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B A Cunningham

Publications and source records attributed to B A Cunningham.

At least 73 records · Page 4Linked to original sources
PubMed1988

Four exons encode a 93-base-pair insert in three neural cell adhesion molecule mRNAs specific for chicken heart and skeletal muscle.

The neural cell adhesion molecule (N-CAM) is detected in chicken brain as three polypeptides of 180 kDa, 140 kDa, and 120 kDa that arise from a single gene by alternative splicing. Heart tissue, however, contains components of 150 kDa, 140 kDa, and 130 kDa; neither the differences in molecular mass among these components nor the difference between neural and cardiac N-CAM could be accounted for by variations in glycosylation alone. A cDNA clone isolated from an embryonic chicken heart library, [lambda N101B, 1.8 kilobases (kb)] contained a 93-base-pair (bp) insert not found in neural N-CAM cDNAs. In the N-CAM gene this sequence mapped within a large region between exons 12 and 13 and was derived from four exons (12A-D) of 15, 33, 42, and 3 bp. Exons 12C and 12D together coded for 15 amino acids very similar to the second half of the muscle-specific insert (MSD1) found in N-CAM cDNA from human muscle cell cultures [Dickson, G., Gower, H. J., Barton, C. H., Prentice, H. M., Elsom, V. L., Moore, S. E., Cox, R. D., Quinn, C., Putt, W. & Walsh, F. S. (1987) Cell 50, 1119-1130]; the sequences of 12A and 12B, however, were much less similar to the corresponding region of the MSD1 sequence. Two oligonucleotides, one specific to exons 12A plus 12B and one specific to exon 12C both recognized mRNA species of 6.4 kb, 4.3 kb, and 3.0 kb in chicken cardiac and skeletal muscle and no mRNA species in smooth muscle or brain. The 3' end of clone lambda N101B contained a sequence coding for a potential phosphatidylinositol linkage signal as does the smallest form of brain N-CAM. In heart cell membranes only the 130-kDa N-CAM polypeptide was released by phospholipase C, suggesting that this form of N-CAM is encoded by clone lambda N101B. The other heart N-CAM species (150 kDa and 140 kDa) may be transmembrane forms that include the 12A-D (and possibly other) inserts. Tissue-specific forms of N-CAM can thus be formed by alternative use of multiple small exons that may alter the conformation of the extracellular region of the molecule. Differential use or switching of these small exons in conjunction with the differential expression of larger exons specifying regions associated with the cell membrane and cytoplasmic domains may signal key events in embryogenesis and histogenesis.

Animals↗
PubMed1988

A cDNA clone for cytotactin contains sequences similar to epidermal growth factor-like repeats and segments of fibronectin and fibrinogen.

Cytotactin is an extracellular glycoprotein that influences neuron-glia interactions. It has been shown to appear in multiple forms that are differentially expressed in neural and non-neural tissues during vertebrate development. We report here the isolation and characterization of a cytotactin cDNA clone (lambda C801) that encodes 933 amino acids, equivalent to about half of a cytotactin polypeptide. Clone lambda C801 is an authentic cytotactin cDNA: it encodes a polypeptide that reacts with a monoclonal anti-cytotactin antibody and its deduced amino acid sequence is identical for 15 amino acids to the directly determined sequence of a CNBr fragment that reacted with the same antibody. Southern blot analyses with fragments of lambda C801 suggested that there may be only one cytotactin gene, but RNA transfer blots detected multiple mRNAs ranging in size from 6.5 to 8.0 kilobases. An 8.0-kilobase message and a Mr 240,000 cytotactin polypeptide were present in embryonic gizzard but not brain, while a 7.2-kilobase message and a Mr 220,000 polypeptide were present in brain but not gizzard. These results indicate that differential splicing of primary transcripts of the cytotactin gene yields various site-specific polypeptides. Sequence analyses of lambda C801 indicated that it specifies a region with extensive similarities to other proteins: the sequence begins with four consecutive epidermal growth factor-like repeats that are followed by eight segments that closely resemble each other and the type III repeats in fibronectin, and it ends with a 66 amino acid sequence similar to part of the beta and gamma chains of fibrinogen. One fibronectin-like repeat contains a single Arg-Gly-Asp sequence. The similarities with all three of these apparently unrelated proteins are extensive, suggesting that cytotactin has an evolutionary and possibly a functional relationship to each.

Amino Acid Sequence↗
PubMed1987

Neural cell adhesion molecule: structure, immunoglobulin-like domains, cell surface modulation, and alternative RNA splicing.

The neural cell adhesion molecule, N-CAM, appears on early embryonic cells and is important in the formation of cell collectives and their boundaries at sites of morphogenesis. Later in development it is found on various differentiated tissues and is a major CAM mediating adhesion among neurons and between neurons and muscle. To provide a molecular basis for understanding N-CAM function, the complete amino acid sequences of the three major polypeptides of N-CAM and most of the noncoding sequences of their messenger RNA's were determined from the analysis of complementary DNA clones and were verified by amino acid sequences of selected CNBr fragments and proteolytic fragments. The extracellular region of each N-CAM polypeptide includes five contiguous segments that are homologous in sequence to each other and to members of the immunoglobulin superfamily, suggesting that interactions among immunoglobulin-like domains form the basis for N-CAM homophilic binding. Although different in their membrane-associated and cytoplasmic domains, the amino acid sequences of the three polypeptides appear to be identical throughout this extracellular region (682 amino acids) where the binding site is located. Variations in N-CAM activity thus do not occur by changes in the amino acid sequence that alter the specificity of binding. Instead, regulation is achieved by cell surface modulation events that alter N-CAM affinity, prevalence, mobility, and distribution on the surface. A major mechanism for modulation is alternative RNA splicing resulting in N-CAM's with different cytoplasmic domains that differentially interact with the cell membrane. Such regulatory mechanisms may link N-CAM binding function with other primary cellular processes during the embryonic development of pattern.

Amino Acid Sequence↗
PubMed1987

Body composition changes in marrow transplant recipients receiving total parenteral nutrition.

Nine patients with acute lymphocytic leukemia in remission, aged 12-35 years, undergoing allogeneic bone marrow transplantation (BMT) were studied for changes in body fluid balance and body composition. Body composition and fluids were assessed the first 4 weeks following BMT, using isotope dilution and anthropometry. Oral and parenteral nutrient intakes were recorded daily. Tracer dilution techniques were used to assess body fluid volumes and estimate body cell, lean body, and body fat masses. Body cell mass was lost (mean -1.62 kg, P less than 0.05) without significant changes in body fat or lean body masses. There was an expansion of the extracellular fluid compartment (mean +0.8 l, P less than 0.05) and a loss in the intracellular fluid compartment (mean -1.3 l, P less than 0.05) with little change in total body water volume. Changes in body weight correlated poorly with body cell mass or fluid volume changes. Change in arm muscle area correlated well with changes in body cell mass (r = 0.61, P less than 0.05) and lean body mass (r = 0.68, P less than 0.05), while that of arm fat area did not reflect its isotope dilution-derived counterpart. Instead, the change in arm fat area was related to shifts in fluid compartments. Prealbumin decreased significantly (mean -9.3 mg/dl, P less than 0.05), while albumin decreased slightly (mean -0.1 mg/dl), and both were related to changes in body cell mass. Nitrogen balance was negative throughout the study and the overall mean was related to the change in body cell mass (r = 0.60, P less than 0.05). Calorie and protein intakes were not associated with the changes in body composition, implying other causal factors.

Adolescent↗
PubMed1987

Organization of the neural cell adhesion molecule (N-CAM) gene: alternative exon usage as the basis for different membrane-associated domains.

The neural cell adhesion molecule, N-CAM, is expressed as at least three polypeptide chain, (ld, sd, and ssd chains) specified by a single gene and derived by alternative splicing and polyadenylation-site selection during RNA processing. We describe here the characterization of seven overlapping genomic phage clones reactive with N-CAM cDNA, indicating that the chicken N-CAM gene is more than 50 kilobases long. Analysis of the gene shows that there are at least 19 exons and that the coding sequences for the ld, sd, and ssd chains are assembled from 18, 17, and 15 exons, respectively. The first 14 exons appear to be common to all three chains and encode the amino-terminal portion of N-CAM, which contains five tandem homologous repeats resembling those seen in the immunoglobulin gene superfamily. In contrast to other genes containing such domains, each of these segments in N-CAM is specified by two exons. The carboxyl-terminal portion of each N-CAM chain is different as a result of the alternative use of exons. A single exon encodes the carboxyl-terminal 26 amino acids of the ssd chain and the 3' untranslated region of its mRNA, ending with a poly(A)-addition site. Two exons encode the transmembrane and cytoplasmic sequences common to the ld and sd chains, and another exon encodes the additional 261 amino acids found in the cytoplasmic domain of the ld chain. The carboxyl-terminal 21 amino acids common to the ld and sd chains and the 3' untranslated region common to their mRNAs are encoded by a single large exon of 3475 base pairs that ends with a second poly(A)-addition site. Sequences from the 13-kilobase intron that separates the exons encoding the amino-terminal and carboxyl-terminal regions of the molecule hybridize to a 2-kilobase poly(A)+ RNA transcript of unknown identity. This description of the chicken N-CAM gene provides a basis for determining the mechanisms that regulate the differential expression of the N-CAM polypeptide chains during development.

Amino Acid Sequence↗
PubMed1987

Cellular expression of liver and neural cell adhesion molecules after transfection with their cDNAs results in specific cell-cell binding.

Mouse L cells, which do not express the known primary cell adhesion molecules (CAMs), were permanently transfected with vectors containing the simian virus 40 early promoter and cDNA sequences encoding chicken liver CAM (L-CAM) or each of the three major polypeptide forms of chicken neural CAM (N-CAM). Transfected cells in culture expressing the Ca2+-dependent L-CAM showed uniform surface expression of the molecule. Unlike untransfected L cells, these cells aggregated readily; the aggregation was inhibited by Fab' fragments of antibodies to L-CAM but not by fragments of anti-N-CAM. These cells spread more efficiently in culture than did their untransfected counterparts, forming small colonies of flattened cells that gradually assumed morphologies resembling closely packed L cells. Transfected L cells expressing either the small or large intercellular domain polypeptide (sd or ld) chains of N-CAM aggregated specifically with each other or bound membrane vesicles from chick brain. Both types of binding were specifically inhibited by Fab' fragments of anti-N-CAM antibodies. These cells, in contrast to those transfected with vectors for L-CAM, showed rounded morphologies and spread inefficiently in culture. L cells transfected with vectors specifying the small surface domain polypeptide (ssd) chain of N-CAM showed no phenotypic changes and no evidence for linkage of ssd chains to the cell membrane by phosphatidylinositol intermediates. Instead, these cells synthesized the molecule and released it into the medium. These findings complete the demonstration that different CAMs have specific roles in ligating the cells that synthesize them, and they provide further evidence that L-CAM and N-CAM bind by homophilic mechanisms. The different phenotypic changes observed for each specific CAM are consistent with the hypothesis that CAM synthesis or differing associations of CAM carboxyl-terminal domains with the cell surface and cortex may lead directly or indirectly to specific alterations in the cells bound together by that CAM.

Antigens, Surface↗
PubMed1987

Sequence analysis of a cDNA clone encoding the liver cell adhesion molecule, L-CAM.

The liver cell adhesion molecule (L-CAM) appears on non-neural epithelial tissues and mediates calcium-dependent adhesion in these tissues both in the embryo and in the adult. It appears on cell surfaces as a glycoprotein of Mr 124,000 but is synthesized as a precursor of Mr 135,000. We have isolated and determined the nucleic acid sequence of a cDNA clone (lambda L320) encoding chicken L-CAM. The 5' end of this clone has an open reading frame extending for 2520 base pairs, followed by an 850-base-pair untranslated region terminating with a polyadenylylation site at its 3' end. Protein sequence analysis of intact L-CAM and of cyanogen bromide fragments of the protein confirmed the reading frame and indicated that lambda L320 encodes the complete sequence of L-CAM as it is expressed on the cell surface as well as the bulk of the precursor. The sequence includes a hydrophobic segment of 31 amino acids, supporting our earlier conclusion that L-CAM is an intrinsic membrane protein. There are five potential asparagine glycosylation sites on the extracellular part of the molecule and an intracellular domain that is phosphorylated in vivo. The mature L-CAM polypeptide consists of 727 amino acids, with a calculated Mr of 79,900 for the carbohydrate-free protein. The L-CAM sequence is not homologous to other known protein sequences, including those of the neural cell adhesion molecule (N-CAM) and other members of the immunoglobulin superfamily, but the L-CAM molecule does contain three contiguous segments (113 amino acids each) that are homologous to each other. The similarities among these segments suggest that at least part of the L-CAM molecule arose by gene duplication.

Amino Acid Sequence↗
PubMed1987

Sex differences in nitrogen balance following marrow grafting for leukemia.

A sex difference in nitrogen balance was investigated in 40 adults, 21 men and 19 women, undergoing chemoradiotherapy and marrow transplantation for leukemia and receiving total parenteral nutrition. Twenty-four hour collections of urine and mixed urine-stool were analyzed for total nitrogen daily through day 14 posttransplant. Nitrogen balance, corrected for changes in blood urea nitrogen, decreased significantly over time (p less than 0.005) in both men and women, but men experienced a greater negative nitrogen balance during the time period (p less than 0.001). Mean daily nitrogen balance in men was -6.0 g for week 1 and -9.2 g for week 2, corresponding to -3.3 g and -5.6 g in women for week 1 (p less than 0.005) and 2 (p less than 0.01), respectively. The differences remained after controlling for stress level and adjusting for total calorie intakes. There were no differences in age, disease status, or nitrogen intakes per kg ideal body weight, and no effects on nitrogen balance by arm muscle area at admission, cyclosporine use, or the branched-chain amino acid content of the parenteral solution. The average rise in 3-methylhistidine excretion was 23% in men and 11% in women. These results suggest higher per kg nutrient needs in males during stress and may indicate differing metabolic responses to stress. The possibility of gender differences should be considered in research evaluating nitrogen metabolism during severe stress.

Adult↗
PubMed1986

Thiocyanate and bromide ions influence the bilayer structural parameters of phosphatidylcholine bilayers.

The influence of monovalent cations and anions on the structural parameters of dipalmitoylphosphatidylcholine (DPPC) bilayers was examined at 25 degrees C using X-ray diffraction. It was shown that monovalent salts, in general, have little effect on lipid packing within the bilayer. However, fully hydrated DPPC bilayers in 1 M KSCN pack in an interdigitated acyl chain phase. This is the first observation of an ion-induced interdigitated bilayer phase in a zwitterionic lipid. In addition, gel state DPPC bilayers in 1 M KBr imbibe approx. 10 A more solvent than bilayers in water. The influence of these same salts on the phase transitions of DPPC bilayers was also examined using high-resolution differential scanning calorimetry. These results are discussed in terms of ion-induced changes in solvent and solvent/bilayer structure.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1986

Ca2+ induced phase separations in phospholipid mixtures.

We have probed the character of the observed phase separation in mixtures of phosphatidylcholines (PC) and/or phosphatidylethanolamines (PE) in the presence of CaCl2 solutions. Egg yolk phosphatidylethanolamine (EYPE) and a 1:1 molar ratio of dioleoylphosphatidylcholine/dioleoylphosphatidylethanolamine (DOPC/DOPE) were observed to undergo phase separation in CaCl2 solutions, as was previously observed for egg yolk phosphatidylcholine (EYPC) (L.J. Lis et al. Biochemistry, 20 (1981) 1771-1777). However, the mixed chain lipid, palmitoyloleoyl-PC, yielded only a single phase in water or CaCl2 solution. We hypothesize that two lipid species are necessary for the observed phase separation to occur, but that the separation itself is not a function of the individual lipid species, but of the mixture.

Calcium↗
PubMed1986

The influence of ion species on phosphatidylcholine bilayer structure and packing.

The effects of various monovalent cations and anions on the bilayer packing and structure of dipalmitoylphosphatidylcholine were studied using X-ray diffraction and differential scanning calorimetry. It was observed from the X-ray diffraction studies that monovalent salts, in general, have no effect on bilayer packing. The results of DSC studies on metal chloride systems are consistent with the interpretation that cations in general and Li+ in particular bind to DPPC bilayers. The effect of potassium salts on pre- and main-transition temperatures suggest that anions, such as Acetate-, also significantly bind to DPPC head groups.

Anions↗
PubMed1986

cDNA clones of the neural cell adhesion molecule (N-CAM) lacking a membrane-spanning region consistent with evidence for membrane attachment via a phosphatidylinositol intermediate.

In embryonic chicken brains, the neural cell adhesion molecule N-CAM is expressed mainly as two polypeptides, the large intracellular-domain polypeptide (ld) (Mr = 160,000) and the small intracellular-domain polypeptide (sd) (Mr = 130,000) chains, that differ in their cytoplasmic domains and that arise by alternative splicing of RNA transcribed from a single gene. There is evidence for a minor N-CAM polypeptide of Mr = 120,000 that is similar to the ld and sd chains for most of its amino-terminal sequence, but which lacks a cytoplasmic domain. We report here the isolation and characterization of a cDNA clone, lambda N151, that appears to encode this third N-CAM polypeptide, which we designate the ssd (small surface-domain) polypeptide chain. The cDNA insert of lambda N151 consists of 2437 base pairs (bp). DNA hybridization and sequencing indicate that the first 1721 bp are nearly identical to the corresponding sequences of clone lambda N208, which encodes the ld chain. Following in the same reading frame, lambda N151 encodes 25 amino acids not present in lambda N208. The rest of lambda N151 consists of a 637-bp noncoding region containing an AATACA polyadenylylation sequence and a 55-bp poly(A) tract. Messenger RNAs complementary to lambda N151 appear later in development than those complementary to the ld and sd chains, and their appearance is correlated with the appearance of the ssd polypeptide. Although the polypeptide encoded by lambda N151 lacks a membrane region that would define a cytoplasmic domain, it does contain at its carboxyl end a relatively hydrophobic stretch of amino acids similar to those seen in precursors of membrane proteins that are attached to membranes via the lipid phosphatidylinositol. We show here that the ssd chain of chicken N-CAM can be released from brain vesicles by treatment with phospholipase C, suggesting that it too may have a phosphatidylinositol anchor. These results define two additional modes by which N-CAM expression can be modulated: by RNA splicing at a new site and by differential membrane attachment of the resulting polypeptide through a lipid intermediate.

Amino Acid Sequence↗
PubMed1986

Sequence of a cDNA clone encoding the polysialic acid-rich and cytoplasmic domains of the neural cell adhesion molecule N-CAM.

Purified fractions of the neural cell-adhesion molecule N-CAM from embryonic chicken brain contain two similar polypeptides (Mr, 160,000 and 130,000), each containing an amino-terminal external binding region, a carbohydrate-rich central region, and a carboxyl-terminal region that is associated with the cell. Previous studies indicate that the two polypeptides arise by alternative splicing of mRNAs transcribed from a single gene. We report here the 3556-nucleotide sequence of a cDNA clone (pEC208) that encodes 964 amino acids from the carbohydrate and cell-associated domains of the larger N-CAM polypeptide followed by 664 nucleotides of 3' untranslated sequence. The predicted protein sequence contains attachment sites for polysialic acid-containing oligosaccharides, four tandem homologous regions of polypeptide resembling those seen in the immunoglobulin superfamily, and a single hydrophobic sequence that appears to be the membrane-spanning segment. The cytoplasmic domain carboxyl terminal to this segment includes a block of approximately equal to 250 amino acids present in the larger but not in the smaller N-CAM polypeptide. We designate these the ld (large domain) polypeptide and the sd (small domain) polypeptide. The intracellular domains of the ld and sd polypeptides are likely to be critical for cell-surface modulation of N-CAM by interacting in a differential fashion with other intrinsic proteins or with the cytoskeleton.

Amino Acid Sequence↗
PubMed1986

Alternatively spliced mRNAs code for different polypeptide chains of the chicken neural cell adhesion molecule (N-CAM).

Rabbit polyclonal antibodies directed against the chicken neural cell adhesion molecule (N-CAM) were used to isolate four overlapping cDNA clones from a chicken cDNA expression library in bacteriophage gamma gt11. These clones collectively accounted for 3.8 kilobases of N-CAM mRNA sequence and hybridized specifically to two 6-7-kilobase brain polyadenylated RNA species that co-migrated with previously identified N-CAM mRNAs. DNA fragments derived from an internal region of the cloned cDNA sequences hybridized to the larger but not to the smaller N-CAM mRNA species, while fragments on either side of this region hybridized to both mRNAs. A cDNA fragment that recognized only the larger mRNA was subcloned into gamma gt11, and the expressed fusion protein was used to affinity-purify rabbit polyclonal antibodies; the antibodies recognized only the larger of the two structurally related N-CAM polypeptides. In contrast, when several cDNA clones that recognized both mRNAs were used to purify antibodies, the antibodies recognized both polypeptides. The results, in conjunction with other data indicating that there is one gene specifying N-CAM, suggest that different N-CAM polypeptides are synthesized from multiple N-CAM messages generated by alternative splicing of transcripts from a single N-CAM gene.

Animals↗
PubMed1986

Cell surface modulation of the neural cell adhesion molecule resulting from alternative mRNA splicing in a tissue-specific developmental sequence.

The neural cell adhesion molecule N-CAM is an intrinsic membrane glycoprotein that is expressed in the embryonic chicken nervous system as two different polypeptide chains encoded by alternatively spliced transcripts of a single gene. Because they differ by the presence or absence of approximately 250 amino acids in their cytoplasmic domains, these polypeptides are designated ld and sd, for large and small cytoplasmic domain, respectively. We report here that the ld-specific sequences comprise a single exon in the chicken N-CAM gene and that developmental expression of the ld and sd chains occurs in a tissue-specific fashion, with the ld chain restricted to the nervous system. Comparison of the nucleotide sequences from an N-CAM genomic clone with cDNA sequences showed that a single exon of 783 base pairs corresponded to the unique cytoplasmic domain of the ld polypeptide. Sequences from this exon were absent from the single N-CAM mRNA detected in several non-neural tissues by RNA blot hybridization, and immunoblot analysis confirmed that antigenic determinants unique to the ld-specific domain were not expressed in these tissues. Immunohistochemical experiments indicated that only the sd chain was expressed on cell surfaces of non-neural tissues throughout embryonic development. The ld chain was found on cell bodies and neurites of differentiated neurons; it first appeared as neurons began to extend neurites and to express the neuron-glia cell adhesion molecule (Ng-CAM) and it was restricted to definite layers in laminar tissues such as the retina and cerebellum. These results suggest that the control of mRNA splicing may affect the regulation of N-CAM function at specific sites within the nervous system and thus influence the control of neural morphogenesis and histogenesis.

Amino Acid Sequence↗
PubMed1986

A prospective study of unexplained nausea and vomiting after marrow transplantation.

We prospectively studied patients with enigmatic nausea and vomiting after allogeneic marrow transplantation to define the causes of this syndrome. Fifty consecutive episodes of persistent vomiting were investigated using physical examination and laboratory tests, endoscopic biopsies and brushings, and clinical follow-up for four weeks. Potential causes of vomiting were identified in 39 of the 50 cases (78%). Fifteen cases had gastrointestinal infections (mainly herpesviruses), 13 had unsuspected acute intestinal graft-versus-host disease (GVHD), 8 had intestinal infection plus acute GVHD, and 3 had other causes (subdural hematomas, bacteremia, and encephalitis). In the remaining 11 cases, no cause of vomiting was found. Endoscopy was necessary for diagnosis in 36 cases and required a combination of methods: routine histology, cytology, viral culture, and immunohistology using monoclonal antibodies to cytomegalovirus (CMV) and herpes simplex virus type 1. Patients with unexplained vomiting or intestinal GVHD had significant improvement of nausea and vomiting over the four-week observation period, but those with CMV did not (P = .01). We conclude that most allogeneic marrow transplant patients with enigmatic nausea and vomiting have gastrointestinal herpesvirus infections, acute GVHD, or both. Untreated CMV infections and persistent GVHD are associated with protracted vomiting in these patients.

Adolescent↗