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B C Sorkin

Publications and source records attributed to B C Sorkin.

12 recordsLinked to original sources
PubMed1998

Short chain carboxylic acids decrease human gingival keratinocyte proliferation and increase apoptosis and necrosis.

Epithelia are key barriers to infections. In periodontal disease, the gingival sulcular epithelium becomes ulcerated. In this report, we test the hypothesis that short-chain carboxylic acids (SCCA) inhibit keratinocyte proliferation, increase necrosis and apoptosis, and may thus promote ulceration. SCCA produced by bacteria are present at millimolar concentrations in the periodontal pockets of subjects with periodontal disease. SCCA concentrations are higher in subjects with severe disease than in those with mild disease, and are not detectable in healthy subjects. Cell proliferation is critical for maintenance of epithelial barrier function. All SCCA tested, when neutralized, decreased epithelial cell proliferation (as measured by 3H-thymidine incorporation) in a dose-dependent manner. We found that epithelial cell viability decreased with increasing SCCA concentrations, accounting at least partly for the decreased 3H-thymidine incorporation. For all conditions we tested, SCCA-induced apoptosis preceded and exceeded necrosis. While the molecular mechanism(s) for these effects remain to be determined, the results indicate that SCCA derived from caries- or periodontal disease-associated bacteria could alter gingival barrier function.

Apoptosis↗
PubMed1998

Adenomatous polyposis coli protein is expressed in alternate stages of the ameloblast life cycle.

Mutations of the adenomatous polyposis coli gene protein (APC) are associated with familial polyposis and also sporadic colon adenomas, both preconditions to cancer formation. Some familial polyposis patients also develop Gardner's syndrome, a condition characterized by supernumerary teeth, mandibular osteomas, and other maladies. We investigated participation of APC in normal tooth development. Using a monoclonal antibody to study APC expression in the forming rat incisor, we found no APC staining in differentiating ameloblasts, then strong staining in secreting ameloblasts and stratum intermedium cells, followed by cells in the transition stage which did not stain. Intense APC staining resumes in maturation-stage ameloblasts and proximal papillary cells. APC staining disappears again in reduced ameloblasts at the conclusion of amelogenesis. APC staining was not seen in any other odontogenic cells. We report a unique system in which APC expression is upregulated and downregulated twice during the normal life cycle of ameloblasts. APC, therefore, is important in the normal maturation of both colonic epithelium and odontogenic epithelium. At this point, we cannot rule out any of the known functions of APC, which include: modulation of cell adhesion by binding to catenin, regulation of beta-catenin as a differentiative signaling molecule, and promotion of microtubule assembly. In this respect, the rat incisor enamel organ provides a unique tissue for studying the regulation and functions of APC.

Adenomatous Polyposis Coli↗
PubMed1996

Cadherin-mediated adhesion is required for normal growth regulation of human gingival epithelial cells.

The cadherins are a family of cell membrane proteins that mediate calcium-dependent cell-cell adhesion. E-cadherin is required for the formation, differentiation, polarization and stratification of epithelia; P-cadherin is also expressed on many epithelia. We report here the first study of cadherin expression in immortalized human gingival epithelial cells (IHGK) and examine the role of cadherins in growth regulation of these cells. We found that the IHGK cells are similar to normal gingival epithelial cells in their cadherin expression and density-dependent inhibition of growth. The IHGK cells proliferate more rapidly at low calcium concentration (0.15 mM) than at physiological concentrations of calcium (1.8 mM) and magnesium (0.65 mM; Ca/Mg medium) suggesting that calcium is required for density-dependent regulation of proliferation. To evaluate the possibility that cadherin function is required for contact inhibition in these cells, we grew them in Ca/Mg medium in the presence of adhesion-blocking anti-cadherin monoclonal antibodies. At anti-E-cadherin concentrations sufficient to disrupt cell-cell adhesion, the proliferation of the IHGK cells was similar to that observed in medium containing 0.2 mM EDTA. Anti-P-cadherin had a much weaker effect on cell proliferation than anti-E-cadherin, and cells grown in medium containing both antibodies grew at intermediate rates. The increased proliferation of the IHGK cells in either low calcium medium or Ca/Mg medium containing adhesion-blocking anti-cadherin antibodies suggests that cadherin-mediated adhesion is required for density-dependent regulation of growth of these cells.

Antibodies, Monoclonal↗
PubMed1993

Identification of the promoter and a transcriptional enhancer of the gene encoding L-CAM, a calcium-dependent cell adhesion molecule.

L-CAM is a calcium-dependent cell adhesion molecule that is expressed in a characteristic place-dependent pattern during development. Previous studies of ectopic expression of the chicken L-CAM gene under the control of heterologous promoters in transgenic mice suggested that cis-acting sequences controlling the spatiotemporal expression patterns of L-CAM were present within the gene itself. We have now examined the L-CAM gene for sequences that control its expression and have found an enhancer within the second intron of the gene. A 2.5-kb Kpn I-EcoRI fragment from the intron acted as an enhancer of a simian virus 40 minimal promoter driving a chloramphenicol acetyltransferase (CAT) reporter gene and produced 14.0-fold induction of CAT activity in MDCK cells. To narrow down the region responsible for enhancer activity and to determine whether the enhancer could function in a cell type-specific manner, a number of smaller restriction fragments from the intron were tested for activity in two chicken cell lines, the LMH hepatoma line, which produces high levels of L-CAM, and the SL-29 fibroblast line, which produces little, if any, L-CAM. Four L-CAM enhancer plasmids containing shorter segments derived from the intron showed enhanced CAT activity levels (between 9.4- and 16.5-fold) in extracts from transfected LMH cells but not from SL-29 cells. DNA sequence analysis of the L-CAM enhancer region revealed putative binding sites for the transcription factors SP1, E2A, and AP-2. In addition, LE-9, the smallest L-CAM enhancer segment (310 bp), contained a consensus binding site for the liver-enriched POU-homeodomain transcription factor, HNF-1. Tests of upstream sequences showed that a 630-bp fragment, corresponding to nearly the entire intergenic region between L-CAM and its neighboring CAM gene, K-CAM, could function as a promoter. In combination with the L-CAM enhancer, this fragment directed cell type-specific expression of the CAT reporter gene in LMH cells at a level comparable to that observed with enhancer constructs using the simian virus 40 minimal promoter. These combined observations define a promoter and an enhancer for the chicken L-CAM gene. They raise the possibility that these cis-acting regulatory sequences may be instrumental in directing specific place-dependent expression of the L-CAM gene in the chicken.

Animals↗
PubMed1991

Genes for two calcium-dependent cell adhesion molecules have similar structures and are arranged in tandem in the chicken genome.

Genomic sequences immediately upstream of the translational start site for the chicken liver cell adhesion molecule (L-CAM) gene contain a second closely related gene, which, because of its location, we have designated the K-CAM gene. Less than 700 base pairs separate the presumed poly(A) site in the K-CAM gene from the translation initiation site for L-CAM. The sizes of exons 4-15 of the K-CAM gene are almost identical to those in the L-CAM gene and the exon/intron junctions occur at exactly equivalent positions in both genes. Exon 16, which includes the 3' untranslated region, is much shorter in the K-CAM gene and intron sizes and sequences are not generally conserved between the two genes. Probes from the K-CAM gene hybridized to a 3-kilobase mRNA that was present at high levels in embryonic skin, at lower levels in kidney, heart, and gizzard, and at still lower levels in brain and liver, as determined by Northern blotting. The sequence of the predicted gene product was nearly identical to that of the chicken B-cadherin cDNA, although the distribution of the K-CAM gene transcript differed from that reported for the cadherin. The proximity and identical overall structure of the K-CAM and L-CAM genes strongly suggest that they arose by gene duplication and raise the possibility that genes for other calcium-dependent CAMs may be located in clusters. Moreover, the tandem arrangement of the genes may have important implications for the regulation of their expression.

Amino Acid Sequence↗
PubMed1989

Identification of two protein kinases that phosphorylate the neural cell-adhesion molecule, N-CAM.

The neural cell-adhesion molecule (N-CAM) is detected as at least 3 related polypeptides generated by alternative splicing of a single gene. In vivo the 2 larger polypeptides are phosphorylated, but the smallest polypeptide, which lacks a cytoplasmic domain, is not. We have found that the 2 larger polypeptides are phosphorylated in vivo on several common phosphorylation sites. Furthermore, the largest polypeptide has additional sites, suggesting that some phosphorylation occurs in that portion of the intracellular region unique to it. In vitro N-CAM is not a substrate for cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase, calcium/calmodulin-dependent protein kinase I, II, or III, protein kinase C, or casein kinase II. However, we have isolated 2 protein kinases from mammalian and avian brain that phosphorylate rodent and chicken N-CAM. On the basis of their chromatographic behavior and substrate specificity, the 2 kinases are glycogen synthase kinase 3 (GSK-3) and casein kinase I (CK I). The 2 kinases phosphorylate N-CAM rapidly, to a high stoichiometry and with a low Km for N-CAM, suggesting that the phosphorylation of N-CAM by these kinases is physiologically relevant. Both enzymes phosphorylate the 2 larger N-CAM polypeptides in vitro in the cytoplasmic domain on threonyl residues that are phosphorylated to a low level in vivo. In addition, the threonyl residues are close to seryl residues phosphorylated to a high level in vivo. Prior phosphorylation at the in vivo sites appears to be a prerequisite for phosphorylation by GSK-3 and CK I. Taken together, the results suggest that N-CAM may be physiologically phosphorylated on 2 sets of interrelated sites, one demonstrable in vivo and one in vitro. Phosphorylation on the "in vivo" sites is resistant to dephosphorylation and may be constitutive, while phosphorylation on the "in vitro" sites is much more labile.

Animals↗
PubMed1988

Structure of the gene for the liver cell adhesion molecule, L-CAM.

The liver cell adhesion molecule, L-CAM, mediates calcium-dependent cell-cell adhesion in early embryos and in nonneural epithelia in adult tissues. Earlier studies of cDNAs for chicken L-CAM established the amino acid sequence of the mature protein. The sequence has now been extended in the 5' direction through the precursor and signal sequences and past a consensus translation initiation site. The combined cDNAs were used to isolate genomic clones covering the entire L-CAM coding sequence. The structural gene for chicken L-CAM contains 16 exons ranging in size from 115 to over 1045 base pairs with an average size of 222 base pairs. Single exons do not correspond to known structural elements such as the signal sequence, precursor segment, internal repeats, or membrane-spanning region of L-CAM. Hybridization of restriction digests of chicken genomic DNA with cDNA and genomic probes indicated that there is a single L-CAM gene in the chicken. In contrast to genes for other cell-cell or cell-substrate adhesion molecules, there is no evidence for alternative splicing of exons in this gene.

Amino Acid Sequence↗
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↗
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

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↗
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↗