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Biomedical subjects

B A Cunningham

Publications and source records attributed to B A Cunningham.

At least 91 records · Page 5Linked to original sources
PubMed1985

Chromosomal location of the gene encoding the neural cell adhesion molecule (N-CAM) in the mouse.

The gene encoding the neural cell adhesion molecule, N-CAM, has been localized on mouse chromosome 9. A BALB/cJ mouse genomic library prepared in lambda bacteriophage EMBL4 was screened by using a cDNA probe, pEC204, that corresponds to the coding region of the chicken N-CAM gene. Four weakly reactive and one strongly reactive recombinant phage were isolated. A region of the latter that was strongly homologous to pEC204 was subcloned to yield a new probe, pEC501. RNA transfer blots and nucleotide sequencing indicated that pEC501 encoded part of the mouse N-CAM gene. This probe defined a unique genetic locus, Ncam, associated with a restriction fragment length polymorphism that allowed the definition of two alleles. The locus could be provisionally assigned either to chromosome 9 or to chromosome 10 by correlating the presence or absence of mouse-specific DNA fragments reactive with the probe in a panel of somatic hybrid cell lines with the presence or absence of the various mouse chromosomes. Analysis of the inheritance of the Ncam-associated DNA polymorphism in recombinant inbred strains of mice revealed close linkage between Ncam and the Lap-1, Sep-1, and Thy-1 loci on chromosome 9. This result suggests an additional linkage between Ncam and the locus for the cerebellar mutation staggerer (sg). The Ncam locus provides an important reference point for mapping the genes for additional cell adhesion molecules as well as genes for other molecules involved in neural development and function.

Animals↗
PubMed1985

Isolation of a cDNA clone for the liver cell adhesion molecule (L-CAM).

Liver cell adhesion molecule (L-CAM) is a calcium-dependent cell adhesion molecule found in very early vertebrate embryos and on liver and other epithelial cells in adults. To describe the genes coding for the molecule and study its synthesis, we have cloned cDNA from poly(A)+ RNA of 10-day embryonic chicken liver using the delta gt11 expression vector. One clone, lambda L301, has been characterized and used in analyses of L-CAM mRNA and genomic DNA. Clone lambda L301 produced a fusion protein that reacted strongly with polyclonal antibodies that recognize L-CAM (Mr 124,000) and its Mr 81,000 NH2-terminal fragment, Ft1, released from liver membranes by trypsin. This result indicates that lambda L301 contains a cDNA insert complementary to protein coding sequence within the two-thirds of the mRNA coding region beginning at the 5' end. The 220-base-pair cDNA insert was isolated and used as a probe in hybridization experiments. RNA transfer blot analysis of poly(A)+ RNA showed a single 4-kilobase mRNA; Southern blot analysis showed multiple components consistent with the presence of one to three L-CAM genes. To test whether different tissues express different forms of L-CAM message, poly(A)+ RNA from eight embryonic organs was analyzed. Only organs that expressed L-CAM protein contained poly(A)+ RNA that hybridized to the lambda L301 probe; in all cases a single band, with the same mobility as that in liver, was observed. The L-CAM mRNA in each tissue was present in proportions similar to those detected previously for the L-CAM protein in these tissues. The combined results suggest that any possible heterogeneity in the L-CAM genes is not reflected in the size of either the mRNA or protein.

Animals↗
PubMed1984

Sulfation and phosphorylation of the neural cell adhesion molecule, N-CAM.

Embryonic chicken brain tissue cultured in media containing 35S-labeled sulfate or 32P-labeled phosphate incorporated 35S or 32P into the neural cell adhesion molecule (N-CAM). The 35S label was located in asparagine-linked carbohydrates on both glycopeptides (molecular weights, 170,000 and 140,000) but not in the sialic acid. The 32P label was detected in phosphoamino acids in the carboxyl-terminal third of both polypeptides, but the ratio of phosphoserine to phosphothreonine differed in the two species. The sulfated saccharides and phosphoamino acids may provide additional sites for functional control of N-CAM.

Animals↗
PubMed1984

Ontogenetic expression of cell adhesion molecules: L-CAM is found in epithelia derived from the three primary germ layers.

Immunofluorescence techniques using specific antibodies against the liver cell adhesion molecule, L-CAM, were used to explore the appearance of L-CAM during early embryogenesis and organogenesis, as well as in adult tissue. Immunoblots of L-CAM from embryonic and adult organs indicated that molecules detected in each tissue were L-CAM, and that the antibodies were not simply detecting cross-reacting molecules. L-CAM was found in low levels on pregastrulation embryos. During gastrulation, the molecule remained present on ectoderm but was not detected on mesodermal and definitive endodermal cells. During neurulation, L-CAM disappeared from the neural ectoderm, in which staining for the neural cell adhesion molecule, N-CAM, had previously been shown to increase markedly. During organogenesis, L-CAM appeared in all endodermal structures, in ectoderm other than neural derivatives, in placodes, in extraembryonic ectoderm and endoderm, and in some mesodermal structures such as Wolffian ducts, oviduct, and kidney epithelium. Other mesodermal derivatives were not stained and the molecule was not detected in hemangioblastic areas of the lateral plate mesoderm nor in splanchnopleural derivatives such as spleen, adrenal glands, and gonads. During embryonic induction, for example, neurulation and in early kidney development, changes in L-CAM distribution were correlated with both locations and times of induction events. Analysis of distribution in the adult revealed that L-CAM was present in the stratum germinativum of the skin, in endodermally derived epithelia, in the female reproductive tract, and in the kidneys. In several fully differentiated glandular organs, L-CAM staining was restricted to basal or apical parts of the cell surface. When correlated with previous results obtained for N-CAM, these findings support the idea that local cell surface modulation of a small number of cell adhesion molecules may regulate other primary processes of development to yield specific patterns, both in early development and in organogenesis. Reflections of these patterns remain in adult life.

Animals↗
PubMed1984

Isolation of cDNA clones for the chicken neural cell adhesion molecule (N-CAM).

Enriched mRNA coding for the neural cell adhesion molecule (N-CAM) was prepared from 9-day embryonic chicken brains by immunoprecipitation of polysomes with antibodies to N-CAM. This mRNA programmed the translation in vitro of N-CAM polypeptide chains in a rabbit reticulocyte lysate system. Two independent N-CAM cDNA clones (designated pEC001 and pEC020) were derived from the enriched RNA. The specificity of pEC001 for N-CAM mRNA was verified by hybrid selection experiments. Both plasmids hybridized to two discrete 6- to 7-kilobase-long RNA species in poly(A)+ RNA from embryonic chicken brain and to lesser amounts of polydisperse material of smaller sizes (probably degradation products of the large RNAs). No hybridization was detected to poly(A)+ RNA from embryonic liver. Southern blotting experiments with pEC001 detected only one hybridizing fragment in chicken genomic DNA digested with several different restriction enzymes, suggesting that sequences corresponding to those within this region of N-CAM mRNA are present at most only a few times, and possibly only once, in the chicken genome.

Animals↗
PubMed1984

Linear organization of the liver cell adhesion molecule L-CAM.

A linear model of the liver cell adhesion molecule L-CAM from embryonic chickens is proposed in terms of its orientation on the cell surface, the number, type, and distribution of carbohydrate moieties, and sites of phosphorylation. L-CAM is isolated from cell membranes as a glycoprotein of Mr = 124,000. A soluble fragment (Ft1) of Mr = 81,000 can be released from cells by digestion with trypsin in the presence of calcium. Radiochemical amino acid sequence analyses indicated that both polypeptides have the same sequence for the first 10 amino acids, suggesting that fragment Ft1 contains the amino terminus of the L-CAM molecule and that the carboxyl-terminal portion of the peptide chain is associated with the cell. Digestions with endoglycosidase H and endoglycosidase F indicated that Ft1 has all of the N-linked carbohydrate groups associated with the larger species, including one high mannose oligosaccharide and three complex oligosaccharides. When hepatocytes were grown in the presence of 32PO4, 32P was detected in phosphoserine and phosphothreonine residues of intact L-CAM, but little or no 32P was detected in Ft1, suggesting that L-CAM is phosphorylated in the carboxyl-terminal region. On CNBr cleavage, the bulk of the 32P was detected in a single fragment of Mr = 20,000. The overall features of the L-CAM molecule incorporated in the model provide a basis for correlating its structure with its cell-cell binding activity and for detailed comparisons with similar molecules described in mammalian species.

Amino Acids↗
PubMed1984

Mapping of three carbohydrate attachment sites in embryonic and adult forms of the neural cell adhesion molecule.

The sialic-rich carbohydrate moiety of the neural cell adhesion molecule (N-CAM) undergoes major structural changes during development and plays a significant role in altering the homophilic binding of the molecule. In order to understand the mechanism of these changes, a cyanogen bromide (CNBr) fragment that contained 90% of the sialic acid of N-CAM was isolated and characterized according to the number of carbohydrate attachment sites and reactivity with specific monoclonal antibodies. The CNBr sialopeptide migrated on SDS PAGE as a broad zone of Mr 42,000-60,000. Upon treatment with neuraminidase, it was converted to a single component of Mr 42,000, and subsequent, limited treatment with endoglycosidase F gave four evenly spaced components of Mr 35,000-42,000, suggesting that it contained three attachment sites for N-linked oligosaccharides. The fragment reacted with monoclonal antibody 15G8, which detects the sialic acid in embryonic N-CAM, and with a monoclonal antibody, anti-(N-CAM) No. 2. Treatment with neuraminidase or with endoglycosidase F destroyed reactivity with 15G8 but not with anti-(N-CAM) No. 2. A similar CNBr sialopeptide was obtained from adult N-CAM; it contained sialic acid, had three N-linked oligosaccharides and reacted with anti-(N-CAM) No. 2 but not with 15G8 monoclonal antibodies. A peptide fragment, Fr2, comprising the NH2 terminal and middle regions of the molecule yielded a CNBr fragment closely similar to the fragment obtained from the whole molecule. The CNBr fragment from Fr2 reacted with monoclonal antibody anti-(N-CAM) No. 2. Fr1, comprising the NH2 terminal region alone, failed to react. These data confirm that the majority of the sialic acid is localized in the middle region of the N-CAM molecule and support the hypothesis that embryonic to adult conversion of N-CAM is the result of differences in sialidase or sialytransferase activity.

Aging↗
PubMed1983

Molecular topography of the neural cell adhesion molecule N-CAM: surface orientation and location of sialic acid-rich and binding regions.

Chemical analyses and binding studies have been correlated to clarify the relationship of structure to function in the neural cell adhesion molecule (N-CAM) from embryonic chicken brain. N-CAM isolated from the cell surface appears to include two closely related polypeptide chains. Treatment with neuraminidase of such preparations of N-CAM bound by antibodies on solid supports yielded components of Mr 140,000 and 170,000. These components each had the same amino-terminal sequence as N-CAM and gave nearly identical profiles on peptide maps. Immunoprecipitation of N-CAM from 9-day brain cells treated with tunicamycin yielded corresponding components of Mr 130,000 and 160,000, suggesting that the differences between these two components of N-CAM are in the polypeptide rather than the carbohydrate portions of the molecules. N-CAM appears to be oriented with the amino terminus extending away from the cell surface and with the bulk of the sialic acid near the middle of the peptide chain. As shown previously, incubation of N-CAM at 37 degrees C generates a fragment (Fr1) of Mr 65,000 that lacks most of the sialic acid. Treatment of membranes with Staphylococcus aureus V-8 protease released a fragment (Fr2) of N-CAM that contained most of the sialic acid; this fragment had an Mr of 108,000 after neuraminidase treatment. Both of these fragments contain the amino-terminal portion of the polypeptide chain. At least a portion of the N-CAM binding site was found to be located in the amino-terminal region of the peptide chain. Most or all of the sialic acid was not directly involved in binding, although it can influence binding, as indicated by the finding that neuraminidase-treated N-CAM (desialylated-N-CAM) bound to cells to a greater extent than untreated N-CAM. The Fr1 and the Fr2 fragments in solution did not bind to cells but were as effective as N-CAM and desialylated-N-CAM as competitors for N-CAM binding to cells. When fixed covalently to beads, N-CAM, desialylated-N-CAM, and the Fr1 and Fr2 fragments bound specifically to cells. In contrast, the N-CAM autolysis products released along with Fr1 neither bound to cells nor competed for N-CAM binding. In addition to suggesting a location for the N-CAM binding region, the accumulated results raise the possibility that valence may play a key role in N-CAM binding.

Animals↗
PubMed1983

Early epochal maps of two different cell adhesion molecules.

N-CAM, the neural cell-adhesion molecule, has previously been found to be expressed during several epochs of development and function, first as an early marker in embryo-genesis, later during organogenesis, and finally in adult life. L-CAM, the liver cell-adhesion molecule, has now been localized in embryonic and adult tissues of the chicken by fluorescent antibody techniques. In the early embryonic epoch, L-CAM and N-CAM appeared in epiblastic and hypoblastic tissues. L-CAM was distributed thereafter across all three germ layers. By the onset of neurulation, however, L-CAM disappeared in the region of the neural plate and N-CAM increased in amount in that region. L-CAM appeared strongly on all budding endodermal structures (liver, pancreas, lung, thyroid, parathyroid, thymus, and bursa of Fabricius) whereas N-CAM appeared most strongly in the neural plate, neural tube, and in cardiac mesoderm but was not found in endodermal derivatives. In placodes, both L-CAM and N-CAM were present until the formation of definitive neural structures, at which time L-CAM disappeared. In kidney precursors, the two CAMs followed a complex reciprocal pattern of appearance and disappearance. For the most part, however, the distributions of the two molecules did not overlap during organogenesis. Like N-CAM, L-CAM persisted in a distinctive pattern of expression in adult tissues. During embryonic development, the two different CAMs were distributed on tissues derived from more than two-thirds of the early embryonic surface. Interpretation of maps summarizing CAM distributions over a defined developmental epoch suggested a key role for both L-CAM and N-CAM in embryonic induction. Consistent with this interpretation and with the fact that the continuity of germ layers is lost when organ rudiments are formed, neither of the CAMs was limited in distribution to a single germ layer. The regions of the early epochal maps that lacked both L-CAM and N-CAM comprised some portions of the splanchnopleure and somatopleure. Certain adult tissues that derive from this lateral plate mesoderm such as smooth muscle also lacked L-CAM and N-CAM. Such observations suggest that at least one more CAM may exist in these and similarly derived tissues.

Animals↗
PubMed1983

Characterization of L-CAM, a major cell adhesion molecule from embryonic liver cells.

We have developed a method for purifying L-CAM, the cell adhesion molecule from embryonic chicken liver cells, and have compared its properties with those of N-CAM, the neural cell adhesion molecule. L-CAM was released from membranes with trypsin, purified by a series of chemical techniques, and used to generate monoclonal antibodies which allowed the identification of the intact L-CAM molecule from membranes. The monoclonal antibodies were used to isolate trypsin-released L-CAM in a single step by affinity chromatography. Material purified by either technique was predominantly a component of M(r) 81,000 on NaDodSO(4)/polyacrylamide gel electrophoresis with a pI of 4.0-4.5. Rabbit antibodies to this component and to the M(r) 81,000 species that had been further purified on NaDodSO(4)/polyacrylamide gel electrophoresis displayed all of the activities of anti-L-CAM. Some of the trypsin-released L-CAM bound specifically to lentil lectin, suggesting that L-CAM is a glycoprotein. The apparent molecular weight of material having L-CAM antigenic determinants depended upon the procedures used to extract membranes; this appears to account for the various values reported previously in the literature. Both the rabbit serum antibodies and the monoclonal antibodies detected the M(r) 81,000 species on immunoblots of unfractionated trypsin-released material. Immunoblots of whole liver cell membranes with the same antibodies revealed a major M(r) 124,000 component, with minor components of M(r) 94,000 and 81,000. Active L-CAM derivatives released by trypsin in the presence of EGTA were detected as a species of M(r) 40,000. L-CAM derivatives obtained by extraction of membranes with EDTA alone appeared as species of M(r) 53,000, 62,000, and 81,000. The combined results suggest that L-CAM on the cell surface is an acidic glycoprotein of M(r) 124,000. In the presence of calcium, the molecule can be released from membranes by trypsin as a soluble M(r) 81,000 fragment; in the absence of calcium, it is released by either endogenous proteases or by trypsin as a variety of smaller fragments.

Antibodies, Monoclonal↗
PubMed1983

Veno-occlusive disease following marrow transplantation.

Veno-occlusive disease is a newly identifiable complication after marrow transplantation that demands further research into its treatment and etiology. Nurses play a key role in the early detection as well as the continual physical and psychologic support needed by the patient throughout the course of this disease.

Ascites↗
PubMed1982

Chemical characterization of a neural cell adhesion molecule purified from embryonic brain membranes.

A neural cell adhesion molecule (N-CAM) was purified in milligram quantities from detergent extracts of embryonic chick brain membranes. N-CAM has an unusual carbohydrate content and structure, is polydisperse in solution, and is associated with proteolytic activity leading to its spontaneous cleavage. The carbohydrate composition of N-CAM includes 13 mol of sialic acid but only 1.4 mol of galactose/100 mol of amino acids, suggesting the presence of a sialic acid to protein linkage not previously observed in higher organisms. N-CAM appears to be an integral membrane protein in that its extraction from membranes required detergent. Although soluble, the purified molecule was aggregated (Mr = 0.5 to 1.2 X 10(6)) and polydisperse in detergent-free solutions. N-CAM from brain also migrated as a broad but continuously stained region from Mr = 200,000 to Mr = 250,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the molecule from retina was similar but had a somewhat faster mobility. Desialation of N-CAM did not significantly change its behavior in solution, but converted both brain and retinal N-CAM to components migrating on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as material of about Mr = 140,000. Despite the apparent heterogeneity, amino acid sequence analysis and comparison of proteolytic fragments suggest that all forms of the glycoprotein are derived from the same polypeptide chain. On prolonged incubation at neutral pH, N-CAM undergoes apparent proteolysis to yield a polypeptide that contains little sialic acid and has a Mr = 65,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a separate sialic acid-rich component, and a variety of small peptides. The 65,000-dalton polypeptide appeared to contain all of the antigenic determinants of intact N-CAM that neutralize the adhesion-blocking ability of anti-retinal cell Fab' fragments.

Amino Acid Sequence↗
PubMed1982

In vitro translation and processing of a precursor form of favin, a lectin from Vicia faba.

Favin, the glucose- and mannose-binding lectin isolated from fava (Vicia faba) beans, consists of two polypeptide chains (alpha, Mr = 5,571; beta, Mr = 20,700). Translation of fava bean mRNA in vitro in a wheat germ-derived system yields a single favin polypeptide chain of Mr = 29,000. This molecule appears to consist of a hydrophobic 29-amino acid residue signal sequence at the NH2 terminus followed by the beta chain sequence; it also includes the alpha chain sequence. These results suggest that the alpha and beta chains arise by post-translational cleavage of a single precursor polypeptide: signal-beta chain-alpha chain. The signal peptide is similar in sequence to those seen in animal and prokaryotic systems, suggesting that translocation mechanisms are highly conserved. Translation of favin mRNA in the presence of dog pancreas microsomal membranes yields at least three polypeptides in addition to the presumed precursor chain. The largest of these molecules is translocated into the lumen of the membrane vesicles and glycosylated but its signal sequence remains intact. The two other species are translocated and glycosylated, but their signal sequences have been removed; they appear to differ from each other in that one begins with the beta chain sequence and the other begins one residue after the NH2-terminal threonine of the beta chain. These three variants could reflect normal features of the processing of the favin precursor but more likely result from aberrant processing of the plant protein by dog pancreas membranes.

Amino Acid Sequence↗
PubMed1982

Amino acid sequence and variant forms of favin, a lectin from Vicia faba.

We have determined the complete amino acid sequence (182 residues) of the beta chain of favin, the glucose-binding lectin from fava beans (Vicia faba), and have established that the carbohydrate moiety is attached to Asn 168. Together with the sequence of the alpha chain previously reported (Hemperly, J. J., Hopp, T. P., Becker, J. W., and Cunningham, B. A. (1979) J. Biol. Chem. 254, 6803-6810), these data complete the analysis of the primary structure of the lectin. We have also examined minor polypeptides that appear in all preparations of favin. Two lower molecular weight species (Mr = 9,500-11,600) appear to be fragments of the beta chain resulting from cleavage following Asn 76, whereas six high molecular weight forms (Mr = 25,000 or greater) appear to include aggregates of the beta chain and possibly some alternative products of chain processing.

Amino Acid Sequence↗
PubMed1981

Distances between structural metal ion, substrates, and allosteric modifier of fructose bisphosphatase.

The binding of two paramagnetic probes within a subunit of fructose bisphosphatase, viz., Mn2+ at a structural site and a nitroxide spin-label at a sulfhydryl site, has permitted the measurement of NMR and electron paramagnetic resonance (EPR) relaxation rates to map the active and allosteric site topography. Distances from these loci to the phosphoryl of fructose 6-phosphate (Fru-6-P) and inorganic phosphate (Pi) and four nuclei of adenosine 5'-phosphate (AMP) (the phosphorus nucleus, H-8, H-2, and H-1') were obtained. These measurements located the Mn2+ approximately equidistant from the two phosphoryl moieties of the product ligands Fru-6-P and Pi and in close proximity to the AMP. The adenosine moiety of the latter is oriented anti. Analysis of EPR data revealed that the nitroxide group is approximately 16 A from the structural Mn2+ site. Calculation of the residence times for the hydrolysis reaction products suggests that their dissociation should not be rate limiting in the overall reaction cycle.

Allosteric Regulation↗