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Biomedical subjects

E A Davidson

Publications and source records attributed to E A Davidson.

At least 19 recordsLinked to original sources
PubMed1995

Primary structure of the canine U1 snRNA.

The nucleotide (nt) sequence of the canine U1 snRNA was determined. It exhibited significant homology (90-98%) with known U1 sequences. The RNA can be folded according to the secondary structure previously proposed for the U1 snRNA. It contained the conserved sequence UUACCUG in loop A (nt 6-12), required for the recognition of the 5' splice site, and the sequence UGCACU in loop B (nt 68-73), required for recognition of the U1-70K protein. The U1 snRNA was localized in the nucleus and its transcription was sensitive to alpha-amanitin, suggesting that it is transcribed by RNA polymerase II. Southern analysis revealed that the canine genome possesses 5-10 copies of U1 snRNA-encoding genes.

Animals↗
PubMed1994

Role of sialic acid on the viscosity of canine tracheal mucin glycoprotein.

The role of sialic acid on the viscosity of canine tracheal mucin (CTM) was investigated. The mucin glycoprotein, purified from canine tracheal mucus, was subjected to mild acid hydrolysis with aqueous acetic acid and autohydrolysis in water, in which approximately 50% drop in the relative viscosity (nr) occurred. Carbohydrate compositional analysis before and after mild acid hydrolysis and autohydrolysis showed the complete removal of glycosidically bound sialic acid residues while all other sugar residues (i.e. galactose, N-acetyl galactosamine and N-acetyl glucosamine) remained unaltered, indicating that sialic acid residues are contributing towards the viscosity of CTM to a greater extent.

Animals↗
PubMed1994

Isolation and characterization of novel mucin-like glycoproteins from cobra venom.

A high molecular weight, heavily glycosylated protein fraction was isolated from cobra venom. It consists of mucin-like glycoproteins (designated as cobra venom mucin) in noncovalent association with several lower molecular weight proteins and glycoproteins. The mucin was purified by CsBr density gradient centrifugation under dissociative conditions. The purified venom mucin comprised about 85% carbohydrate and 15% protein and was rich in Thr, Ser, Pro, Gly, Glu, Asp, and Ala. The mucin was resolved into two or more distinct classes of mucin-like glycoproteins which differ in their amino acid compositions and/or carbohydrate content. Unlike other mucins, cobra venom mucin does not form highly viscous solutions. It appears to keep several venom proteins and glycoproteins soluble by noncovalent interactions. Cobra venom mucin contains both O- and N-linked oligosaccharides; 1 N-linked chain for every 8-10 O-linked oligosaccharides. The O-linked chains are novel structures with high molar proportions of fucose, galactose, and N-acetylglucosamine relative to N-acetylgalactosamine; they have a very low sialic acid content and lack sulfate esters. The majority of the O-linked oligosaccharides are unusually large and contain 15 to as many as 50 sugar residues. The O-linked oligosaccharides are poly-N-acetyllactosaminyl chains consisting of -3Gal beta 1-4GlcNAc beta 1- and -3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1- repeats and thus they contain inner Le(X) antigenic determinants. These oligosaccharides terminate with novel alpha-galactosylated Le(X) and Le(a) epitopes. Due to the abundance of terminal alpha-galactosyl residues, cobra venom mucin reacts with anti-alpha-Gal antibodies that are normally present in human serum.

Amino Acids↗
PubMed1994

Structural features of water-soluble novel polysaccharide components from the leaves of Tridax procumbens Linn.

Two water-soluble polysaccharide fractions, WSTP-IA and WSTP-IB were purified from the leaves of Tridax procumbens Linn. with graded ethanol precipitation followed by mild delignification and size-exclusion chromatography. WSTP-IA contained L-Araf and D-Galp in approximately 1:3 molar proportions, and WSTP-IB contained only D-Galp as the major sugar component. The results of methylation linkage analysis, and 1H and 13C NMR studies on the native and modified polysaccharides, indicated that WSTP-IA is an L-arabino-D-galactan with a beta-(1-->6)-D-galactan main chain in which at least one in every two D-Galp residues carries single residues of either L-Araf (alpha-/beta-) or beta-D-Galp end-group as substituents at O-3. WSTP-IB is a linear beta-(1-->6)-D-galactan. This is the first report of polysaccharides containing a beta-(1-->6)-D-galactan main chain isolated from plant sources.

Arabinose↗
PubMed1994

Factor X-activating glycoprotein of Russell's viper venom. Polypeptide composition and characterization of the carbohydrate moieties.

There is contradictory information regarding the molecular weight and polypeptide chain composition of RVV-X, a glycoprotein in Russell's viper venom that is capable of activating factor X to Xa. We show that RVV-X is a 92,880-Da glycoprotein. It consists of three disulfide-linked polypeptide chains, one heavy chain (alpha-chain, M(r) 57,600) and two light chains (beta- and gamma-chains, M(r) 19,400 and 16,400, respectively). The two light chains seen on SDS-polyacrylamide gel electrophoresis under reducing conditions are two distinct components of the molecule, rather than a heterogeneous mixture of a single chain as previously reported (Takeya, H., Nishida, S., Miyata, T., Kawada, S., Saisaka, Y., Morita, T., and Iwanaga, S. (1992) J. Biol. Chem. 267, 14109-14117). The following evidence supports this conclusion. (i) The two light chains of RVV-X are present in equal proportion. (ii) The estimated molecular weight of an alpha 1 beta 1 gamma 1-structure closely matches the molecular weight determined by matrix-assisted laser desorption mass spectrometry. (iii) The amino acid compositions and NH2-terminal sequences of the beta- and gamma-chains are different. (iv) Although both the beta- and gamma-chains contain one N-linked oligosaccharide chain each, they are glycosylated differentially. RVV-X contains six N-linked oligosaccharides, four in the alpha-chain and one in each of the beta- and gamma-chains. The carbohydrate structures are different from those known for other snake venom glycoproteins, and they resemble closely those in various mammalian glycoproteins. The majority of the oligosaccharides are complex bi-, tri-, and tetraantennary structures, with a small proportion of multiantennary and high mannose-type structures. Two notable structural features of RVV-X oligosaccharides are as follows. (i) Sialic acid residues are linked to beta-galactosyl residues solely by alpha 2,3-linkages, and (ii) bisecting N-acetylglucosamine residues are present in the majority of the oligosaccharides.

Amino Acid Sequence↗
PubMed1994

Non-sequence-specific antimalarial activity of oligodeoxynucleotides.

The effects of exogenously applied oligodeoxynucleotides on Plasmodium falciparum proliferation was investigated. A fluorescence-activated cell sorter assay was employed to measure parasitemia after administration of either phosphodiester or phosphorothioate oligodeoxynucleotides. We report sequence-independent antimalarial activity preferentially with phosphorothioate congeners with IC50 values in the 1-2 microM range. Phosphorothioate oligodeoxynucleotides which were antisense, sense or nonsense to Plasmodium mRNA, as well as homopolymers (30-mers containing all A or T bases) were equally effective inhibitors of parasitemia. The antimalarial activity was dependent upon oligomer length, concentration, and time of addition to the cultures but was independent of the parasite strain tested. Four P. falciparum strains, including a multi-drug-resistant strain (MDR-K), a drug-sensitive strain (FCR-3), a erythrocyte membrane sialic acid-independent strain (7G8) and a strain isolated from a cerebral malaria patient (CM-87) were equally susceptible to treatment with a phosphorothioate oligomer. Inhibition of red cell invasion is primarily responsible for the observed decrease in proliferation as determined by a study of parasite maturation in the presence of a 30-mer nonsense phosphorothioate oligodeoxynucleotide.

Animals↗
PubMed1994

Mucin synthesis in immortalized canine tracheal epithelial cells.

To study the regulation of mucin synthesis in canine tracheal epithelial cells, it is desirable to establish a cell line which synthesizes mucin continuously. We adopted the approach of immortalizing canine tracheal epithelial cells using a vector encoding the human papillomavirus (type 18) E6 and E7 genes. The E6 and E7 genes are essential and sufficient for the immortalization of human genital keratinocytes, as well as human tracheal epithelium. Primary epithelial cells from dog trachea were transfected with a vector containing HPV18 genes E6 and E7. The resultant cells (CT1) were cloned and maintained in selective medium supplemented with growth factors and hormones. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated the expression of the canine tracheal mucin (CTM) mRNA in these cells. The half-life of the CTM mRNA was found to be 45-60 min. Incorporation of labelled precursor (glucosamine) indicated that high-molecular-weight mucin glycoprotein was synthesized by these immortalized cells, which reacted with the antiserum to the native CTM. Equilibrium gradient centrifugation analysis showed that the buoyant density of the mucin synthesized in CT1 cells (1.486 g/ml) was similar to the reported value for native CTM (1.5 g/ml). Mucin which was isolated from immortalized cells was not a proteoglycan as chondroitinase treatment had no effect. These results suggest that CT1 cells synthesize a mucin glycoprotein which exhibits properties similar to native CTM. When characterized by immunostaining with a pool of monoclonal antibodies, these cells showed common epithelial antigens related to keratin expression. The CT1 cell line represents a unique resource for studying mucin biosynthesis and regulation.

Animals↗
PubMed1994

New approach towards deglycosylation of sialoglycoproteins and mucins.

A modified procedure for chemical deglycosylation of glycoproteins containing sialylated and/or O-linked oligosaccharides, using anhydrous trifluoromethane sulfonic acid (TFMSA) is described. Although sialic acid residues are acid labile, it has been known that anhydrous TFMSA does not effectively remove carbohydrate side chains from glycoproteins if they are sialylated. In this procedure, sialic acid residues were removed by mild acid hydrolysis and the desialylated glycoprotein was treated with anhydrous TFMSA reagent under conditions which remove all the carbohydrate residues except the core D-GalNAc linked to serine/threonine. The core D-GalNAc residues were removed by reacting the glycoprotein with periodate followed by a second treatment with anhydrous TFMSA; this procedure gave a completely deglycosylated protein. The protein thus obtained was soluble in aqueous buffers and useful for biochemical and biophysical studies. The method was successfully employed to isolate polypeptides from alpha 1-acid glycoprotein (N-linked), fetuin, canine tracheal mucin and gastric mucin.

Amino Acids↗
PubMed1994

Mucin coding sequences are remarkably conserved.

Mucins are complex glycoproteins expressed by glandular epithelia and the carcinoma which develop from these tissues. The core protein is aberrantly glycosylated in cancers, and some antibodies show specificity in their reactions with the cancer-associated mucins, which also contains epitopes recognized by T-cells from pancreatic and breast cancer patients. Based on the PCR amplification of the mucin coding sequences, hybridization analysis and determination of the sequence divergence we present the evidence that mucin coding sequences are conserved in a number of species. A broad series of organisms were examined for analogous sequences. Data show that mucin-type sequences are present in a variety of mammals, but less apparent in chicken and yeast. Divergence increased in the order human, monkey, rabbit/rat/cow, mouse; chicken and yeast exhibited minimal homology. Furthermore, nucleotide sequences not included in the tandem repeats, a common feature of mucin core structure, are more conserved than the flanking sequences which also suggests that the flanking sequences may be functionally significant while repeats are structurally important. The hybridization bands showed different restriction patterns (suggesting for the existence of the restriction fragment length polymorphism). Northern analysis indicates message polydispersity, commonly seen with this class of RNA. The major features of the protein appear broadly conserved in the different mammalian species examined. The evolutionary significance of the above studies has been discussed.

Animals↗
PubMed1994

Canine U2 snRNA gene: nucleotide sequence, characterization and implications in RNA processing and cancer biology.

Abnormal RNA processing (splicing) may lead a cell to become cancerous. Transcription of a gene starts in the nucleus where genomic DNA is converted to precursor RNA by removing introns and joining exons. Splicing, mediated by small nuclear RNA (snRNA) and nuclear proteins, is tightly regulated during growth and development. U2 snRNAs are small, stable RNAs located in the nucleus of eukaryotic cells that recognize the branch point of the intron-exon junction. We describe here the organization of DNA sequences complementary to canine U2 snRNA. From a genomic library we isolated one recombinant containing the U2 gene. Southern analysis revealed that the canine species possesses only 3 to 5 U2 snRNA genes or very closely related sequences. The size of the U2 gene is 125 nt whereas in rat, Drosophila, trypanosome and yeast it is 189, 234, 141, and 192 nt respectively. The nucleotide sequence showed 82, 78, 72 and 95% homology with rat, Drosophila, yeast, and trypanosome U2 snRNA, respectively. The sensitivity of U2 snRNA towards alpha-amanitin suggests that it is transcribed by RNA polymerase II. The conserved nucleotide sequences which have been implicated in heterogeneous nuclear RNA splicing have been identified. The implications of the knowledge gained through above studies in cancer biology are discussed.

Animals↗
PubMed1992

Structural features of carbohydrate moieties in snake venom glycoproteins.

The structures of the carbohydrate moieties of glycoproteins in snake venoms are largely unknown. In the present study, we have analyzed venoms of several species of snakes as well as plasma and tissue glycoproteins from one species of cobra (Naja naja kaouthia) by lectin affinity staining of Western blots. The data demonstrate that glycoproteins in cobra venom invariably contain terminal alpha-galactosyl residues with negligible proportions of sialic acids. Interestingly, however, terminal alpha-galactosyl residues are present in significantly lower proportions in cobra tissues such as brain, liver, lung, kidney, spleen, muscle, and totally absent in cobra plasma glycoproteins. In sharp contrast to cobras, venom glycoproteins of other snakes do not contain terminal alpha-galactosyl residues but do contain terminal 2,3- and/or 2,6-linked sialic acids as well as beta-galactosyl residues. Cobra venom also contains high molecular weight heavily glycosylated proteins bearing poly-N-acetyllactosaminyl oligosaccharides, the majority of which appear to be linked to the protein core via O-glycosidic bonds.

Animals↗
PubMed1990

Cloning and cDNA sequence of a bovine submaxillary gland mucin-like protein containing two distinct domains.

A lambda gt11 cDNA library prepared from bovine submaxillary gland mRNA was screened with polyclonal anti-apo-bovine submaxillary mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core protein. One of the positive clones had a 1.8 kilobase (kb) cDNA insert and coded for an incomplete protein. A 2.0-kb cDNA clone was isolated by rescreening the library with the 1.8-kb cDNA. Nucleotide sequencing of the full-length 2.0-kb cDNA revealed an open reading frame that coded for a 563-amino acid protein. A striking feature of the cloned protein is the skewed distribution of the amino acids, most notably that of the hydroxy amino acids and cysteine. The amino-terminal domain of 339 residues is very rich in threonine, serine, and glycine and poor in cysteine, aspartic acid, tyrosine, phenylalanine, and tryptophan. In contrast, the carboxyl-terminal domain of 224 residues is rich in cysteine, aspartic acid, tyrosine, lysine, and asparagine and relatively poor in threonine, serine, and glycine. A search of the protein data bank for homologies to the deduced amino acid sequence revealed statistically significant matches to several proteins, including the porcine submaxillary apomucin fragment. The cysteine-rich domain by itself was not statistically homologous with any of the registered polypeptide sequences. RNA blot analysis using DNA probes corresponding to the mucin-like and cysteine-rich regions detected a nearly identical pattern of transcripts, demonstrating that the characterized clones are not artifacts of cDNA library construction. The blots also showed the presence of polydisperse transcripts in bovine submaxillary gland but no detectable hybridization signals in liver or brain RNA.

Amino Acid Sequence↗
PubMed1989

Modification of sialyl residues of glycoconjugates by reductive amination. Characterization of the modified sialic acids.

The sialic acid residues of alpha 1-acid glycoprotein and fetuin were modified by introduction of an amino residue, such as glycine and [3H]glycine. This modification involved (a) the selective periodate oxidation of the exocyclic carbon atoms of the sialic acid residue generating an aldehyde group at C-7, and (b) the reduction of the Schiff base formed with an amino compound by use of sodium cyanoborohydride. Thin layer chromatography, high pressure liquid chromatography, and amino acid composition data of the modified glycoprotein showed that the conversion was essentially quantitative. The glycine-modified sialic acids were isolated by mild acid hydrolysis and identified by g.l.c.-m.s. and n.m.r. spectroscopy, thus confirming that the quantitative modification produced a glycine-aminated C-7 sialic acid analog. Strong acid hydrolysis of the glycine-modified sialic acid yielded a fragment that had chromatographic characteristics similar to those of glycine.

Chemical Phenomena↗
PubMed1988

Protein components of human tracheobronchial mucin: partial characterization of a closely associated 65-kilodalton protein.

A high-density mucin glycoprotein was isolated from human tracheobronchial secretions substantially free of contaminating protein, low-density glycoprotein, proteolytic enzymes, and lipid. A closely associated 65-kDa protein was discovered while investigating the effect of 2-mercaptoethanol treatment on the purified mucin glycoprotein. It has been established that the 65-kDa protein is neither alpha 1-antichymotrypsin nor human serum albumin, two proteins of similar molecular weight which are found in crude tracheobronchial secretions. This protein lacks cross-reactivity with antibodies directed against serum components and is presumably comparable to the 65-kDa protein similarly isolated from canine tracheal pouch secretions [Ringler et al. (1987) Biochemistry 26, 5322-5328]. Although both the presence of sulfhydryl groups and the ability to be reassociated with the mucin molecule have been established, it is not clear whether its association is due to direct disulfide bonding, hydrophobicity, or entrapment. It was found that 14C-methylated methemoglobin was an inappropriate substrate for measurement of proteolytic activity in mucin preparations due to inherent entrapment and clearance capabilities of mucin molecules.

Amino Acids↗
PubMed1988

Purification and partial characterization of a malignancy-associated glycoprotein.

The isolation from cancer patient serum of a glycoprotein (Cc) associated with the presence of a variety of malignancies was previously reported. Although preliminary chemical and physical data indicated that Cc was different from identified circulating glycoproteins, subsequent immunological studies suggested that it was closely related to alpha 1-acid glycoprotein. Further analysis revealed the presence of two components in some Cc preparations and prompted a re-examination of the isolation and characterization data. In the present study, Cc was purified by a modified protocol which involved the use of pleural fluid obtained from individuals with cancer, and an alpha 1-acid glycoprotein antibody column to remove contaminating alpha 1-acid glycoprotein. Typically, the material not retained by the antibody column gave a single band with Mr 53,000 when subjected to sodium dodecyl sulfate-polyacrylamide electrophoresis. Amino terminal analysis revealed that the protein contained a blocked amino terminus, and carbohydrate analysis indicated that complex, asparagine-linked saccharide units were present. The product could be distinguished from alpha 1-acid glycoprotein and other previously described circulating glycoproteins by several criteria, including molecular weight, isoelectric point, and amino acid and carbohydrate composition. One of three preparations isolated had an apparent Mr of 59,000. Carbohydrate analysis as well as deglycosylation studies showed that the change in molecular weight was due to increased glycosylation.

Adsorption↗