Expression cloning of Forssman glycolipid synthetase: a novel member of the histo-blood group ABO gene family.

A phenotypic cloning approach was used to isolate a canine cDNA encoding Forssman glycolipid synthetase (FS; UDP-GalNAc:globoside alpha-1,3-N-acetylgalactosaminyltransferase; EC 2.4.1.88). The deduced amino acid sequence of FS demonstrates extensive identity to three previously cloned glycosyltransferases, including the enzymes responsible for synthesis of histo-blood group A and B antigens. These three enzymes, like FS, catalyze the addition of either N-acetylgalactosamine (GalNAc) or galactose (Gal) in alpha-1,3-linkage to their respective substrates. Despite the high degree of sequence similarity among the transferases, we demonstrate that the FS cDNA encodes an enzyme capable of synthesizing Forssman glycolipid, and demonstrates no GalNAc or Gal transferase activity when closely related substrates are examined. Thus, the FS cDNA is a novel member of the histo-blood group ABO gene family that encodes glycosyltransferases with related but distinct substrate specificity. Cloning of the FS cDNA will allow a detailed dissection of the roles Forssman glycolipid plays in cellular differentiation, development, and malignant transformation.

Chemical camouflage of antigenic determinants: Stealth erythrocytes

In a number of clinical circumstances it would be desirable to artificially conceal cellular antigenic determinants to permit survival of heterologous donor cells. A case in point is the problem encountered in transfusions of patients with rare blood types or chronically transfused patients who become allosensitized to minor blood group determinants. We have tested the possibility that chemical modification of the red blood cell (RBC) membrane might serve to occlude antigenic determinants, thereby minimizing transfusion reactions. To this end, we have covalently bound methoxy(polyethylene glycol) (mPEG) to the surface of mammalian RBC via cyanuric chloride coupling. Human RBC treated with this technique lose ABO blood group reactivity as assessed by solution–phase antisera agglutination. In accord with this, we also find a profound decrease in anti-blood group antibody binding. Furthermore, whereas human monocytes avidly phagocytose untreated sheep RBC, mPEG-derivatized sheep RBC are ineffectively phagocytosed. Surprisingly, human and mouse RBC appear unaffected by this covalent modification of the cell membrane. Thus, mPEG-treated RBC are morphologically normal, have normal osmotic fragility, and mPEG-derivatized murine RBC have normal in vivo survival, even following repeated infusions. Finally, in preliminary experiments, mPEG-modified sheep RBC intraperitoneally transfused into mice show significantly improved (up to 360-fold) survival when compared with untreated sheep RBC. We speculate that similar chemical camouflage of intact cells may have significant clinical applications in both transfusion (e.g., allosensitization and autoimmune hemolytic disease) and transplantation (e.g., endothelial cells and pancreatic β cells) medicine.

The Ras/Rac1/Cdc42/SEK/JNK/c-Jun Cascade Is a Key Pathway by Which Agonists Stimulate DNA Synthesis in Primary Cultures of Rat Hepatocytes

The ability of signaling via the JNK (c-Jun NH2-terminal kinase)/stress-activated protein kinase cascade to stimulate or inhibit DNA synthesis in primary cultures of adult rat hepatocytes was examined. Treatment of hepatocytes with media containing hyperosmotic glucose (75 mM final), tumor necrosis factor α (TNFα, 1 ng/ml final), and hepatocyte growth factor (HGF, 1 ng/ml final) caused activation of JNK1. Glucose, TNFα, or HGF treatments increased phosphorylation of c-Jun at serine 63 in the transactivation domain and stimulated hepatocyte DNA synthesis. Infection of hepatocytes with poly-l-lysine–coated adenoviruses coupled to constructs to express either dominant negatives Ras N17, Rac1 N17, Cdc42 N17, SEK1−, or JNK1− blunted the abilities of glucose, TNFα, or HGF to increase JNK1 activity, to increase phosphorylation of c-Jun at serine 63, and to stimulate DNA synthesis. Furthermore, infection of hepatocytes by a recombinant adenovirus expressing a dominant-negative c-Jun mutant (TAM67) also blunted the abilities of glucose, TNFα, and HGF to stimulate DNA synthesis. These data demonstrate that multiple agonists stimulate DNA synthesis in primary cultures of hepatocytes via a Ras/Rac1/Cdc42/SEK/JNK/c-Jun pathway. Glucose and HGF treatments reduced glycogen synthase kinase 3 (GSK3) activity and increased c-Jun DNA binding. Co-infection of hepatocytes with recombinant adenoviruses to express dominant- negative forms of PI3 kinase (p110α/p110γ) increased basal GSK3 activity, blocked the abilities of glucose and HGF treatments to inhibit GSK3 activity, and reduced basal c-Jun DNA binding. However, expression of dominant-negative PI3 kinase (p110α/p110γ) neither significantly blunted the abilities of glucose and HGF treatments to increase c-Jun DNA binding, nor inhibited the ability of these agonists to stimulate DNA synthesis. These data suggest that signaling by the JNK/stress-activated protein kinase cascade, rather than by the PI3 kinase cascade, plays the pivotal role in the ability of agonists to stimulate DNA synthesis in primary cultures of rat hepatocytes.