RUVBL1/TIP49a/Pontin52 is a recently identified multi-functional protein with 2 ATP binding (WALKER) sites, which is essential for cell proliferation. We recovered and identified RUVBL1/TIP49a as a tubulin-binding protein from Triton X-100 lysates of U937 promonocytic cells by protein affinity chromatography and tryptic peptide microsequencing. Performing co-immunoprecipitation using newly generated RUVBL1/TIP49a-specific antibodies (mAb and rabbit polyclonal Ab) and RUVBL1/TIP49a-GST fusion protein-pull down assays we demonstrate co-precipitation of alpha- and gamma tubulin with RUVBL1/TIP49a. Confocal immunoflourescence microscopy reveals that RUVBL1/TIP49a was present not only in the nucleus, as expected, but was also concentrated at the centrosome and at the mitotic spindle in colocalization with tubulin. The topology of RUVBL1/TIP49a at the mitotic spindle varied, depending on the mitotic stage. The protein was localized at the centrosome and at the polar and astral microtubules in metaphase, and was detectable at the zone of polar tubule interdigitation in anaphase B and telophase. During cytokinesis the protein reappeared at the area of decondensing chromosomes. Whereas preincubation of U937 cells with colcemid resulted in inhibition of mitotic spindle formation with subsequent loss of RUVBL1/TIP49a mitotic spindle staining, no relevant influence of colcemid on RUVBL1/TIP49a-tubulin binding was observed. An agonistic effect of RUVBL1/TIP49a on in vitro tubulin assembly is demonstrated. Our results reveal a new functional aspect of RUVBL1/TIP49a.

B cells express complement receptors (CRs) that bind activated fragments of C3 and C4. Immunized CR knockout (KO) mice have lower antibody titers and smaller germinal centers (GCs), demonstrating the importance of CR signals for the humoral immune response. CR ligands were thought to be generated via complement fixation mediated by preexisting “natural” IgM or early Ab from inefficiently activated B cells. This concept was recently challenged by a transgenic (Tg) mouse model that lacks circulating antibody but still retains membrane IgM (mIgM) and mounts normal immune responses. To test whether CR ligands could be generated by the B cell receptor (BCR) itself, we generated similar mice carrying a mutated mIgM that was defective in C1q binding. We found that B cells from such mutant mice do not deposit C3 on B cells upon BCR ligation, in contrast to B cells from mIgM mice. This has implications for the immune response: the mutant mice have smaller GCs than mIgM mice, and they are particularly deficient in the maintenance of the GC response. These results demonstrate a new BCR-dependent pathway that is sufficient and perhaps necessary to provide a CR1/2 ligand that promotes efficient B cell activation.

Individuals born with a low birth weight (LBW) have an increased prevalence of type 2 diabetes, but the mechanisms responsible for this association are unknown. Given the important role of insulin resistance in the pathogenesis of type 2 diabetes, we examined insulin sensitivity in a rat model of LBW due to intrauterine fetal stress. During the last 7 days of gestation, rat dams were treated with dexamethasone and insulin sensitivity was assessed in the LBW offspring by a hyperinsulinemic euglycemic clamp. The LBW group had liver-specific insulin resistance associated with increased levels of PEPCK expression. These changes were associated with pituitary hyperplasia of the ACTHsecreting cells, increased morning plasma ACTH concentrations, elevated corticosterone secretion during restraint stress, and an ϳ70% increase in 24-h urine corticosterone excretion. These data support the hypothesis that prenatal stress can result in chronic hyperactivity of the hypothalamic-pituitary-adrenal axis, resulting in increased plasma corticosterone concentrations, upregulation of hepatic gluconeogenesis, and hepatic insulin resistance. gluconeogenesis; adrenocorticotropic hormone; corticosterone; type 2 diabetes ACCORDING TO THE BARKER HYPOTHESIS, harmful events taking place during the fetal period can induce life-long changes in different organs predisposing to development of disease (1). In accordance with this hypothesis, individuals born with a birth weight of less than 5.5 lbs [low birth weight (LBW)] are insulin resistant (3,12,15,17,22,28,30,35) and have increased prevalence of type 2 diabetes (10, 15, 28). Additionally, these patients are also prone to exhibit impaired growth in childhood and early adulthood, which may be related to alterations in the growth hormone/IGF-I homeostasis (16,18,20). The mechanisms responsible for these changes associated with LBW are unknown.Recently, fetal stress and high plasma levels of glucocorticoids have been suggested (5, 27) to lead to hypothalamicpituitary-adrenal (HPA) axis hyperactivity, which after birth may result in chronically excessive adrenal glucocorticoid secretion, and an increased risk for the development of type 2 diabetes. However, these findings have been contrasted by other studies that have found no change (32) or decreased HPA axis activity (21) in LBW rat models.To examine the mechanism and potential role of the HPA axis in causing LBW-associated insulin resistance, we assessed insulin-stimulated liver and muscle glucose metabolism, total IGF-I, and IGF-binding proteins (IGFBPs), as well as HPA axis activity in a rat model of stress-induced LBW. MATERIALS AND METHODS Animals.From day 7 of gestation, pregnant female Sprague-Dawley rats (Charles River, Wilmington, MA) were housed singly under temperature (22-23°C)-and light-controlled (12:12-h light-dark cycle) conditions. On day 14 of gestation, rats were randomized into three groups: control, dexamethasone treatment, or foster mother group. From day 14 to day 21 of gestation, a daily subcutaneously injection of d...

Rossbacher J, Wagner L, Pasternack MS. Inhibitory Effect of Haptoglobin on Granulocyte Chemotaxis, Phagocytosis and Bactericidal Activity. Scand J Immunol 1999;50:399±404 Human haptoglobin (Hp) is synthesized at hepatic and extrahepatic sites as an acute-phase reactant protein (APP). We investigated the effects of Hp on granulocyte function. The chemotaxis of freshly isolated human granulocytes and differentiated HL-60 cells in response to the bacterial tripeptide, f-met-leuphe, was inhibited in the presence of a physiological concentration of Hp, but chemotaxis in the presence of the proin¯ammatory cytokine interleukin-8 (IL-8) was not inhibited. Phagocytosis of viable Escherichia coli, as well as¯uoresceinated nonviable E. coli, was inhibited. Hp also reduced granulocyte intracellular bactericidal activity against E. coli. The observed inhibitory effects of Hp on granulocyte function are similar to those reported for C-reactive protein and suggest that APPs dampen the acute in¯ammatory response.

Mice lacking C3, C4 or complement receptor 1/2 (Cr) have defective germinal centers (GC). The requirement for C4 implicates complement fixation by immune complexes (IC) via the classical pathway. Yet, transgenic (Tg) mice that lack circulating antibody but still express membrane IgM (mIgM) have normal GC responses. We showed previously that cross-linking mIgM leads to the deposition of C3 on the B cell surface and that disruption of this pathway diminishes GC responses. Here, we investigate the role of Cr in this process by generating mIgM-Tg mice that lack Cr and serum Ig. These mIgM/Cr -/-mice have smaller, transient GC, with incomplete B cell receptor downregulation and peanut agglutinin up-regulation, compared to mIgM/Cr wt counterparts. BM chimera experiments showed that Cr on B cells is required for normal GC responses. These results establish that Cr ligands generated at the B cell surface are sufficient for normal GC responses and function by signaling Cr on B cells. Unexpectedly, chimera experiments also showed a critical role for Cr on follicular dendritic cells (FDC), even in the absence of IC, indicating novel functions for FDC-expressed Cr beyond the capture of C3-coated IC.