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J B Lowe

Publications and source records attributed to J B Lowe.

At least 127 records · Page 7Linked to original sources
PubMed1992

Isolation of a novel human alpha (1,3)fucosyltransferase gene and molecular comparison to the human Lewis blood group alpha (1,3/1,4)fucosyltransferase gene. Syntenic, homologous, nonallelic genes encoding enzymes with distinct acceptor substrate specificities.

Biochemical and genetic evidence indicates that the human genome may encode four or more distinct GDP-fucose:beta-D-N-acetylglucosaminide 3-alpha-L-fucosyltransferase (alpha(1,3)fucosyltransferase) activities. Genes encoding two of these activities have been previously isolated. These correspond to an alpha(1,3/1,4)fucosyltransferase thought to represent the human Lewis blood group locus and an alpha(1,3)fucosyltransferase expressed in the myeloid lineage. We report here the molecular cloning and expression of a third human alpha(1,3)fucosyltransferase gene, homologous to but distinct from the two previously reported human fucosyltransferase genes. When expressed in transfected mammalian cells, this gene determines expression of a fucosyltransferase capable of using N-acetyllactosamine to form the Lewis x epitope, and alpha(2,3)sialyl-N-acetyllactosamine to construct the sialyl Lewis x moiety. This enzyme shares 91% amino acid sequence identity with the human Lewis blood group alpha(1,3/1,4)fucosyltransferase, yet exhibits only trace amounts of alpha(1,4)fucosyltransferase activity. Polymerase chain reaction analyses were used to demonstrate that the gene is syntenic to the Lewis locus on chromosome 19. These analyses also excluded the possibility that this DNA segment represents an allele of the Lewis locus that encodes alpha(1,3)fucosyltransferase but not alpha(1,4)fucosyltransferase activity. These results are consistent with the hypothesis that this gene encodes the human "plasma type" alpha(1,3)fucosyltransferase, and suggest a molecular basis for a family of human alpha(1,3)fucosyltransferase genes.

Alleles↗
PubMed1992

Preventing fetal alcohol exposure: a cognitive behavioral approach.

Fetal alcohol exposure is a serious social and public health problem. However, most prevention programs in this area are solely informational. Important gaps in both understanding processes which underlie alcohol consumption during pregnancy and in the design of interventions to prevent fetal alcohol exposure are discussed. We argue that an important group missed by current interventions are women for whom abstinence is difficult, but who are not physically addicted. Cognitive behavioral interventions are proposed as appropriate for this target group. Recommendations for future directions in the area of fetal alcohol exposure are discussed.

Adult↗
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↗
PubMed1991

Interaction of fatty acids with recombinant rat intestinal and liver fatty acid-binding proteins.

Intestinal enterocytes contain two homologous fatty acid-binding proteins, intestinal fatty acid-binding protein (I-FABP)2 and liver fatty acid-binding protein (L-FABP). Since the functional basis for this multiplicity is not known, the fatty acid-binding specificity of recombinant forms of both rat I-FABP and rat L-FABP was examined. A systematic comparative analysis of the 18 carbon chain length fatty acid binding parameters, using both radiolabeled (stearic, oleic, and linoleic) and fluorescent (trans-parinaric and cis-parinaric) fatty acids, was undertaken. Results obtained with a classical Lipidex-1000 binding assay, which requires separation of bound from free fatty acid, were confirmed with a fluorescent fatty acid-binding assay not requiring separation of bound and unbound ligand. Depending on the nature of the fatty acid ligand, I-FABP bound fatty acid had dissociation constants between 0.2 and 3.1 microM and a consistent 1:1 molar ratio. The dissociation constants for L-FABP bound fatty acids ranged between 0.9 and 2.6 microM and the protein bound up to 2 mol fatty acid per mole of protein. Both fatty acid-binding proteins exhibited relatively higher affinity for unsaturated fatty acids as compared to saturated fatty acids of the same chain length. cis-Parinaric acid or trans-parinaric acid (each containing four double bonds) bound to L-FABP and I-FABP were displaced in a competitive manner by non-fluorescent fatty acid. Hill plots of the binding of cis- and trans- parinaric acid to L-FABP showed that the binding affinities of the two sites were very similar and did not exhibit cooperativity. The lack of fluorescence self-quenching upon binding 2 mol of either trans- or cis-parinaric acid/mol L-FABP is consistent with the presence of two binding sites with dissimilar orientation in the L-FABP. Thus, the difference in binding capacity between I-FABP and L-FABP predicts a structurally different binding site or sites.

Carrier Proteins↗
PubMed1991

Molecular cloning, expression, and uses of mammalian glycosyltransferases.

Lineage specific expression patterns of mammalian oligosaccharides are regulated largely by glycosyltransferases. Several distinct glycosyltransferase genes have recently been isolated by gene transfer and traditional cloning approaches. These each maintain a type II transmembrane topology, that yields a COOH-terminal, Golgi-resident catalytic domain. However, significant primary sequence similarities between cloned glycosyltransferases have been found only between enzymes with substantial functional similarity. These cloned DNA segments, and their cognate enzymes, have been used to determine glycosyltransferase expression patterns, characterize the molecular basis for mutant glycosyltransferase alleles, and identify oligosaccharide ligands for members of the Selectin family of cell adhesion molecules. They have also shown promise for use in the preparative synthesis of defined oligosaccharide molecules, and for transgenic animal approaches designed to explore the function(s) of oligosaccharides in mammalian organisms.

Animals↗
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

Altered membrane structure in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein.

Mouse L cell fibroblasts were transfected with cloned cDNA encoding rat liver fatty acid binding protein (L-FABP) also known as sterol carrier protein. Stable transfectant cell lines were selected and expression of L-FABP determined using Western blot analysis. The nontransfected controls and low expression cells did not differ significantly in any of the properties examined. All cell lines showed similar doubling times but cells expressing high levels of L-FABP attained 2-fold higher cell saturation density and differed significantly in their lipid metabolism as indicated by 1) higher cholesterol ester and phospholipid content, and 2) decreased sterol/phospholipid ratio. The observed changes in the lipid composition predicted a lower degree of membrane-lipid order (higher fluidity) in the plasma membranes of cells expressing high levels of L-FABP. Therefore, fluorescent molecule, 1,6-diphenyl-1,3,5-hexatriene, and multifrequency (1-250 MHz) phase and modulation fluorometry were used to probe the effect of L-FABP expression on membrane structure. Steady-state polarization and limiting anisotropy of diphenylhexatriene were significantly lower in the isolated plasma membrane vesicles from the high expression clones. The observed changes in L-cells as a result of de novo expression of L-FABP are consistent with the ability of this protein to bind sterols and fatty acids, stimulate sterol esterification, and stimulate phospholipid biosynthesis. This evidence is supportive of a physiologic role for L-FABP in modulating cellular lipid metabolism and membrane structure.

Animals↗
PubMed1990

Frameshift and nonsense mutations in a human genomic sequence homologous to a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase cDNA.

We have previously isolated a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase (alpha(1,3)-GT) cDNA (Larsen, R. D., Rajan, V. P., Ruff, M. M., Kukowska-Latallo, J., Cummings, R. D., and Lowe, J. B. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8227-8231). This enzyme constructs the terminal alpha(1,3)-galactosyl linkage within the epitope Gal alpha 1----3Gal. This epitope is expressed by New World monkeys and many nonprimate mammals but generally not by Old World primates, anthropoid apes, or man. To investigate the molecular basis for the apparent species-specific absence of this enzyme and its oligosaccharide product, we have sequenced a human genomic DNA fragment homologous to the murine alpha(1,3)-GT cDNA. This fragment contains a 703-nucleotide region that shares 82% identity with a region of the murine cDNA encoding part of the enzyme's catalytic domain. The human sequence, however, has suffered deletion of single nucleotides at two separate positions, relative to the murine sequence. These frameshift mutations disrupt the translational reading frame that would otherwise maintain a 76% amino acid sequence identity between the human sequence and the murine alpha(1,3)-GT. Moreover, nonsense mutations exist within this disrupted reading frame that would truncate the human polypeptide, relative to the murine enzyme. We therefore propose that this human sequence represents a pseudogene and cannot determine expression of Gal alpha 1----3Gal epitopes on human cells.

Amino Acid Sequence↗
PubMed1990

Transfer and expression of a murine UDP-Gal:beta-D-Gal-alpha 1,3-galactosyltransferase gene in transfected Chinese hamster ovary cells. Competition reactions between the alpha 1,3-galactosyltransferase and the endogenous alpha 2,3-sialyltransferase.

The cDNA encoding a murine UDP-Gal:beta-D-Gal-alpha 1,3-galactosyltransferase has recently been cloned and sequenced using a transient expression method (Larsen, R.D., Rajan, V.P., Ruff, M.M., Kukowska-Latallo, J., Cummings, R.D., and Lowe, J.B. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8227-8231). This report describes the construction and analysis of a Chinese hamster ovary (CHO) cell line in which in vitro expression alpha 1,3-galactosyltransferase activity has been achieved via transfer and expression of the murine alpha 1,3-galactosyltransferase gene. A primary aim of this research was to explore the role of the alpha 1,3-galactosyltransferase in regulating glycoprotein and glycolipid biosynthesis. CHO cells were cotransfected with murine genomic DNA fragments from F9 cells and plasmid DNA containing a resistance gene to the antibiotic G418. Cells resistant to G418 were then selected for expression of surface glycoconjugates containing terminal alpha 1,3-galactosyl residues by isolating cells bound to immobilized Griffonia simplicifolia-I-B4, a lectin which binds to alpha 1,3-galactosyl residues. A positive, stable transfectant clone, designated Clone 3, was obtained and analyzed for expression of the murine of alpha 1,3-galactosyltransferase. Fluorescence-activated cell sorting demonstrated that Clone 3, but not parental, CHO cells bound significant amounts of fluorescein isothiocyanate-labeled G. simplicifolia-I-B4. Southern and Northern blot analyses using the murine alpha 1,3-galactosyltransferase cDNA demonstrated that clone 3, but not parental, CHO cells contain murine alpha 1,3-galactosyltransferase genomic DNA sequences, and express a homologous transcript that comigrates with the authentic 3.6 kilobase alpha 1,3-galactosyltransferase murine mRNA. Enzyme assays confirmed that clone 3, but not parental CHO cells, contained the alpha 1,3-galactosyltransferase activity and that the level of activity is comparable to that found in F9 cells. [3H]Galactose-labeled glycopeptides and glycolipids were obtained from metabolically radiolabeled parental and Clone 3 cells and were analyzed for the presence of terminal alpha 1,3-galactosyl residues. Complex-type, Asn-linked oligosaccharides from both parental and Clone 3 cells contain the repeating disaccharide [3Gal beta 1, 4GlcNAc beta 1]n or poly-N-acetyllactosamine sequences, but only the poly-N-acetyllactosamine chains from clone 3 cells contained the terminal sequence Gal alpha 1,3Gal beta 1,4GlcNAc beta 1-R.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Molecular cloning, sequence, and expression of a human GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferase cDNA that can form the H blood group antigen.

We have previously used a gene-transfer scheme to isolate a human genomic DNA fragment that determines expression of a GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferase [alpha(1,2)FT; EC 2.4.1.69]. Although this fragment determined expression of an alpha(1,2)FT whose kinetic properties mirror those of the human H blood group alpha(1,2)FT, their precise nature remained undefined. We describe here the molecular cloning, sequence, and expression of a human cDNA corresponding to these human genomic sequences. When expressed in COS-1 cells, this cDNA directs expression of cell surface H structures and a cognate alpha(1,2)FT activity with properties analogous to the human H blood group alpha(1,2)FT. The cDNA sequence predicts a 365-amino acid polypeptide characteristic of a type II transmembrane glycoprotein with a domain structure analogous to that of other glycosyltransferases but without significant primary sequence similarity to these or other known proteins. To directly demonstrate that the cDNA encodes an alpha(1,2)FT, the COOH-terminal domain predicted to be Golgi-resident was expressed in COS-1 cells as a catalytically active, secreted, and soluble protein A fusion peptide. Southern blot analysis showed that this cDNA identifies DNA sequences syntenic to the human H locus on chromosome 19. These results strongly suggest that this cloned alpha(1,2)FT cDNA represents the product of the human H blood group locus.

Amino Acid Sequence↗
PubMed1990

A cloned human cDNA determines expression of a mouse stage-specific embryonic antigen and the Lewis blood group alpha(1,3/1,4)fucosyltransferase.

The stage-specific embryonic antigen SSEA-1 is a cell-surface oligosaccharide molecule expressed with temporal precision during the murine preimplantation period and implicated in adhesive events involving the process of compaction. We used a mammalian transient expression system to isolate a cloned human cDNA that determines expression of the SSEA-1 molecule. The cDNA sequence predicts a type II transmembrane protein with a domain structure similar to mammalian glycosyltransferases, but without primary sequence similarity to these enzymes. The carboxy-terminal domain of this protein was shown to be catalytically active as a fucosyltransferase when expressed in COS-1 cells as a portion of a secreted protein A fusion peptide. The enzyme is an exceptional glycosyltransferase in that it can use both type I and type II oligosaccharides as acceptor substrates to generate subterminal Fuc alpha(1,4)- and Fuc alpha(1,3)-linkages, respectively, in a manner analogous to the human Lewis blood group fucosyltransferase. Southern blot analysis shows that the cDNA corresponds to sequences syntenic to the Lewis locus on chromosome 19. These results indicate that this cDNA is the product of the human Lewis blood group locus, provide genetic confirmation of the hypothesis that this enzyme can catalyze two distinct transglycosylation reactions, and outline an approach to the isolation of other sequences that determine expression of developmentally regulated oligosaccharide antigens.

Amino Acid Sequence↗
PubMed1990

Efficiency in reading with closed-circuit television for low vision.

A closed circuit television (CCTV) magnifier was used to measure the reading rates of nine fluent low-vision subjects. Performance was found to be dependent on field width, character size, and the number of characters displayed in line. A field width of 70 degrees and a mean of 24 characters were commensurate with the fastest reading rate. The optimal character size that elicited the fastest reading was determined for each subject. In seven of the nine subjects, this differed from the character size selected by chance at their initial CCTV sessions, prior to these experiments. A clinical procedure is recommended, which avoids inappropriate choice, and guides the examiner in achieving directly the most efficient CCTV reading conditions for each individual patient.

Adult↗
PubMed1989

A cloned human DNA restriction fragment determines expression of a GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase in transfected cells. Evidence for isolation and transfer of the human H blood group locus.

We have described previously a gene transfer system for the isolation of human DNA sequences that determine expression of a mammalian GDP-fucose: beta-D-galactoside-2-alpha-L-fucosyltransferase (alpha-(1,2)-fucosyltransferase) (Ernst, L. K., Rajan, V. P., Larsen, R. D., Ruff, M. M., and Lowe, J. B. (1989) J. Biol. Chem. 264, 3436-3447). With this system, we found that de novo expression of the fucosyltransferase in mouse recipient cells was associated with the transfer and stable genomic integration of characteristic human DNA restriction fragments. We report here the results of experiments designed to determine the genetic origin of the fucosyltransferase determined by these sequences. First, we characterize the fucosyltransferases found in these mouse transfectants and in the human cell line used as a DNA donor. We compare their properties to those displayed by the human H and Secretor blood group fucosyltransferases. We find that the enzymes in the transfected cells have properties similar or identical to those of the human H alpha-(1,2)-fucosyltransferase. However, their properties differ significantly from the properties of the human Secretor alpha-(1,2)-fucosyltransferase and are also distinct from the properties of a murine alpha-(1,2)-fucosyltransferase. To confirm further that these transfected human sequences determine the H phenotype of the transfectants, we cloned the two human EcoRI restriction fragments common to each H-expressing secondary transfectant. The larger of these two fragments directs de novo expression of an alpha-(1,2)-fucosyltransferase when transfected into COS-1 cells. The pH activity profile of this alpha-(1,2)-fucosyltransferase and its apparent Michaelis constants for substrate and acceptor mirror those we determined for the human H alpha-(1,2)-fucosyltransferase. We conclude that genetic information sufficient to determine expression of this alpha-(1,2)-fucosyltransferase resides within the 3.4-kilobase pair human EcoRI restriction fragment and that this most likely represents the human H blood group locus.

ABO Blood-Group System↗
PubMed1989

Expression and initial characterization of a recombinant human thrombospondin heparin binding domain.

Thrombospondin (TSP) is a trimeric glycoprotein of Mr 420,000. It was originally described as a major component of human platelet alpha granules and is essential for the secondary phase of platelet aggregation. TSP is also synthesized and secreted by a variety of nucleated cells where it functions in processes involving growth and adhesion of cells to the extracellular matrix. Many of these processes are heparin-inhibitable and are mediated by a proteolytic fragment of TSP called the heparin binding domain (HBD). In order to facilitate the analysis of the structure and function(s) of this domain, we have expressed this molecule in Escherichia coli. A fragment of a TSP cDNA that encodes the heparin binding domain was inserted into the prokaryotic expression vector pJBL6. In bacterial cells grown at 42 degrees C, this vector directs the synthesis of a 24,000-Da polypeptide. Milligram quantities of this protein were purified to homogeneity from E. coli lysates. The structure of the recombinant HBD was confirmed by protein sequencing. The protein was further characterized by analysis of its conformation and function. The recombinant HBD binds [3H]heparin with a Kd of 71 nM, almost identical to that of TSP-derived HBD (80 nM). Additionally, the recombinant HBD is able to compete for TSP binding to 11B carcinoma cells. These studies indicate that the recombinant HBD is synthesized and purified in a native configuration and is functionally equivalent to thrombospondin-derived HBD. They further indicate that glycosylation of the thrombospondin HBD is not necessary for its interaction with heparin and that sequences essential to this interaction reside within the first 229 amino acids of secreted thrombospondin.

Base Sequence↗
PubMed1989

Stable expression of blood group H determinants and GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase in mouse cells after transfection with human DNA.

We report here the application of a genetic approach to identify and isolate human DNA sequences controlling the expression of a GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase [alpha-1,2)fucosyltransferase). Mouse L cells were chosen as host cells for this scheme since they express the necessary substrate and acceptor molecules for surface display of blood group H Fuc alpha 1----2 G al linkages constructed by (alpha-1,2) fucosyltransferases. However, they do not express cell surface blood group H structures nor detectable (alpha-1,2)fucosyltransferase activity. We therefore asked if (alpha-1,2)fucosyltransferase activity could be expressed and detected in these cells after transfection with human DNA sequences. These cells were transfected with genomic DNA isolated from a human cell line (A431) that expresses (alpha-1,2)fucosyltransferase. A panning procedure and fluorescence-activated cell sorting were used to isolate a mouse transfectant cell line that expresses cell surface H Fuc alpha 1----2 Gal linkages and a cognate (alpha-1,2)fucosyltransferase. Southern blot analysis showed that the genome of this cell line contains several hundred kilobase pairs of human DNA. Genomic DNA from this primary transfectant was used to transfect mouse L cells, and several independent, H-expressing secondary transfectants were isolated by immunological selection. Each expresses an (alpha-1,2)fucosyltransferase. Southern blot analysis demonstrated that the genome of each secondary transfectant contains common, characteristic human DNA restriction fragments. These results show that transfected human DNA sequences determine expression of the (alpha-1,2)fucosyltransferases in the mouse transfectants, that these sequences represent a single locus, and that they are within or linked to specific human restriction fragments identifiable in each secondary transfectant. These sequences may represent a human (alpha-1,2)fucosyltransferase gene.

ABO Blood-Group System↗