Effect of amphotericin B on the lipids of yeast cells of Sporothrix schenckii

Yeast cells of five strains of Sporothrix schenckii were obtained for partial analysis of lipid composition. Quantitative analysis of lipids and sterols were completed, as well as qualitative analysis of sterols by thin layer chromatography and by ultraviolet spectra. These determinations were made on cells cultured in the absence and presence of amphotericin B at sub-MIC (minimum inhibitory concentration) levels. Marked alterations in lipid content were observed in the amphotericin B-treated cells. The major alterations were the reduction of total lipid (18.7–57.6%) and sterols (48.5–96.7%) after exposure to the polyenic antibiotic. It is concluded that amphotericin B altered the lipid profiles, especially sterols of S. schenckii.     

Model for studying virus attachment. II. Binding of biotinylated human T cell leukemia virus type I to human blood mononuclear cells potential targets for human T cell leukemia virus type I infection.

Purified human T cell leukemia virus type I (HTLV-I) was biotinylated and used to study its attachment to human PBMC. The use of biotinylated HTLV-I (biot-HTLV-I) in conjunction with mouse mAb specific for selected cell-surface molecules and flow cytometric analysis allowed us to positively identify virus-binding cells among a heterogeneous blood mononuclear cell population. Biot-HTLV-I efficiently bound not only to T cells, but also to B cells and monocytes. Preincubation of monocytes with excess of unlabeled HTLV-I significantly reduced the attachment of biot-HTLV-I. HTLV-I not only bound to, but also infected, B cells, as suggested by: i) in situ hybridization of a 35S-labeled full length HTLV-I DNA probe with EBV-transformed B cells, previously cocultured with HTLV-I-producing (G11MJ) T cells, and ii) hybridization of the same nick-translated 32P-labeled DNA probe with blotted DNA from similar HTLV-I-infected EBV-transformed B cells. HTLV-I infection did not affect the ability of B cells to secrete IgG. These findings suggest that HTLV-I cannot only infect cells of the T lineage, but can also infect B cells.     

Cancer brings forward oviposition in the fly Drosophila melanogaster

Hosts often accelerate their reproductive effort in response to a parasitic infection, especially when their chances of future reproduction decrease with time from the onset of the infection. Because malignancies usually reduce survival, and hence potentially the fitness, it is expected that hosts with early cancer could have evolved to adjust their life‐history traits to maximize their immediate reproductive effort. Despite the potential importance of these plastic responses, little attention has been devoted to explore how cancers influence animal reproduction. Here, we use an experimental setup, a colony of genetically modified flies Drosophila melanogaster which develop colorectal cancer in the anterior gut, to show the role of cancer in altering life‐history traits. Specifically, we tested whether females adapt their reproductive strategy in response to harboring cancer. We found that flies with cancer reached the peak period of oviposition significantly earlier (i.e., 2 days) than healthy ones, while no difference in the length and extent of the fecundity peak was observed between the two groups of flies. Such compensatory responses to overcome the fitness‐limiting effect of cancer could explain the persistence of inherited cancer‐causing mutant alleles in the wild.     

Effects of dimethyl sulfoxide (DMSO) on microfilament organization, cellular adhesion, and growth of cultured mouse B16 melanoma cells

Cell shape is involved in a variety of cellular activities including proliferation, adhesion, migration, and transformation. Agents known to promote differentiation, such as retinoic acid, butyrate, and dibutyryl cyclic AMP, induce marked alterations in cell shape which are often accompanied by changes in cell functions. In this paper we study the effects of the differentiating polar solvent dimethyl sulfoxide (DMSO) on cytoskeleton, adhesion, and growth properties of cultured mouse B16 melanoma cells. DMSO induced a progressive reorganization of the cytoskeleton which was fully developed in 4 days of continuous exposure to the agent. DMSO-treated cells developed thick and regularly oriented microfilament bundles of the stress fiber type ending at vinculin-rich areas of focal contact between the ventral membrane and the substratum (interference reflection microscopydark adhesion plaques). Such a rearrangement of the cytoskeleton resulted in increased adhesion to the substratum and inhibition of cell growth in comparison to control untreated cells. Cells which became highly flattened and tightly adherent after exposure to DMSO for 4 days progressively reverted their phenotype to that of control untreated cells within 3 days of DMSO withdrawal. Namely, they lost stress fibers and adhesion plaques, became rounded and less adherent, and increased their growth rate. These results indicate that DMSO can change the transformed appearance of B16 mouse melanoma cells to a phenotype which is typical of a variety of nontransformed cells in culture.     

Crystal structure of a human autoimmune complex between IgM rheumatoid factor RF61 and IgG1 Fc reveals a novel epitope and evidence for affinity maturation

Rheumatoid factors (RF) are autoantibodies that recognize epitopes in the Fc region of immunoglobulin (Ig) G and that correlate with the clinical severity of rheumatoid arthritis (RA). Here we report the X-ray crystallographic structure, at 3 A resolution, of a complex between the Fc region of human IgG1 and the Fab fragment of a monoclonal IgM RF (RF61), derived from an RA patient and with a relatively high affinity for IgG Fc. In the complex, two Fab fragments bind to each Fc at epitopes close to the C terminus, and each epitope comprises residues from both Cgamma3 domains. A central role in the unusually hydrophilic epitope is played by the side-chain of Arg355, accounting for the subclass specificity of RF61, which recognizes IgG1,-2, and -3 in preference to IgG4, in which the corresponding residue is Gln355. Compared with a previously determined complex of a lower affinity RF (RF-AN) bound to IgG4 Fc, in which only residues at the very edge of the antibody combining site were involved in binding, the epitope bound by RF61 is centered in classic fashion on the axis of the V(H):V(L) beta-barrel. The complementarity determining region-H3 loop plays a key role, forming a pocket in which Arg355 is bound by two salt-bridges. The antibody contacts also involve two somatically mutated V(H) residues, reinforcing the suggestion of a process of antigen-driven maturation and selection for IgG Fc during the generation of this RF autoantibody.     

Simulation of the mutation F76del on the von Hippel–Lindau tumor suppressor protein: Mechanism of the disease and implications for drug development

The von Hippel–Lindau tumor suppressor protein (pVHL) plays a central role in the oxygen‐sensing pathway by regulating the degradation of the hypoxia‐inducible factor (HIF‐1α). The capture of HIF‐1α by pVHL is regulated by an oxygen‐dependent hydroxylation of a specific conserved prolyl residue. The VHL gene is mutated in the von Hippel–Lindau cancer predisposition syndrome, which is characterized by the development of highly vascularized tumors and is associated with constitutively high levels of HIF‐1α. The disturbance of the dynamic coupling between HIF‐1α and pVHL bearing the commonly found mutation F76del was experimentally confirmed but the mechanism of such complex disruption is still not clear. Performing unbiased molecular dynamics simulations, we show that the F76del mutation may enlarge the HIF binding pocket in pVHL and induce the formation of an internal cavity in the hydrophobic core of the β‐domain, which can lead to a partial destabilization of the β‐sheets S1, S4, and S7 and a consequent loss of hydrogen bonds with a conserved recognition motif in HIF. The newly formed cavity has a significant druggability score and may be a suitable target for stabilizing ligands. Studies of this nature may help to fill the information gap between genotype–phenotype correlations with details obtained at atomic level and provide basis for future development of drug candidates, such as pharmacological chaperones, with the specific aim of reverting the dysfunction of such pathological protein complexes found in patients with VHL. Proteins 2013. © 2012 Wiley Periodicals, Inc.