The hydrodynamic diameters of native rotavirus particles, bovine RF and simian SA11 strains, were determined by quasielastic light scattering. By using this method and agarose gel electrophoresis, the Ca 2؉ dissociation constant, K Ca , governing the transition from triple-layer particles (TLPs) to double-layer particles (DLPs), was shown to increase, at constant pH, as the temperature and/or the ionic strength of the incubation medium increased. We report the novel observation that, under physiological conditions, K Ca values for both RF and SA11 rotaviruses were well above the intracytoplasmic Ca 2؉ concentrations of various cells, which may explain why TLP uncoating takes place within vesicles (possibly endosomes) during the entry process. A correlation between TLP uncoating and cell membrane permeabilization was found, as shown by the release of carboxyfluorescein (CF) from CF-loaded intestinal brush-border membrane vesicles. Conditions stabilizing the virion in the TLP form inhibited CF release, whereas conditions favoring the TLP-to-DLP transformation activated this process. We conclude that membrane permeabilization must be preceded by the loss of the outer-capsid proteins from trypsinized TLP and that physiological ionic strength is required for permeabilization to take place. Finally, the paper develops an alternative explanation for the mechanism of rotavirus entry, compatible with the Ca 2؉ -dependent endocytic pathway. We propose that there must be an iterative process involving tight coupling in time between the lowering of endosomal Ca 2؉ concentration, virion decapsidation, and membrane permeabilization, which would cause the transcriptionally active DLPs to enter the cytoplasm of cells.Recent advances in cryoelectron microscopy and icosahedral image reconstruction techniques have provided a detailed view of the three-dimensional structure of rotavirus, a nonenveloped member of the Reoviridae family (25,29). Rotavirus has a triple-layered spherical structure. The core is composed of the VP2 innermost capsid protein containing a "subcore" formed by the genomic double-stranded RNA and minor proteins VP1 and VP3 (8, 9). The double-layered particle (DLP) is formed by the VP6 intermediate-capsid protein surrounding the core. Finally, the triple-layered particle (TLP) results from addition of the outermost shell formed by proteins VP7 and VP4. In the presence of trypsin, VP4 is cleaved in situ into two smaller proteins, VP5* and VP8*, conferring infectivity to the virus (12).The critical Ca 2ϩ concentration ([Ca 2ϩ ]) governing the TLP-to-DLP transformation has been shown to be strain dependent. By using agar electrophoresis, Ruiz et al. (22) have shown that complete loss of the outer-capsid proteins from TLPs (TLP decapsidation) takes place in the nanomolar range of [Ca 2ϩ ] (e.g., 600 nM for the RF strain and 10 to 20 nM for the SA11 strain) when a Ca-EGTA buffer containing 10 mM MOPS (morpholinepropanesulfonic acid) and 100 mM KCl at room temperature is used. Nevertheless, it should be emphasized...

The effect of arachidonic acid (C20:4) on the production of secretory type II phospholipase A 2 (sPLA 2 -II) by guinea-pig alveolar macrophages was investigated. We show that incubation of these cells with 1±30 mm of arachidonic acid inhibits the synthesis of sPLA 2 -II in a concentration-dependent manner with an IC 50 of < 7.5 mm. The inhibition by low concentrations (5 mm) of arachidonic acid was partially reduced by pretreatment of alveolar macrophages with cyclooxygenase or cytochrome P450 inhibitors (aspirin and 1-aminobenzotriazole, respectively), but not by lipoxygenase inhibitor, BW A4C. However, these inhibitors failed to interfere with the effect of high concentrations (30 mm) of arachidonic acid, suggesting that the latter may act on the expression of sPLA 2 -II, at least in part, independently of eicosanoid generation. Indeed, a similar inhibitory effect on sPLA 2 -II activity and mRNA expression was observed with other unsaturated fatty acids such as eicosapentaenoic (C20:5) and oleic (C18:1) acids, but not with the saturated fatty acid, palmitic acid (C16:0). In addition, arachidonic acid partially reduced the secretion of tumor necrosis factor a, an important intermediate in the induction of sPLA 2 -II synthesis by guinea-pig alveolar macrophages. However, addition of recombinant tumor necrosis factor a failed to reverse the inhibitory effect of arachidonic acid on sPLA 2 -II expression, suggesting that this process occurs downstream of tumor necrosis factor a secretion. We conclude that the expression of sPLA 2 -II in alveolar macrophages is down-regulated at the transcriptional level by arachidonic acid either directly or via its cyclooxygenase and cytochrome P450-derived metabolites.

Ovarian cancer is the leading cause of death among gynecologic diseases in the USA and Europe. High-grade serous carcinoma (HGSC) of the ovary, the most aggressive type of ovarian cancer, is typically diagnosed at advanced stages when the 5-year survival is dismal. Since the cure rate for stage I HGSC is high, early detection of localized initial disease may improve patient outcomes. Serous tubal intraepithelial carcinoma (STIC) is considered to be a precursor lesion of HGSC. Discovery of biomarkers associated with STIC could aid in the development of an HGSC screening algorithm. Using immunohistochemical staining, we have demonstrated overexpression of UCHL1, ADAMTS13, and GAPDH in patients' STIC lesions, but not in cancer-free fallopian tubes. We additionally demonstrated a marked increase of T cells in perineoplastic stroma surrounding STIC lesions (largely CD4 + cells), but not in normal fallopian tubes and HGSC. FOXP3 + T regulatory cells are absent in STIC lesions but are present in HGSC. These observations indicate the microenvironment surrounding a STIC lesion may be immune promoting in contrast to the immune suppressive microenvironment of invasive carcinoma. In summary, we have identified UCHL1, ADAMTS13, and GAPDH as novel potentially useful markers associated with early stages of HGSC tumorigenesis and possibly contribute to STIC immunogenicity. The lack of immune suppression in the STIC microenvironment indicates that the immune system can still recognize and keep STIC controlled at this stage of the tumor development.