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J C Byrd

Publications and source records attributed to J C Byrd.

At least 145 records · Page 8Linked to original sources
PubMed1988

Mucin-like antigens in a human pancreatic cancer cell line identified by murine monoclonal antibodies SPan-1 and YPan-1.

The antigenic determinant recognized by monoclonal antibody SPan-1 is greatly elevated in sera of patients with pancreatic cancer but not in sera of normal individuals. Here we describe the mucin-like characteristics of the SPan-1 antigen isolated from culture medium and xenografts of the human pancreatic cancer cell line SW-1990. YPan-1, another pancreatic cancer associated monoclonal antibody, also reacts with the SPan-1 antigen. The SPan-1/YPan-1 antigens have densities of 1.4-1.5 g/ml and elute in the void volume of Sepharose CL-2B columns. They are resistant to degradation by chondroitinase ABC, nitrous acid, and hyaluronidase but susceptible to protease digestion and reductive beta-elimination. All these characteristics suggest that the SPan-1 and YPan-1 determinants are carried on mucinous antigens. Both SPan-1 and YPan-1 immunoreactivities are unaffected by boiling or by alkylation and reduction of the mucins while they are abolished by mild periodate oxidation or neuraminidase and are markedly decreased by wheat germ agglutinin. Thus, their antigenic determinants are composed principally of carbohydrates with sialic acid, an absolute requirement for reactivity. However, the epitope specificities of SPan-1 and YPan-1 are different since YPan-1 does not compete with SPan-1 for binding to antigen. Moreover, YPan-1 and SPan-1 can be distinguished from several other sialic acid requiring, cancer associated antibodies such as B72.3, CSLEX-1, DU-PAN-2, OC-125, and 19-9 by either their epitope characteristics or their tissue reactivity patterns.

Animals↗
PubMed1988

Structural characterization of intact, branched oligosaccharides by high performance liquid chromatography and liquid secondary ion mass spectrometry.

We report results of a mass-spectrometric-based strategy for determining the detailed structural features of N-linked oligosaccharides from glycoproteins. The method was used to characterize a series of intact, high mannose oligosaccharides isolated from human immunoglobulin M (IgM). The IgM was purified from a patient with Waldenstrom's macroglobulinemia. The strategy included releasing the oligosaccharides by digestion of the purified glycoprotein with endoglycosidase H, separating the released oligosaccharides by high resolution gel filtration, and derivatizing the resulting reducing termini with the uv-absorbing moiety, ethyl p-aminobenzoate. This particular derivative facilitates HPLC detection and provides centers for protonation and deprotonation enhancing liquid secondary ion mass spectra. Positive and negative ion spectra contained molecular species of similar abundance. However, fragment ion peaks yielding sequence information were significantly more prominent in the negative ion mass spectra. Furthermore, it was obvious that the fragmentation patterns differed substantially for linear and branched oligomers. For linear oligosaccharides, a smooth envelope of fragment ions was observed; from low to high mass there was an ordered decrease in ion abundance from both the reducing and nonreducing termini. This pattern of fragment ions was not observed for branched oligosaccharides since in these cases fragments at certain masses could not arise by single bond cleavages. Therefore, these fragments were either significantly reduced in abundance or absent as compared with identical fragments formed from linear molecules. Importantly, 200 pmol of an oligosaccharide could be derivatized, separated, and detected by mass spectrometry, allowing identification of previously unreported minor components of the IgM oligosaccharides. Therefore, this experimental strategy is particularly useful for the purification and detailed structural characterization of low abundance oligosaccharides isolated from heterogeneous biological samples.

Carbohydrate Conformation↗
PubMed1988

Pancreatic cancer mucin from xenografts of SW1990 cells: isolation, characterization, and comparison to colon cancer mucin.

Mucin has been purified from nude mouse xenografts of SW1990 human pancreatic cancer cells. The mucin was eluted at the void volume of Sepharose CL-4B and was of density greater than 1.3 in CsCl gradients. The isolated mucin had a high content of threonine, serine, and proline, with 31% of the amino acid residues O-glycosylated. The average oligosaccharide composition was NeuAc1.8Fuc0.7Gal2.0GlcNAc1.7GalNAc1.4. Polyclonal rabbit antibodies prepared against the purified mucin recognized primarily mucin polypeptide, and there was extensive immunological cross-reaction between SW1990 pancreatic cancer mucin and LS174T colon cancer mucin. However, using carbohydrate-specific monoclonal antibodies, the two mucins were found to differ. SW1990 mucin had more Lewis, sialyl Lewis, and sialyl Lewis activity, while the colon cancer mucin had more sialyl T antigen. Since pancreatic mucins, whether from normal pancreas or pancreatic cancer, have not previously been well characterized, the availability of SW1990 pancreatic cancer mucin may be useful as a model for studying the expressing of organ-specific or cancer-associated antigens.

Animals↗
PubMed1987

Characterization of quantitative mucin variants from a human colon cancer cell line.

Colonic mucins are high molecular weight glycoproteins produced by goblet cells of colonic epithelium. Some studies have indicated that patients with colonic cancers that produce high amounts of mucin have a poorer prognosis than patients whose tumors produce low amounts of mucin. At present, however, the role of mucin in affecting the behavior of colon cancer cells is not well understood. To further elucidate the relationship between cellular mucin content and the growth characteristics and morphology of tumor cells, we utilized a replica plating technique and immunoscreening method to identify and purify variant clones of the human colon cancer cell line LS174T that produce high and low levels of mucin. This procedure enabled us to isolate two high mucin-containing variants (HM3 and HM7) and one low mucin-containing variant (LM12). These variants exhibited different morphology. Both high mucin variants tended to form cell aggregates and suspended cells with adjoining mucoid threads. The low mucin variant formed spread monolayers on the substratum with the formation of cell processes. Metabolic labeling using [3H]glucosamine demonstrated that high mucin variants synthesized 2-fold more mucin in the cell layer and secreted 3-fold more mucin into the culture medium than the low mucin variant. The colony-forming efficiency in semisolid agar for these variants positively correlated with their mucin content. High mucin variant cells when injected into athymic nude mice formed tumors 2-fold larger than those of the parental cells while the low mucin variant formed tumors only one-half as large as those of the parental cell line. These mucin variants should provide a useful model for understanding the biological behavior of mucinous colon cancer cells in vivo and in vitro.

Animals↗
PubMed1987

Two types of transglutaminase in the PC12 pheochromocytoma cell line. Stimulation by sodium butyrate.

Transglutaminases are a class of enzymes capable of covalently cross-linking both intracellular and extracellular proteins. The activity of tissue transglutaminase is known to decrease precipitously following neoplastic transformation, and it has been hypothesized that transglutaminase may be involved in growth regulation. We have found that the differentiation promoter sodium butyrate is able to cause a marked increase in transglutaminase activity in PC12 pheochromocytoma cells in a time- and dose-dependent manner. This increased transglutaminase activity is associated with growth arrest, as well as with striking morphological changes including increased cell adhesion. The transglutaminase induced by sodium butyrate appears to be tissue transglutaminase, based on its cytosolic localization, thermal lability at basic pH, and elution profile on anion-exchange chromatography. Untreated PC12 cells contain only small amounts of transglutaminase which resembles epidermal transglutaminase, an enzyme previously described only in skin. In contrast to sodium butyrate, nerve growth factor did not stimulate tissue transglutaminase in PC12 cells, although it, too, caused growth arrest. It is hypothesized that transglutaminase may be involved in certain morphological changes accompanying cellular differentiation and neoplastic transformation, rather than in growth regulation per se.

Adrenal Gland Neoplasms↗
PubMed1987

Effects of sodium butyrate on human colonic adenocarcinoma cells. Induction of placental-like alkaline phosphatase.

We have examined the effects of the "differentiating agent," sodium butyrate, on the induction of alkaline phosphatase in human colonic tumor cell line LS174T. Culture of these cells in the presence of 2 mM butyrate caused this activity to increase from less than 0.0001 unit/mg of protein to greater than 0.7 unit/mg of protein over an 8-day period. This induction proceeded in a nonlinear fashion with a lag time of 2-3 days occurring before enzymatic activity began to rise. These increases in activity were accompanied by elevations in the content of a placental-like isozyme of alkaline phosphatase as demonstrated by "Western" immunoblots. Dome formation, indicative of differentiation in cultured cells, also required 3 days treatment with butyrate before becoming evident. The rate of biosynthesis of the enzyme, examined using metabolic labeling with L-[35S]methionine and immunoprecipitation, was found to increase continuously between days 2 and 6 of butyrate treatment. "Northern" blot analysis indicated that treatment of these cells with butyrate caused greater than 20-fold induction of a 2700-base mRNA that hybridized to a cDNA probe for placental alkaline phosphatase. The mRNA for alkaline phosphatase produced by these cells upon butyrate treatment was approximately 300-400 bases smaller than the mRNA for alkaline phosphatase found in placenta. Human small intestine also contained two mRNAs that hybridized relatively weakly with the placental alkaline phosphatase probe. These results indicate that a placental alkaline phosphatase-like protein and mRNA are induced by butyrate in LS174T cells with a time course consistent with cellular differentiation preceding induction.

Adenocarcinoma↗
PubMed1987

Differentiation of PC12 pheochromocytoma cells by sodium butyrate.

PC12 pheochromocytoma cells cease dividing and show increased cell-cell and cell-substratum adhesion in response to treatment with sodium butyrate. These changes are accompanied by the rapid appearance of neuron-specific enolase, an APUD cell marker. However, neurofilament proteins, markers for neuronal differentiation, are not induced. These results suggest that sodium butyrate induces differentiation in the PC12 cell line, perhaps along the chromaffin cell pathway.

Adrenal Gland Neoplasms↗
PubMed1987

Proenkephalin gene expression in the PC12 pheochromocytoma cell line: stimulation by sodium butyrate.

The differentiation promoter sodium butyrate increases the content of Met5-enkephalin-Arg6-Gly7-Leu8 (Met5-enk-RGL)-immunoreactive peptides in PC12 pheochromocytoma cells, which, unlike mature adrenomedullary chromaffin cells, contain exceedingly low levels of opioid peptides. These butyrate-induced enkephalin-immunoreactive peptides, which are specific products of the proenkephalin gene, consist principally of two high mol wt forms of amino-terminally extended Met5-enk-RGL. These high mol wt peptides, with apparent mol wt of 20,000 and 10,000, are approximately the same size as the two major immunoreactive peptides found in adult New England Deaconess Hospital rat adrenal. The low mol wt Met5-enk-RGL-immunoreactive peptide found in butyrate-treated cells is similar in size to authentic Met5-enk-RGL, which is not found in the adrenal medulla of the adult rat. When PC12 cells are grown as a tumor in vivo, the amount of Met5-enk-RGL-immunoreactive peptide increased only slightly above the level found in control cells grown in vitro and consisted exclusively of the highest mol wt immunoreactive species. In PC12 cells, the butyrate-stimulated elevation in the content of Met5-enk-RGL-immunoreactive peptides may involve changes in transcription, since the peptide increase is preceded by a 2- to 3-fold increase in the level of proenkephalin mRNA. These results suggest that the PC12 cell line may be useful for investigating those factors that control the initial expression and processing of proenkephalin-derived peptides during embryogenesis.

Adrenal Gland Neoplasms↗
PubMed1986

Epinephrine synthesis in the PC12 pheochromocytoma cell line.

PC12 is a rat pheochromocytoma cell line which has been increasingly used as a model system for both neural differentiation and chromaffin cell function. PC12 cells have been reported to synthesize dopamine and norepinephrine, but not epinephrine. We have found that PC12 cells do synthesize small amounts of epinephrine and that dexamethasone increases both epinephrine content as well as phenylethanolamine-N-methyltransferase (PNMT) activity. These results suggest that the PC12 cell line may be useful in the investigation of the regulation of PNMT.

Adrenal Gland Neoplasms↗
PubMed1985

Rat intestinal nucleotide-sugar pyrophosphatase. Localization, partial purification, and substrate specificity.

The nucleotide-sugar pyrophosphatase activity of rat small intestine was studied using GDP-[14C]Man as substrate. The highest specific activities in the gastrointestinal tract were in the proximal small intestine, with a preferential localization in villus tip cells. Purified brush-border membranes were highly enriched in nucleotide-sugar pyrophosphatase. After the enzyme was solubilized with detergent and purified 180-fold, it hydrolyzed FAD and p-nitrophenyl-5'-thymidylate, as well as nucleotide sugars. That the same enzyme, a 5'-nucleotide phosphodiesterase, is responsible for nucleotide-sugar pyrophosphatase, phosphodiesterase I, and FAD pyrophosphatase activities is indicated by: co-migration in electrophoresis, parallel thermal inactivation, competitive inhibition studies, and similar regional, cellular, and subcellular localizations.

Animals↗
PubMed1982

Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing.

Synthesis of the N-linked oligosaccharides of Saccharomyces cerevisiae glycoproteins has been studied in vivo by labeling with [2-3H]mannose and gel filtration analysis of the products released by endoglycosidase H. Both small oligosaccharides, Man8-14GlcNAc, and larger products, Man greater than 20GlcNAc, were labeled. The kinetics of continuous and pulse-chase labeling demonstrated that Glc3Man9GlcNAc2, the initial product transferred to protein, was rapidly (t1/2 congruent to 3 min) trimmed to Man8GlcNAc2 and then more slowly (t1/2 = 10-20 min) elongated to larger oligosaccharides. No oligosaccharides smaller than Man8GlcNAc2 were evident with either labeling procedure. In confirmation of the trimming reaction observed in vivo, 3H-labeled Man9-N-acetylglucosaminitol from bovine thyroglobulin and [14C]Man9GlcNAc2 from yeast oligosaccharide-lipid were converted in vitro by broken yeast cells to 3H-labeled Man8-N-acetylglucosaminitol and [14C]Man8GlcNAc2. Man8GlcNAc and Man9GlcNAc from yeast invertase and from bovine thyroglobulin were purified by gel filtration and examined by high field 1H-NMR analysis. Invertase Man8GlcNAc (B) and Man9GlcNAc (C) were homogeneous compounds, which differed from the Man9GlcNAc (A) of thyroglobulin by the absence of a specific terminal alpha 1,2-linked mannose residue. The Man9GlcNAc of invertase (C) had an additional terminal alpha 1,6-linked mannose and appeared identical in structure with that isolated from yeast containing the mnn1 and mnn2 mutations (Cohen, R. E., Zhang, W.-j., and Ballou, C. E. (1982) J. Biol. Chem. 257, 5730-5737). It is concluded that Man8GlcNAc2, formed by removal of glucose and a single mannose from Glc3Man9GlcNAc2, is the ultimate product of trimming and the minimal precursor for elongation of the oligosaccharides on yeast glycoproteins. The results suggest that removal of a particular terminal alpha 1,2-linked mannose from Man9GlcNAc2 by a highly specific alpha-mannosidase exposes the nascent Man-alpha 1,6-Man backbone for elongation with additional alpha 1,6-linked mannose residues, according to the following scheme: (formula, see text).

Carbohydrate Conformation↗
PubMed1981

Isolation and partial characterization of the oligosaccharide moieties of rat preputial beta-glucuronidase.

The oligosaccharide alditols of rat preputial gland beta-glucuronidase have been isolated by reductive alkaline hydrolysis of the enzyme followed by gel filtration. Since the product was about decasaccharide in size, and as beta-glucuronidase contains about 20 sugar residues per subunit, the enzyme must contain two oligosaccharides per subunit. Two major subfractions were obtained. Chemical and enzymatic studies on the initial oligosaccharide alditol fraction and on the separated subfractions indicated the presence of a mannosyl-chitobiosyl core and permitted formulation of the following structures for the two different oligosaccharide alditols: Man3 leads to alpha [(alpha Man, GlcNAc) leads to Man] leads to beta GlcNAc leads to beta GlcNAc-ol Man 3 leads to 4 leads to alpha[Man2 leads to alpha Man] leads to beta GlcNAc leads to beta GLcNAc-ol.

Animals↗
PubMed1980

Effect of glucosylation of lipid intermediates on oligosaccharide transfer in solubilized microsomes from Saccharomyces cerevisiae.

Glc3Man9GlcNAc2-P-P-dolichol and Man9GlcNAc2-P-P-dolichol isolated from Saccharomyces cerevisiae are substrates for the N-glycosylation of endogenous proteins in Triton X-100-solubilized yeast microsomes. The solubilized oligosaccharide transferase requires Mn2+ for activity; neither Mg2+ nor Ca2+ is an effective substitute. The pH optimum of the transfer reaction is between 6.5 and 7.5. Unlike animal systems, which utilize glucosylated oligosaccharide-lipid to a much greater extent than the unglucosylated species as a donor in the transferase rection, yeast extracts transfer more than 70% of Glc3Man9GlcNAc2 and Man9GlcNAc2 from their respective oligosaccharide-lipids to proteins. However, the rate of N-glycosylation in vitro is approximately 25-fold faster with Glc3Man9GlcNAc2-P-P-dolichol than with Man9GlcNAc2-P-P-dolichol. The apparent Km value for the glucosylated species is 75 nM, while that for the unglucosylated glycolipid is 55 nM.

Cations, Divalent↗
PubMed1979

Wolman's disease: ultrastructural evidence of lipid accumulation in central and peripheral nervous systems.

We report the first case of Wolman's disease in which the fine structure of either the peripheral or the central nervous system has been examined. We confirm ultrastructurally the presence of lipid within endothelial and pericytic cells. Several cell types previously believed to be uninvolved in this storage process demonstrate lipid inclusions characteristic of Wolman's disease: perineural, endoneurial and Schwann cells of peripheral nerve, and oligodendrocytes and astrocytes of the central nervous system.

Astrocytes↗
PubMed1979

Evaluation of the new API 20C strip for yeast identification against a conventional method.

The new API 20C yeast identification system together with appropriate microscopic morphology determinations achieved a 97% correlation with a rapid conventional method. Whereas a group composed of Candida, Torulopsis, Saccharomyces, and Rhodotorula was identified with ease (98% overall correlation), a second group, containing Cryptococcus, Trichosporon, and Geotrichum species, appeared to give the system the most difficulty (90% correlation). Within this group particular difficulty was encountered in identifying varieties of Cryptococcus albidus, C. terreus, C. laurentii, Trichosporon beigelli, and Geotrichum spp. as to species. The API 20C system should be incubated the full 72 h prescribed by the manufacturer. However, when used in conjunction with appropriate morphological tests, presumptive identifications of some Candida and Torulopsis species may be made at 24 to 48 h. To facilitate identifications of the more difficult group of yeasts, ancillary tests for determining nitrate reductase, urease, and phenol oxidase activities should be considered as additions to the strip. Incorporating the phenol oxidase test would be especially important for identification of Cryptococcus neoformans, a yeast which should be identified as quickly and as accurately as possible. The API 20C system with computer assistance has proved to be an easy-to-inoculate, versatile, and fairly rapid method of yeast identification, giving results comparable to those obtained by conventional methodologies.

Candida↗