Lisbeth Berrueta

THE APC-ASSOCIATED PROTEIN EB1 ASSOCIATES WITH COMPONENTS OF THE DYNACTIN COMPLEX AND CYTOPLASMIC DYNEIN INTERMEDIATE CHAIN

Human EB1 is a highly conserved protein that binds to the carboxyl terminus of the human adenomatous polyposis coli (APC) tumor suppressor protein [1], a domain of APC that is commonly deleted in colorectal neoplasia [2]. EB1 belongs to a family of microtubule-associated proteins that includes Schizosaccharomyces pombe Mal3 [3] and Saccharomyces cerevisiae Bim1p [4]. Bim1p appears to regulate the timing of cytokinesis as demonstrated by a genetic interaction with Act5, a component of the yeast dynactin complex [5]. Whereas the predominant function of the dynactin complex in yeast appears to be in positioning the mitotic spindle [6], in animal cells, dynactin has been shown to function in diverse processes, including organelle transport, formation of the mitotic spindle, and perhaps cytokinesis [7] [8] [9] [10]. Here, we demonstrate that human EB1 can be coprecipitated with p150(Glued), a member of the dynactin protein complex. EB1 was also found associated with the intermediate chain of cytoplasmic dynein (CDIC) and with dynamitin (p50), another component of the dynactin complex, but not with dynein heavy chain, in a complex that sedimented at approximately 5S in a sucrose density gradient. The association of EB1 with members of the dynactin complex was independent of APC and was preserved in the absence of an intact microtubule cytoskeleton. The molecular interaction of EB1 with members of the dynactin complex and with CDIC may be important for microtubule-based processes.

Effect of Human Immunodeficiency Virus Type 1 on Intracellular Activation and Superoxide Production by Neutrophils

The immunopathogenesis of AIDS is associated with the development of opportunistic infections by intracellular pathogens that can invade and reproduce freely because of impaired cellular functions. Neutrophils from asymptomatic human immunodeficiency virus (HIV) type 1-infected persons and from symptomatic patients with AIDS were found to retain normal phagocytosis activity while producing significantly less superoxide than neutrophils from HIV-1-negative subjects, when stimulated through Fc receptors or protein kinase C. After priming with a synthetic HIV-1 envelope peptide and stimulation via the Fc receptor, the neutrophils from HIV-1-negative controls had suppressed superoxide production, reduced phosphorylation of two unidentified cellular proteins, and increased expression of a third phosphoprotein. These results suggest that HIV-1 can produce direct functional damage of neutrophils through binding of envelope components to the cell membrane.

CD40/CD40L expression in leukocytes from chronic granulomatous disease patients.

Chronic granulomatous disease (CGD) is an inherited disorder caused by defects in the NADPH oxidase complex, which generates superoxide, the precursor of hydrogen peroxide (H2O2) and other reactive oxygen derivatives with microbicidal activity. Because CGD patients are at risk of chronic inflammatory manifestations, including inflammatory bowel disease and autoimmune diseases, and it is not clear whether these pathologies are exclusively secondary to altered superoxide production, or whether distinct immunologic defects are involved, we explored cell proliferation, lymphocyte cell counts, immunoglobulin levels, presence of autoimmune antibodies and expression of costimulatory molecules in leukocytes from CGD patients. We found that CGD patients have a diminished phytohemagglutinin‐induced proliferation of blood mononuclear cells. Following stimulation with PMA plus ionomycin, a reduced percentage of CD40L expression in T lymphocytes and a diminished expression of CD40 molecules in neutrophils were observed on leukocytes from these patients. Our results suggest an altered interplay between elements of innate and adaptive immunity in CGD patients, which may be reflected in an increased susceptibility to opportunistic infections.