Continuum Theory for Gas-Solid-Liquid MediaL Theory Development

Gustafson, Robert J.; Mase, George E.; Segerlind, L. J.

doi:10.13031/2013.35725

Cited by 13 publications

(21 citation statements)

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“…HNO # deamination and reduction was carried out as described previously [15]. Mild acid hydrolysis was carried out using conditions (1 M HCl ; 100 mC ; 6 min) used to hydrolyse GlcNAc-1-PO % linkages [16]. To determine the nature of the fatty acid-protein linkage, VSP4A1 (5-10 µg) purified from trophozoites metabolically labelled with [9,10-$H]palmitic acid were dissolved in 80 µl of either 1 M hydroxylamine\HCl, pH 7n5, or 10 mM Tris\HCl, pH 7n5 and incubated for 2 h at room temperature.…”

Section: Release Of Labelled Glycans and Palmitic Acidmentioning

confidence: 99%

“…This may indicate the presence of a small amount of free glucose in the original VSP4A1 sample or the substitution of the protein with monomeric Glc which would be released as glucitol after β-elimination. Linkage to the protein via a phosphodiester bond [16] is unlikely as very little label (approx. 10 %) was released after mild acid hydrolysis that cleaves either Glc-1-PO % or GlcNAc-1-PO % linkages (results not shown).…”

Section: H]gal-labelled Glycans Released By Reductive β-Eliminationmentioning

confidence: 99%

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The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein

Papanastasiou

McConville

Ralton

et al. 1997

Biochemical Journal

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The variant-specific surface proteins (VSPs) of the ancient protist Giardia duodenalis (syn. : Giardia intestinalis, Giardia lamblia) are cysteine-and threonine-rich polypeptides that can vary considerably in sequence and size. In the present study, we have purified a VSP (VSP4A1, formerly called CRISP-90) from a cloned Giardia isolate, derived from a sheep, by Triton X-114 phase partitioning and anion-exchange chromatography. Analysis of the purified VSP4A1 showed that this protein is posttranslationally modified with both glycans and lipid. The glycans of VSP4A1 were detected and partially characterized by (1) compositional analysis, which indicated the presence of GlcNAc and Glc (0n5 and 1n0 mol\mol of protein respectively), and (2) the specific labelling of VSP4A1 with galactosyltransferase\ UDP-[$H]Gal. The glycans were released by β-elimination, suggesting that they are O-linked to the protein. Bio-Gel P4 INTRODUCTIONProtozoan parasites of the genus Giardia belong to the diplomonad flagellates which most likely represent one of the earliest branches of eukaryotic evolution. Giardia duodenalis-type organisms (syn. : Giardia lamblia, Giardia intestinalis) colonize the small intestine of a wide variety of different vertebrate hosts and are the causative agents of the most common protozoal intestinal infection of humans world wide [1]. Analyses of Giardia using a number of complementary techniques have demonstrated that the greatest degree of diversity among isolates of this organism resides in their major surface antigens. These comprise a group of novel polypeptides that are called variant-specific surface proteins (VSPs) and are components of a coat covering the entire cell surface of the parasite [2][3][4]. Although these polypeptides can vary considerably in both sequence and size, they share several common structural features [5][6][7][8]. These include a high content of cysteine (approx. 12 mol %) and threonine (approx. 11 mol %), the presence of multiple copies of the tetrapeptide CXXC motif and a highly conserved C-terminal sequence. The biological function of these proteins is still unclear, but is believed to be associated with the survival of the parasite in the vertebrate small intestine.Previous studies in our laboratory have shown that a cloned isolate of Giardia obtained from a sheep (clone O2-4A1) expresses a VSP (VSP4A1, formerly called CRISP-90) that migrated under Abbreviations used : GC/MS, gas chromatography-mass spectrometry ; GU, glucose unit ; HPAEC, high-pH anion-exchange chromatography ; HPTLC, high-performance thin-layer chromatography ; O-GlcNAc, O-linked GlcNAc ; pI, isoelectric point ; LDAO, N,N-dimethyldodecylamine N-oxide ; TBS, Tris-buffered saline ; TMS, trimethylsilyl ; VSP, variant-specific surface protein.‡ Present address : Cancer Immunotherapy Laboratory, Ecole Pratique des Hautes Etudes, Faculty of Medicine, 7 Boulevard Jeanne d'Arc, 21000 Dijon, France.§ To whom correspondence should be addressed.chromatography of the released galactosylated glycans and further composi...

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Section: Release Of Labelled Glycans and Palmitic Acidmentioning

confidence: 99%

Section: H]gal-labelled Glycans Released By Reductive β-Eliminationmentioning

confidence: 99%

The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein

Papanastasiou

McConville

Ralton

et al. 1997

Biochemical Journal

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“…Similarly, DNA copy-number losses in this region were identified by comparative genomic hybridization (CGH) in tumors of the breast, liver, colon, lung, ovary, bladder and prostate, as well as in osteosarcomas, mesotheliomas, B-cell lymphomas and mantle cell lymphoma (Martinez-Climent et al, 2001;Zitzelsberger et al, 1997). Chromosome 8 transfer into colon and prostate tumor cell lines provided functional evidence for the presence of tumor suppressor genes on chromosome 8p21-22 (Gustafson et al, 1996;Cabeza-Arvelaiz et al, 2001).…”

Section: Introductionmentioning

confidence: 97%

DLC-1 gene inhibits human breast cancer cell growth and in vivo tumorigenicity

et al. 2003

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The human DLC-1 (deleted in liver cancer 1) gene was cloned from a primary human hepatocellular carcinoma (HCC) and mapped to the chromosome 8p21-22 region frequently deleted in common human cancers and suspected to harbor tumor suppressor genes. DLC-1 was found to be deleted or downregulated in a significant number of HCCs. We expanded our investigations to other cancers with recurrent deletions of 8p22, and in this study examined alterations of DLC-1 in primary human breast tumors, human breast, colon, and prostate tumor cell lines. Genomic deletion of DLC-1 was observed in 40% of primary breast tumors, whereas reduced or undetectable levels of DLC-1 mRNA were seen in 70% of breast, 70% of colon, and 50% of prostate tumor cell lines To see whether DLC-1 expression affects cell growth and tumorigenicity, two breast carcinoma cell lines lacking the expression of endogenous gene were transfected with the DLC-1 cDNA. In both cell lines, DLC-1 transfection caused significant growth inhibition and reduction of colony formation. Furthermore, introduction of the DLC-1 cDNA abolished the in vivo tumorigenicity in nude mice, suggesting that the DLC-1 gene plays a role in breast cancer by acting as a bona fide tumor suppressor gene.

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“…We are interested in studying the role of carbohydrate modifications in this organism (2). One of these is the addition of GlcNAc-1-P to serine residues, which has been well documented to occur on a cysteine proteinase called proteinase I found in vegetative cells (3)(4)(5). Although antibodies against GlcNAc-1-P recognize various proteins in the cells and in secretions of cells grown in axenic medium 1 the identity of these proteins is unknown.…”

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confidence: 99%

Identification of Two Novel Dictyostelium discoideum Cysteine Proteinases That Carry N-Acetylglucosamine-1-P Modification

Souza¹,

Hirai²,

Mehta³

et al. 1995

Journal of Biological Chemistry

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Dictyostelium discoideum makes multiple developmentally regulated lysosomal cysteine proteinases. One of these, a lysosomal enzyme called proteinase I, contains a cluster of GlcNAc-␣-1-P-Ser residues. We call this phosphoglycosylation. To study its function, a cDNA library from vegetative cells was screened, and two novel cysteine proteinase clones were characterized (cprD and cprE). Each of them has highly conserved regions expected for cysteine proteinases, but unlike any other, each has a serine-rich domain containing three distinct motifs, poly-S, SGSQ, and SGSG. cprD and cprE cDNAs were overexpressed in Dictyostelium and the active enzymes identified. cprD codes for a protein of approximately 36 kDa (CP4), which is recognized by monoclonal antibodies against GlcNAc-1-P and fucose. cprE corresponds to a 29-kDa protein, which is recognized by antibodies against GlcNAc-1-P. mRNA for both enzymes is present in the vegetative phase and increases during growth on bacteria but decreases throughout development. When the formation of the fruiting body is complete the mRNA for both messages is detected again but in very low levels. Having cloned cDNAs for proteins that carry GlcNAc-1-P should allow us to probe the function of the carbohydrate in these putative lysosomal enzymes.Dictyostelium discoideum is an eukaryotic amoeba that grows as single cells, but when the bacterial food source is removed, the cells initiate a complex multicellular developmental program. Cells aggregate and differentiate into several different types and, in the end, 85% of them are converted into spores setting atop a cellular stalk (1). We are interested in studying the role of carbohydrate modifications in this organism (2). One of these is the addition of GlcNAc-1-P to serine residues, which has been well documented to occur on a cysteine proteinase called proteinase I found in vegetative cells (3-5). Although antibodies against GlcNAc-1-P recognize various proteins in the cells and in secretions of cells grown in axenic medium 1 the identity of these proteins is unknown. To study the function of GlcNAc-1-P on a defined protein, we decided to clone members of the cysteine proteinase family expressed in vegetative cells.Previous studies in Dictyostelium identified two developmentally regulated members of this gene family, cprA (CP1) and cprB (CP2) (6 -8), but none have been identified in vegetative cells. Since cysteine proteinases are highly conserved in all eukaryotes, we used the active site consensus sequence of cysteine proteinases and the cprA and cprB cDNAs to clone two novel vegetative cysteine proteinases, cprD and cprE. They have the predicted conserved regions but also have an unusual serine-rich domain not previously found in any known cysteine proteinase that could be the site of GlcNAc-1-P addition. The cDNA clones were overexpressed and the active enzymes were shown to have GlcNAc-1-P. EXPERIMENTAL PROCEDURESMaterials-Radionucleotides were purchased from DuPont NEN and ICN Biomedicals, Inc. The random primed labeling kit an...

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Continuum Theory for Gas-Solid-Liquid MediaL Theory Development

Cited by 13 publications

References 0 publications

The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein

The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein

DLC-1 gene inhibits human breast cancer cell growth and in vivo tumorigenicity

Identification of Two Novel Dictyostelium discoideum Cysteine Proteinases That Carry N-Acetylglucosamine-1-P Modification

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Continuum Theory for Gas-Solid-Liquid MediaL Theory Development

Cited by 13 publications

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The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein

The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein

DLC-1 gene inhibits human breast cancer cell growth and in vivo tumorigenicity

Identification of Two Novel Dictyostelium discoideum Cysteine Proteinases That Carry N-Acetylglucosamine-1-P Modification

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Continuum Theory for Gas-Solid-Liquid MediaL Theory Development