Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans

Chronic infection with hepatitis B virus (HBV) is associated with the majority of hepatocellular carcinoma (HCC). The diagnosis of HCC is usually made in the late stages of the disease, when treatment options are limited and prognosis is poor. We therefore have developed a method of glycoproteomic analysis in an attempt to discover serum markers that can assist in the early detection of HBV-induced liver cancer. Briefly, a comparative method for analysis of oligosaccharides released from serum glycoproteins and for recovery and identification of proteins with aberrant glycosylation, as a function of cancer diagnosis, is described. The model we have used is the woodchuck (Marmota monax), which shares similarities in the glycosylation pattern associated with liver proteins in human HCC. In this report, we show that woodchucks diagnosed with HCC have dramatically higher levels of serum-associated core alpha-1,6-linked fucose, as compared with woodchucks without a diagnosis of HCC. The coupling of this methodology with 2D gel proteomics has permitted the identification of several glycoproteins with altered glycosylation as a function of cancer. One such glycoprotein, Golgi Protein 73 (GP73), was found to be elevated and hyperfucosylated in animals with HCC. Further, the study showed GP73 to be elevated in the serum of people with a diagnosis of HCC, providing a validation of our approach. The potential of this technology for biomarker discovery and the implications of increased levels of GP73 in liver cancer are discussed.     

Interaction of neuropeptides with human mast cells

Mast cells of human skin, but not lung, adenoids, tonsils, or intestine, release histamine in response to substance P, vasoactive intestinal polypeptide, and somatostatin. The substance P receptor of skin mast cells is not of the NK-1, NK-2 or NK-3 subtypes of smooth muscle. Time course and calcium dependency of release by peptides differed from anti-IgE. With anti-IgE, the molar ratios of histamine:PGD2:LTC4 generated by skin mast cells was 1,000:25:2, whereas with substance P these ratios were 1,000:1:0.1. Similar results were obtained with the other neuropeptides. The ability of peptides to stimulate skin mast cell histamine release suggests a mechanism whereby their release from dermal nerve endings is coupled to changes in microvasculature.     

Purification and characterization of enterotoxigenic El Tor-like hemolysin produced by Vibrio fluvialis

The halophilic bacterium Vibrio fluvialis is an enteric pathogen that produces an extracellular hemolysin. This hemolysin was purified to homogeneity by using sequential hydrophobic-interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. It has a molecular weight of 63,000 and an isoelectric point of 4.6, and its hemolytic activity is sensitive to heat, proteases, and preincubation with zinc ions. The hemolysin lyses erythrocytes of the eight different animal species that we tested, is cytotoxic against Chinese hamster ovary cells in tissue culture, and elicits fluid accumulation in suckling mice. Lysis of erythrocytes occurs by a temperature-dependent binding step followed by a temperature- and pH-dependent lytic step. Fourteen of the first 20 N-terminal amino acid residues (Val-Ser-Gly-Gly-Glu-Ala-Asn-Thr-Leu-Pro-His-Val-Ala-Phe-Tyr-Ile-Asn-Val-Asn-Arg) are identical to those of the El Tor hemolysin of Vibrio cholerae and the heat-labile hemolysin of Vibrio mimicus. This homology was further confirmed by PCR analysis using a 5' primer derived from the amino-terminal sequence of the hemolysin and a 3' primer derived from the El Tor hemolysin structural gene. The hemolysin also reacts with antibodies to the El Tor-like hemolysin of non-O1 V. cholerae.