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Biomedical subjects

R M Marks

Publications and source records attributed to R M Marks.

At least 37 records · Page 2Linked to original sources
PubMed1996

Cloning of the cDNA for rabbit L-selectin and expression of recombinant protein with a kinase target site to facilitate radiolabeling.

The cDNA encoding rabbit L-Selectin has been cloned from a cDNA library, utilizing a PCR-derived probe. It encodes a peptide of 377 amino acids, including a signal peptide of 38 amino acids. Sequence analysis demonstrated extensive homology with L-Selectin's from other species. Recombinant rabbit L-Selectin protein was expressed in eukaryotic cells in a chimeric construct incorporating the entire extracellular portion of the protein coding region, a phosphokinase target site to allow high activity radiolabeling with 32P, and the constant region of the heavy chain of human IgG1 to facilitate purification and detection. Amino-terminal peptide sequencing of recombinant L-Selectin confirmed that the signal peptide had been removed at the expected site.

Amino Acid Sequence↗
PubMed1996

Demonstration of binding of dengue virus envelope protein to target cells.

The nature of the initial interaction of dengue virus with target cells and the extent to which this interaction defines tropism are unknown. Infection of some cells may involve antidengue antibody-mediated immune adherence to cells bearing immunoglobulin Fc receptors; however, this mechanism does not explain primary infection or the infection of cells without Fc receptors. We hypothesized that dengue virus envelope protein mediates initial binding to target cells. To test this hypothesis, a recombinant chimeric form of dengue type 2 virus envelope protein was used as a probe to investigate binding to the surfaces of potential target cells. Envelope protein was expressed amino terminal to the heavy-chain constant region of human immunoglobulin G containing the Fc receptor binding motif; the binding mediated by envelope determinants was distinguishable from the binding mediated by immunoglobulin Fc determinants. We found that the recombinant chimera bound to Vero, CHO, endothelial, and glial cells through envelope protein determinants and to monocytes and U937 cells by Fc-Fc receptor interactions. The highest level of binding was to Vero cells; binding was dose and time dependent and saturable. Examination of partial-length recombinant envelope proteins indicated that the binding motif was expressed between amino acids 281 and 423. Recombinant envelope protein inhibited infection of Vero cells by dengue virus, indicating the functional significance of the interaction of envelope protein and target cells in infectivity. These results suggest that envelope protein binding to a non-Fc receptor could explain the cell and tissue tropism of primary dengue virus infection.

Animals↗
PubMed1995

Role of B61, the ligand for the Eck receptor tyrosine kinase, in TNF-alpha-induced angiogenesis.

B61, a cytokine-inducible endothelial gene product, is the ligand for the Eck receptor protein tyrosine kinase (RPTK). Expression of a B61-immunoglobulin chimera showed that B61 could act as an angiogenic factor in vivo and a chemoattractant for endothelial cells in vitro. The Eck RPTK was activated by tumor necrosis factor-alpha (TNF-alpha) through induction of B61, and an antibody to B61 attenuated angiogenesis induced by TNF-alpha but not by basic fibroblast growth factor. This finding suggests the existence of an autocrine or paracrine loop involving activation of the Eck RPTK by its inducible ligand B61 after an inflammatory stimulus, the net effect of which would be to promote angiogenesis, a hallmark of chronic inflammation.

Animals↗
PubMed1995

Postoperative blood salvage in total knee arthroplasty using the Solcotrans autotransfusion system.

One hundred forty-four patients who underwent primary total knee arthroplasty were examined in a prospective controlled study to determine the efficacy and safety of a postoperative wound drainage autotransfusion system (Solcotrans, Smith & Nephew Richards, Memphis, TN). The patients were divided into two groups: control group 1 comprised 88 (61%) patients who either received a Hemovac disposable drainage system (63 patients) or the Solcotrans system and had inadequate drainage for autotransfusion (25 patients). Experimental group 2 comprised 56 (39%) patients who received a Solcotrans drainage system and were autotransfused. The Solcotrans proved itself safe. No sepsis, transfusion reactions, or coagulopathies were associated with autotransfusion, which averaged 524 mL. There were no significant differences between groups 1 and 2 when comparing preoperative and postoperative hemoglobins and hematocrits. The Solcotrans system did not lower homologous blood requirements. Only 1.6% (2 patients) of all patients who autodonated at least 2 units of autologous blood (122 patients) were in need of a homologous blood transfusion in the postoperative period. Thus, although safe, the Solcotrans system was not proven effective in the management of primary total knee arthroplasty patients.

Blood Transfusion, Autologous↗
PubMed1995

The management of neuroarthropathic fracture-dislocations in the diabetic patient.

Appropriate management for the diabetic patient with a fracture or sprain depends on recognition of "at-risk" factors. For patients with stable, minimally displaced injuries, conservative modalities (prolonged immobilization and non-weight-bearing) are sufficient. For patients with unstable or displaced fracture-dislocations, and whose general condition does not contraindicate surgery, open reduction and internal fixation, at times combined with external fixation, is recommended. Initial aggressive management can avoid or minimize the disastrous sequelae of a destructive neuroarthropathic process and can effect a biomechanically sound plantigrade, braceable, and shoeable lower extremity.

Adult↗
PubMed1995

Ultraviolet light and 8-methoxypsoralen inhibit expression of endothelial adhesion molecules.

OBJECTIVE: Having previously found that treating small areas of synovitis within the knees of patients with rheumatoid arthritis (RA) with 8-methoxypsoralen (8-MOP) and laser-derived ultraviolet A (PUVA) resulted in decreases in adhesion molecule expression, we sought to determine the effect of PUVA on expression of vascular cellular adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and E-selection by human umbilical vein endothelial cells (HUVEC). METHODS: Expression of VCAM-1, ICAM-1, and E-selectin on the surface of HUVEC was measured using specific antibodies and flow cytometry or a fluorescence plate reader, following treatment of cells with 8-MOP and UVA, before and after tumor necrosis factor (TNF) stimulation. RESULTS: PUVA led to significant dose dependent decreases in the expression of VCAM-1 and E-selectin that had been induced with TNF before PUVA treatment. Pretreatment with PUVA was also able to prevent subsequent TNF induction of VCAM-1 expression. TNF-induced ICAM-1 expression was not decreased by PUVA, however, and pretreatment only partially decreased ICAM-1 expression. CONCLUSION: The in vivo effects of PUVA may be explained, in part, by down regulation of adhesion molecule expression. The relative resistance of ICAM-1 to PUVA suggests some specificity to the effect on adhesion molecule expression.

Cells, Cultured↗
PubMed1994

Fas ligation triggers apoptosis in macrophages but not endothelial cells.

The reticuloendothelial system includes macrophages and endothelial cells. These cells are produced and destroyed in vivo with a precision that implies the existence of homeostatic mechanisms. The stimuli for endothelial cell proliferation and monocyte production are becoming well characterized. However, the mechanisms involved in eliminating these cells are poorly understood. One mechanism involved in cellular elimination is apoptosis, which can be triggered in some cells by ligation of the Fas molecule. In this report we have investigated whether macrophages and endothelial cells express the Fas molecule, and whether Fas transmits an apoptosis-inducing signal in these cells. We demonstrate that macrophages express Fas and readily undergo apoptosis when cultured with anti-Fas. In contrast, while endothelial cells can express the Fas molecule, Fas ligation is insufficient to induce apoptosis. These results suggest differential regulation of Fas function among cells of the reticuloendothelial system, and imply different mechanisms of homeostasis.

Antigens, Surface↗
PubMed1992

Cloning of a novel tumor necrosis factor-alpha-inducible primary response gene that is differentially expressed in development and capillary tube-like formation in vitro.

TNF is a proinflammatory cytokine that has pleiotropic effects on cells and tissues, mediated in large part by alterations in target tissue gene expression. We have used the technique of differential hybridization to identify several primary response genes induced by TNF in human umbilical vein endothelial (HUVE) cells, a cell type that is profoundly activated by cytokine treatment. One of these cDNA, designated B94, detects a rapidly and transiently induced 4-kb transcript in TNF-treated HUVE cells, and this transcript is superinduced in the concomitant presence of cycloheximide. Other proinflammatory stimuli including IL-1 beta and LPS are also able to induce B94 mRNA expression. Nuclear run-on experiments demonstrate that TNF induction of B94 transcript occurs primarily at the level of transcriptional activation. Further, B94 is shown to be a single copy gene that is evolutionarily conserved. The gene is localized to the q32 region of chromosome 14, a region that is often rearranged in lymphoid neoplasms. B94 transcript expression is also found to be regulated during mouse development and in an in vitro model of endothelial capillary tube formation. Developmental regulation occurs most prominently in mouse embryonic liver and kidney, and a second smaller form of B94 transcript is detected in the placenta and testes. B94 and other TNF-responsive transcripts are also induced during capillary tube formation suggesting overlap between genes induced by TNF and those induced during angiogenesis. Sequence analysis of the B94 cDNA reveals an open reading frame encoding a 73-kDa polypeptide that has no homology to any known protein. Polyclonal antisera directed against the carboxyl-terminal portion of the B94 protein immunoprecipitates a protein of the predicted molecular mass both from COS cells transfected with a B94 expression vector and from TNF-treated HUVE cells.

Amino Acid Sequence↗
PubMed1992

Characterization of a novel tumor necrosis factor-alpha-induced endothelial primary response gene.

The response of endothelial cells to the cytokine tumor necrosis factor-alpha (TNF) is complex, involving the induction and suppression of multiple genes and gene products. Differential screening of a TNF-stimulated, cycloheximide-treated human umbilical vein endothelial cell library has resulted in the cloning of several novel cDNAs whose protein products are involved in the primary response of the endothelium to TNF. One of these cDNAs, designated B12, is further characterized here. B12 is encoded by a 3.5-kilobase transcript and is induced rapidly and transiently by TNF. Transcript expression is found to be developmentally regulated in a tissue-specific manner, with B12 message being differentially expressed in the heart and liver during mouse embryogenesis. The open reading frame of B12 predicts a 316-amino acid sequence rich in charged residues, particularly at the carboxyl terminus, and has neither significant homology to other known proteins nor to any extent sequence motifs. B12 is found to be a highly conserved single-copy gene which is located in the q22----q23 region of human chromosome 17. Polyclonal antibodies raised against a large portion of the B12 open reading frame immunoprecipitate a 36-kilodalton polypeptide from wheat germ lysates programmed to translate in vitro transcribed B12 mRNA. The B12 protein is further shown to be induced in human umbilical vein endothelial cells by TNF, and the protein is shown to be rapidly degraded.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of a human fucosyltransferase gene that determines expression of the Lewis x and VIM-2 epitopes but not ELAM-1-dependent cell adhesion.

We have used the human Lewis blood group fucosyltransferase cDNA and cross-hybridization procedures to isolate a human gene that encodes a distinct fucosyltransferase. Its DNA sequence predicts a type II transmembrane protein whose sequence is identical to 133 of 231 amino acids at corresponding positions within the catalytic domain of the Lewis fucosyltransferase. When expressed by transfection in cultured cell lines, this gene determines expression of a fucosyltransferase capable of efficiently utilizing N-acetyllactosamine to form the Lewis x determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc). By contrast, biochemical and flow cytometry analyses suggest that the enzyme cannot efficiently utilize the type II acceptor NeuNAc alpha 2----3Gal beta 1----4GlcNAc, to form the sialyl Lewis x determinant. In Chinese hamster ovary cells, however, the enzyme can determine expression of the alpha 2----3-sialylated, alpha 1----3-fucosylated structure known as VIM-2, a putative oligosaccharide ligand for ELAM-1. Cell adhesion assays using VIM-2-positive, sialyl Lewis x-negative transfected Chinese hamster ovary cells indicate that surface expression of the VIM-2 determinant is not sufficient to confer ELAM-1-dependent adhesive properties upon the cells. These results demonstrate that substantial structural similarities can exist between mammalian glycosyltransferases with closely related enzymatic properties, thus facilitating isolation of their cognate genes by cross-hybridization methods. The results further suggest that cell surface expression of the VIM-2 determinant is not necessarily sufficient to mediate ELAM-1-dependent cell adhesion.

Amino Acid Sequence↗
PubMed1990

ELAM-1--dependent cell adhesion to vascular endothelium determined by a transfected human fucosyltransferase cDNA.

Adhesion of circulating leukocytes to the vascular endothelium during inflammation is mediated in part by their interaction with the endothelial-leukocyte adhesion molecule ELAM-1. ELAM-1, a member of the LEC-CAM family of cell adhesion molecules, expresses an N-terminal carbohydrate recognition domain (CRD) homologous to various calcium-dependent mammalian lectins. However, the contribution of the CRD to cell adhesion and its carbohydrate binding specificity have not been elucidated. This study demonstrates that transfection of a human fucosyltransferase cDNA into nonmyeloid cell lines confers ELAM-1--dependent endothelial adhesion. Binding activity correlates with de novo cell surface expression of the sialylated Lewis x tetrasaccharide, whose biosynthesis is determined by the transfected fucosyltransferase cDNA. We propose that specific alpha(1,3)fucosyltransferases regulate cell adhesion to ELAM-1 by modulating cell surface expression of one or more alpha(2,3)sialylated, alpha(1,3)fucosylated lactosaminoglycans represented by the sialyl Lewis x carbohydrate determinant.

Animals↗
PubMed1990

Tumor necrosis factor-alpha induction of novel gene products in human endothelial cells including a macrophage-specific chemotaxin.

Cytokines such as tumor necrosis factor alpha (TNF) profoundly affect endothelial cell function, promoting for example interaction with leukocytes and inducing a procoagulant phenotype. Changes of this nature are likely to be central to the proinflammatory effects of TNF. In order to elucidate molecular mechanisms by which TNF alters endothelial cell function we utilized differential plaque hybridization to identify TNF-responsive genes. Forty TNF-inducible cDNAs were identified which on cross-hybridization were found to arise from six unique genes. DNA sequencing of these cDNAs revealed two encoded known cytokine-induced genes, endothelial leukocyte adhesion molecule 1 and neutrophil chemotactic factor. One of the cDNAs encodes a recently described monocyte-specific chemotactic factor not previously associated with endothelium. The production of a monocyte chemotaxin by cytokine-activated endothelium has important implications for understanding the role of the vessel wall in disease states such as atherosclerosis and may also in part explain the indirect angiogenic activity of TNF. The three other cDNAs are completely novel as judged by data bank searches of partial DNA sequences and remain unidentified. On exposure of endothelial cells to TNF there is a rapid and substantial increase in levels of mRNA encoding the six genes, which are further superinduced by cycloheximide. Thus these represent primary response genes as their induction does not depend on protein synthesis. Interleukin-1 beta and lipopolysaccharide are also potent inducers. Nuclear run-on studies revealed that in most cases induction by TNF is mediated largely at the transcriptional level.

Biological Factors↗
PubMed1990

A novel immediate-early response gene of endothelium is induced by cytokines and encodes a secreted protein.

We have previously described the cloning of a group of novel cellular immediate-early response genes whose expression in human umbilical vein endothelial cells is induced by tumor necrosis factor alpha in the presence of cycloheximide. These genes are likely to participate in mediating the response of the vascular endothelium to proinflammatory cytokines. In this study, we further characterized one of these novel gene products named B61. Sequence analysis of cDNA clones encoding B61 revealed that its protein product has no significant homology to previously described proteins. Southern analysis suggested that B61 is an evolutionarily conserved single-copy gene. B61 is primarily a hydrophilic molecule but contains both a hydrophobic N-terminal and a hydrophobic C-terminal region. The N-terminal region is typical of a signal peptide, which is consistent with the secreted nature of the protein. The mature form of the predicted protein consists of 187 amino acid residues and has a molecular weight of 22,000. Immunoprecipitation of metabolically labeled human umbilical vein endothelial cell preparations revealed that B61 is a 25-kilodalton secreted protein which is markedly induced by tumor necrosis factor.

Amino Acid Sequence↗
PubMed1990

Iloprost inhibits neutrophil-induced lung injury and neutrophil adherence to endothelial monolayers.

We hypothesized that Iloprost, a long-acting prostacyclin analog, would inhibit neutrophil (PMN)-induced lung injury and decrease PMN adherence to vascular endothelium. Human PMNs infused into isolated buffer-perfused rat lungs subsequently stimulated with phorbol myristate acetate (PMA) resulted in lung injury as assessed by the accumulation of [125I]bovine serum albumin (125I-BSA) in lung parenchyma and alveolar lavage fluid. Addition of Iloprost to the lung perfusate, prior to activation of the PMNs, reduced lung injury as assessed by a decrease in the accumulation of 125I-BSA in the lung. This protective effect was not due to the vasodilatory effect of Iloprost. Protection by Iloprost was not linked to a reduction in PMA-induced PMN superoxide production since Iloprost did not reduce the amount of superoxide released into lung perfusate. In vitro, Iloprost caused a dose-dependent inhibition of PMA-stimulated PMN adherence to endothelial cells. Iloprost did not affect the number of Mo1 adhesion molecules constitutively expressed or the number of receptors expressed on the PMNs following PMA. Addition of cAMP or dibutyryl cAMP to the endothelial cells mimicked the effects of Iloprost, diminishing PMA-stimulated PMN adhesion. In separate experiments, addition of the phosphodiesterase inhibitor IBMX to Iloprost resulted in a greater inhibition of PMA-stimulated PMN adherence, while addition of an adenylate cyclase inhibitor, SQ 22,536, or cAMP antibodies with the Iloprost abolished Iloprost's inhibitory effect on PMN adhesion. Thus, Iloprost inhibits PMA-activated PMN-induced lung injury despite continued superoxide production. Iloprost inhibition of PMN adhesion is dependent on cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1989

The antimitogenic action of tumor necrosis factor is associated with increased AP-1/c-jun proto-oncogene transcription.

Tumor necrosis factor alpha (TNF) mediates its in vivo antineoplastic effect primarily through its action on the endothelial surfaces of tumor vessels. Among other changes, endothelial cells increase the expression of surface procoagulant molecules which allow for the genesis of microthrombi and the eventual anoxic killing of the tumor tissue. TNF is also a potent antimitotic factor for endothelial cells. We investigated the molecular basis for these diverse actions of TNF by differential screening of a cDNA library prepared from TNF and cycloheximide-treated human umbilical vein endothelial (HUVE) cells. One of the cDNA clones identified encodes the transcription factor and proto-oncogene AP-1/c-jun. The expression of AP-1/c-jun following TNF treatment is mediated largely at the transcriptional level. Neither c-myc nor c-fos transcripts were induced by TNF. Since AP-1/c-jun has been previously identified as one of the earliest transcripts expressed in quiescent cells that have been stimulated by growth factors, our results suggest that mitogenic and antimitogenic stimuli evoke distinct yet overlapping sets of molecular responses and that the coordinate expression of AP-1/c-jun and c-fos is not mandatory. AP-1/c-jun transcript induction is thus one of the initial events mediated by TNF treatment of HUVE cells.

Blotting, Northern↗