Freeness of adjoint linear systems on threefolds with terminal Gorenstein singularities or some quotient singularities

We generalize the result of Kawamata concerning the strong version of Fujita's freeness conjecture for smooth 3-folds to some singular cases, namely, Gorenstein terminal singularities and quotient singularities of type 1/r(1,1,1) and of type 1/r(1,1,-1). We generalize furthermore the result of that to projective threefolds with only canonical singularities for canonical and not terminal singularities. It turns out that the estimates in the first three cases are better than the one for the smooth case, which it is not in the fourth case. We also give explicit examples which show the estimate in the fourth case is necessarily worse than the one for the smooth case.Comment: 21 pages, Late

Detection a Fibrous Component in Keratohyalin Granules of Newborn Rat Epidermis

Keratohyalin granules of frozen newborn rat epidermis were stained histologically and immunohistochemically and studied by light and electron microscopy. They showed a variety of reactivity to eosin, hematoxylin, uranyl acetate and rabbit monospecific antikeratin IgG. We found that good preservation of ultrastructure can be obtained from quickly frozen skin, and filamentous components remain after extraction of tissue substances by 0.14 m NaCl in 0.1 m-Tris-HCl buffer, pH 8.0. Furthermore, filaments in keratohyalin granules became immunologically reactive with rabbit anti-keratin IgG after extraction. Poststain with uranyl acetate further facilitated ultrastructural demonstration of filaments in the granules. These results indicate that filaments are one of the constituents of keratohyalin granules and the staining property changes occur in tonofilaments as they associate with other components to form keratohyalin granules

Unusual aggregation property of recombinantly expressed cancer-testis antigens in mammalian cells

Transient expression of human intracellular proteins in human embryonic kidney (HEK) 293 cells is a reliable system for obtaining soluble proteins with biologically active conformations. Contrary to conventional concepts, we found that recombinantly expressed intracellular cancer-testis antigens (CTAs) showed frequent aggregation in HEK293 cells. Although experimental subcellular localization of recombinant CTAs displayed proper cytosolic or nuclear localization, some proteins showed aggregated particles in the cell. This aggregative property was not observed in recombinant housekeeping proteins. No significant correlation was found between the aggregative and biophysical properties, such as hydrophobicity, contents of intrinsically disordered regions and expression levels, of CTAs. These results can be explained in terms of structural instability of CTAs, which are specifically expressed in the testis and aberrantly expressed in cancer cells and function as a hub in the protein–protein network using intrinsically disordered regions. Hence, we speculate that recombinantly expressed CTAs failed to form this protein complex. Thus, unfolded CTAs formed aggregated particles in the cell

Engineering Cancer/Testis Antigens With Reversible S-Cationization to Evaluate Antigen Spreading

Serum autoantibody to cancer/testis antigens (CTAs) is a critical biomarker that reflects the antitumor immune response. Quantitative and multiplexed anti-CTA detection arrays can assess the immune status in tumors and monitor therapy-induced antitumor immune reactions. Most full-length recombinant CTA proteins tend to aggregate. Cysteine residue-specific S-cationization techniques facilitate the preparation of water-soluble and full-length CTAs. Combined with Luminex technology, we designed a multiple S-cationized antigen-immobilized bead array (MUSCAT) assay system to evaluate multiple serum antibodies to CTAs. Reducible S-alkyl-disulfide-cationized antigens in cytosolic conditions were employed to develop rabbit polyclonal antibodies as positive controls. These control antibodies sensitively detected immobilized antigens on beads and endogenous antigens in human lung cancer-derived cell lines. Rabbit polyclonal antibodies successfully confirmed the dynamic ranges and quantitative MUSCAT assay results. An immune monitoring study was conducted using the serum samples on an adenovirus-mediated REIC/Dkk-3 gene therapy clinical trial that showed a successful clinical response in metastatic castration-resistant prostate cancer. Autoantibody responses were closely related to clinical outcomes. Notably, upregulation of anti-CTA responses was monitored before tumor regression. Thus, quantitative monitoring of anti-CTA antibody biomarkers can be used to evaluate the cancer-immunity cycle. A quality-certified serum autoantibody monitoring system is a powerful tool for developing and evaluating cancer immunotherapy