from yolk sacs or fetal livers isolated from CD34-null embryos also showed a decreased number of these precursor cells. In spite of these diminished embryonic hematopoietic progenitor numbers, the CD34-null mice developed normally, and the hematopoietic profile of adult blood appeared typical. However, the colony-forming activity of hematopoietic progenitors derived from both bone marrow and spleen is significantly reduced in adult CD34-deficient animals, and these CD34-deficient progenitors also appear to be unable to expand in liquid cultures in response to hematopoietic growth factors. Even with these apparent progenitor cell deficiencies, CD34-null animals showed kinetics of erythroid, myeloid, and platelet recovery after sublethal irradiation that are indistinguishable from wild-type mice. These data strongly suggest that CD34 plays an important role in the formation of progenitor cells during both embryonic and adult hematopoiesis. However, the hematopoietic sites of adult CD34-deficient mice may still have a significant reservoir of progenitor cells that allows for normal recovery after nonmyeloablative peripheral cell depletion.

Animals↗
PubMed1995

CD34+ endothelial cell lines derived from murine yolk sac induce the proliferation and differentiation of yolk sac CD34+ hematopoietic progenitors.

Embryonic hematopoiesis is initiated in part in the blood islands of the yolk sac. Previous confocal microscopic analysis has shown that the CD34 antigen, a mucin-like cell surface glycoprotein that is expressed by hematopoietic progenitors and all endothelial cells of the adult and embryo, is also found on a subset of luminal hematopoietic-like cells in the yolk sac blood islands as well as on the vascular endothelium lining these early hematopoietic locations. We show here that, as in all other hematopoietic sites thus far examined, immunoaffinity-purified CD34+ nonadherent cells from murine yolk sacs contain the vast majority of erythroid and myeloid progenitor cell colony forming activity. To examine the developmental interactions between these CD34+ hematopoietic progenitor cells of the yolk sac and the CD34+ yolk sac endothelium, we have immunaffinity-purified adherent endothelial cells from day 10.5 yolk sacs using CD34 antiserum and produced cell lines by transformation with a retrovirus expressing the polyoma middle T antigen. Analysis of these cell lines for CD34, von Willebrand's factor, FLK 1 and FLT 1 expression, and capillary growth in Matrigel indicates that they appear to be endothelial cells, consistent with their original phenotype in vivo. Coculture of yolk sac CD34+ hematopoietic cells on these endothelial cell lines results in up to a 60-fold increase in total hematopoietic cell number after approximately 8 days. Analysis of these expanded hematopoietic cells showed that the majority were of the monocyte/macrophage lineage. In addition, examination of the cultures showed the rapid formation of numerous cobblestone areas, a previously described morphologic entity thought to be representative of early pluripotential stem cells. Scrutiny of the ability of these endothelial cell lines to expand committed progenitor cells showed up to a sixfold increase in erythroid and myeloid colony-forming cells after 3 to 6 days in culture, consistent with the notion that these embryonic endothelial cells mediate the expansion of these precursor cells. Polymerase chain reaction analyses showed that most of the cell lines produce FLK-2/FLT-3 ligand, stem cell factor, macrophage colony-stimulating factor, leukemia-inhibitory factor, and interleukin-6 (IL-6), whereas there is a generally low or not measurable production of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, IL-1, IL-3, transforming growth factor beta-1, erythropoietin, or thrombopoietin. The output of mature hematopoietic cells from these cocultures can be modified to include an erythroid population by the addition of exogenous erythropoietin. These data suggest that endothelial cell lines derived form the yolk sac provide an appropriate hematopoietic environment for the expansion and differentiation of yolk sac progenitor cells into at least the myeloid and erythroid lineages.

Animals↗
PubMed1995

The sialomucin CD34 is expressed on hematopoietic cells and blood vessels during murine development.

The processes of angiogenesis and hematopoiesis require a high degree of coordination during embryogenesis. Whereas much is understood about the development of the vascular system in avian embryos, little information has been attained in mammals, predominantly because there are no specific markers for either blood vessels or hematopoietic cells in any developing mammalian system. We have recently shown that murine CD34 (mCD34) is expressed on the vascular endothelium in all organs and tissues of the adult mouse as well as on a small percentage of presumably hematopoietic stem cells in the bone marrow and fetal liver. Here we show that mCD34 is also expressed on the endothelium of blood vessels and on a subset of hematopoietic-like cells throughout murine development. mCD34 is first observed on the yolk sac endothelium of day 7.5 embryos and on a subset of hematopoietic cells within these yolk sacs. mCD34 expression is maintained on vessels and hematopoietic cells in all organs and tissues throughout embryogenesis. In addition, mCD34 is localized on growth conelike filopodial processes that appear at the budding edge of newly sprouted capillaries. Double staining of capillaries for mCD34 and laminin shows that these growth conelike processes seem to be free of laminin, whereas the formed capillaries seem to be coated with this extracellular matrix protein. Analysis of vessels in developing brain shows that these filopodial processes seem to be directed toward the ventricular epithelium, a previously described site of vascular endothelial growth factor synthesis. Finally, we show that the vascular structures of developing murine embryoid bodies also express mCD34. These data suggest that mCD34 is a useful marker for the analysis of the development of the blood vascular system in murine embryos.

Animals↗
PubMed1995

Selectin-carbohydrate interactions and the initiation of the inflammatory response.

The orderly migration of various white blood cell types to inflammatory sites is a highly regulated process that involves a diversity of adhesion and signaling molecules. This cellular influx is initiated by relatively low affinity interactions that allow for leukocytes to roll along the vascular surface. This rolling phenomenon is mediated by adhesive interactions between lectin containing adhesion molecules, termed selectins, on both the vascular endothelium and leukocytes, and carbohydrate ligands immobilized on mucin-like scaffolds. This adhesion allows for a rapid recognition of various cell types under the conditions of vascular flow, with the result that inflammatory cells are specifically decelerated adjacent to sites of inflammation. This review focuses on the various biochemical aspects of the interactions between the selectins and their cognate carbohydrate ligands, with an emphasis on the importance of these adhesive events to the inflammatory response.

Animals↗
PubMed1994

Global vascular expression of murine CD34, a sialomucin-like endothelial ligand for L-selectin.

Extravasation of leukocytes into organized lymphoid tissues and into sites of inflammation is critical to immune surveillance. Leukocyte migration to peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and Peyer's patches (PP) depends on L-selectin, which recognizes carbohydrate-bearing, sialomucin-like endothelial cell surface glycoproteins. Two of these ligands have been identified at the molecular level. One is the potentially soluble mucin, GlyCAM 1, which is almost exclusively produced by high endothelial venules (HEV) of PLN and MLN. The second HEV ligand for L-selectin is the membrane-bound sialomucin CD34. Historically, this molecule has been successfully used to purify human pluripotent bone marrow stem cells, and limited data suggest that human CD34 is present on the vascular endothelium of several organs. Here we describe a comprehensive analysis of the vascular expression of CD34 in murine tissues using a highly specific antimurine CD34 polyclonal antibody. CD34 was detected on vessels in all organs examined and was expressed during pancreatic and skin inflammatory episodes. A subset of HEV-like vessels in the inflamed pancreas of nonobese diabetic (NOD) mice are positive for both CD34 and GlyCAM 1, and bind to an L-selectin/immunoglobulin G (IgG) chimeric probe. Finally, we found that CD34 is present on vessels of deafferentiated PLN, despite the fact that these vessels are no longer able to interact with L-selectin or support lymphocyte binding in vitro or trafficking in vivo. Our data suggest that the regulation of posttranslational carbohydrate modifications of CD34 is critical in determining its capability to act as an L-selectin ligand. Based on its ubiquitous expression, we propose that an appropriately glycosylated form of vascular CD34 may act as a ligand for L-selectin-mediated leukocyte trafficking to both lymphoid and nonlymphoid sites.

Animals↗
PubMed1994

A liquid-phase binding analysis for L-selectin. A strong dependency on highly clustered sulfate groups.

L-selectin, together with E- and P-selectins, forms a newer group of cell adhesion molecules which are believed to interact with carbohydrate ligands [Lasky, L. A., Singer, M. S., Yednoch, T. A., Dowbenko, D., Fennie, C., Rodriguez, H., Nguyen, T., Stachel, S. & Rosen, S. D. (1989) Cell 56, 1045-1055]. Using radiolabeled fucoidan as a reference ligand, we have developed a new liquid-phase microcentrifugation assay where fine differences in binding affinity can be compared accurately. We found that glucan sulfates strongly inhibited the binding of fucoidan by murine L-selectin-IgG chimera. The efficacy of inhibition is extremely dependent on the size (up to 12 kDa) and the sulfate density (up to two sulfate groups/glucose molecule) of the glucan sulfates. The nature of the inter-glucose linkages is also important. These data suggest that the binding by L-selectin prefers certain clustering and proper spatial arrangement of the anionic groups.

Anticoagulants↗
PubMed1994

A sweet success.

Explore the source record for details and available documents.

Animals↗
PubMed1994

Sialomucin ligands for selectins: a new family of cell adhesion molecules.

The specific migration of various leukocyte subsets to areas undergoing pathogenic attack is one of the cornerstones of the immune system. Assorted adhesion molecules, chemokines, non-protein signalling molecules, and their respective receptors are utilized during this process. The combinatorial matrix formed by this diversity of agents determines the cell type that migrates to a given class of inflammatory site. The selectins are a family of adhesion molecules that elicit the rolling response that initiates the inflammatory cascade. Selectins contain a type C lectin domain at their N-termini which recognizes sialylated, fucosylated carbohydrate ligands of the sialyl Lewis X (sLex) type. Recently, it has been demonstrated that these carbohydrate ligands are presented by a new class of adhesion molecules that have mucin-like structure and have been termed the sialomucin adhesion family. The function of this new family of adhesion molecules is to act as a scaffold that presents selectin carbohydrate ligands in a clustered, tissue specific manner to allow for higher avidity interactions between leukocytes and endothelial cells during the inflammatory process. In this manner, the appropriate type of leukocyte rolls adjacent to an inflammatory site in a temporally correct manner. This review will focus on the molecular and cellular biology of the sialomucin adhesion family and will discuss the possible implications of these findings on the development of specific inhibitors of selectin function.

Animals↗
PubMed1993

Expression of GlyCAM-1, an endothelial ligand for L-selectin, is affected by afferent lymphatic flow.

The interaction of naive, L-selectin-bearing lymphocytes with counterreceptors on the surface of high endothelial venules (HEV) is the initial step in the extravasation of these cells from the bloodstream into the peripheral lymph node. Recently, two sulfated glycoprotein ligands, 50 and 90 kDa, respectively, have been identified as ligands for L-selectin using an L-selectin-IgG chimera. cDNA cloning of one of these molecules, the 50-kDa sulfated glycoprotein (glycosylation-dependent cell adhesion molecule 1 [GlyCAM-1]), has shown it to be a mucinlike scaffold that presents a carbohydrate ligand(s) to the lectin domain of L-selectin. Herein, we analyze the factors that might regulate the expression of these ligands. Ligation of afferent lymphatics results in a complete loss of the mRNA for GlyCAM-1. In addition, L-selectin-mediated adhesion, as inferred by binding of an L-selectin-IgG chimera, is also lost on interruption of afferent flow. It thus appears that a soluble and/or cellular component(s) of afferent lymph regulates the expression of GlyCAM-1 mRNA and the resultant HEV adhesiveness for lymphocytes.

Animals↗
PubMed1993

Binding of L-selectin to the vascular sialomucin CD34.

The adhesive interactions between leukocyte L-selectin and the endothelium are involved in the migration of lymphocytes through peripheral lymph nodes and of neutrophils to sites of inflammation. A recombinant L-selectin stains high endothelial venules (HEVs) in lymph nodes and recognizes sulfated carbohydrates found on two endothelial glycoproteins, Sgp50 and Sgp90. Amino acid sequencing of purified Sgp90 revealed a protein core identical to that CD34, a sialomucin expressed on hematopoietic stem cells and endothelium. A polyclonal antiserum to recombinant murine CD34 stains peripheral lymph node endothelium and recognizes Sgp90 that is functionally bound by L-selectin. Thus, an HEV glycoform of CD34 can function as a ligand for L-selectin.

3T3 Cells↗