G protein-coupled receptor kinase 5 modifies cancer cell resistance to paclitaxel

Molecular and Cellular Biochemistry - The critical role of the intrarenal renin-angiotensin system (RAS) in the development of kidney disease has been well demonstrated in animal and cell-culture...     

Glycomodification and characterization of anti-colorectal cancer immunotherapeutic monoclonal antibodies in transgenic tobacco

Anti-colorectal cancer mAb CO17-1A (IgG_2a) recognizes the antigen GA733, which is highly expressed on the surface membrane of human colorectal carcinoma cells. In this study, a transgenic tobacco system for the production of mAb CO17-1A was developed. The mAb construct included a KDEL sequence, an endoplasmic reticulum (ER) retention signal attached to the C-terminus of the heavy chain, to target accumulation of mAb into ER. An immunoblot showed significantly enhanced levels of expression of the plant-derived mAbK (mAb^PK) CO17-1A compared to mAb^P CO17-1A mAb without the KDEL sequence. An ELISA assay using human colorectal carcinoma cells confirmed that expression of mAb^PK was also significantly higher than that of mAb^P. Glycosylation analysis revealed that mAb^P had plant-specific glycans; whereas, mAb^PK primarily had oligomannose glycans. FACS showed that the Fc domains of both mAb^PK and mammalian-derived mAb (mAb^M) had similar binding activity to the FcγRI receptor (CD64). However, the Fc domains of the mAb^P had slightly lower binding activity to the Fc_γRI receptor than both mAb^PK and mAb^M. The antibody-dependent cell cytotoxicity of mAb^PK, against human colorectal cancer cells, was as efficient as mAb^M; whereas mAb^P was very low. These results suggest that KDEL localized and accumulated mAb^P in the ER and eventually enhanced the expression of mAb^P with oligomannose glycan and similar anti-cancer biological activity to the parental mAb^M.     

The effect of 5-fluorouracil on cells and regenerating protoplasts ofSaccharomyces cerevisiae

The regeneration of the yeast cell-wall was studied using 5-fluorouracil and yeast protoplasts. Protein synthesis in yeast cells (Saccharomyces cerevisiae) was kept reduced in the presence of this inhibitor at a rate corresponding to that before inhibition and was independent on the concentration of the inhibitor (10 or 100 μg/ml). The inhibition of the RNA synthesis was incomplete and dependent on the concentration of the inhibitor. Synthesis of thymidine and DNA was not inhibited as indicated by the growth tests. On the basis of the obtained data it may be concluded that fluorouracil inhibits only thede novo and the induced protein synthesis while permitting protein synthesis that has already been started before inhibition. Fluorouracil was then applied during the regeneration of yeast protoplasts. The results obtained have shown that fluorouracil does not inhibit the synthesis of the yeast cell wall but that the normal course of cell division is impaired by fluorouracil. The low efficiency of the fluorouracil inhibition of the cell wall synthesis indicates that processes leading to the regeneration of the cell wall are in fact only a continuation of those taking place under normal growth conditions.     

Panosialins, inhibitors of an α1,3-fucosyltransferase Fuc-TVII, suppress the expression of selectin ligands on U937 cells

Panosialins A and B were isolated as inhibitors of an 1,3-fucosyltransferase, Fuc-TVII, which is a key enzyme in the biosynthesis of selectin ligands, from culture broth of Streptomyces sp. Panosialins A and B inhibited the Fuc-TVII activity with IC50 values of 4.8 and 5.3 g/ml, respectively. Panosialin A suppressed expression of selectin ligands on U937 cells, and inhibited the cell adhesion to immobilized E-selectin-immunoglobulin. Panosialins are the first reported Fuc-TVII inhibitors which can suppress the biosynthesis of selectin ligands and then inhibit selectin-mediated cell adhesion.     

Adult alveolar epithelial cells express multiple subtypes of voltage-gated K+ channels that are located in apical membrane

Whole cell perforated patch-clampexperiments were performed with adult rat alveolar epithelial cells.The holding potential was 60 mV, and depolarizing voltage stepsactivated voltage-gated K⁺ (Kv) channels. Thevoltage-activated currents exhibited a mean reversal potential of 32mV. Complete activation was achieved at 10 mV. The currents exhibitedslow inactivation, with significant variability in the time coursebetween cells. Tail current analysis revealed cell-to-cell variabilityin K⁺ selectivity, suggesting contributions of multiple Kv-subunits to the whole cell current. The Kv channels also displayedsteady-state inactivation when the membrane potential was held atdepolarized voltages with a window current between 30 and 5 mV.Analysis of RNA isolated from these cells by RT-PCR revealed thepresence of eight Kv -subunits (Kv1.1, Kv1.3, Kv1.4, Kv2.2, Kv4.1,Kv4.2, Kv4.3, and Kv9.3), three -subunits (Kv1.1, Kv2.1, andKv3.1), and two K⁺ channel interacting protein (KChIP)isoforms (KChIP2 and KChIP3). Western blot analysis with available Kv-subunit antibodies (Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3) showedlabeling of 50-kDa proteins from alveolar epithelial cells grown inmonolayer culture. Immunocytochemical analysis of cells from monolayersshowed that Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3 were localized to theapical membrane. We conclude that expression of multiple Kv -, -,and KChIP subunits explains the variability in inactivation gating andK⁺ selectivity observed between cells and that Kv channelsin the apical membrane may contribute to basal K⁺ secretionacross the alveolar epithelium.

     

Bcl-rambo induces apoptosis via interaction with the adenine nucleotide translocator

The Bcl-2 family proteins plays a central role in apoptosis. The pro- or anti-apoptotic activities of Bcl-2 family are dependent on the Bcl-2 homology (BH) regions. Bcl-rambo, a new pro-apoptotic member, is unusual in that its pro-apoptotic activity is independent of its BH domains. However, the mechanism underlying Bcl-rambo-induced apoptosis is largely unknown. Mitochondrial localization is indispensable for the pro-apoptotic function of Bcl-rambo. Bcl-rambo interacts physically with the adenine nucleotide translocator (ANT), suppresses the ADT/ATP-dependent translocation activity of ANT. Collectively, our data indicate Bcl-rambo is a pro-apoptotic member of the Bcl-2 family, induces the permeability transition via interaction with ANT.

Structured summary of protein interactions:

Bcl-Rambo and HSP60colocalize by fluorescence microscopy (View interaction)Bcl-rambobinds to ANT1 by pull down (View interaction)     

Isolation of dextran‐hydrolyzing intestinal bacteria and characterization of their dextranolytic activities

The aim of this study was to isolate dextran‐hydrolyzing bacteria from the human intestines and to identify their dextranolytic enzymes. For this, dextranase‐producing microorganisms were screened from fecal samples by using blue dextran‐containing media. Colonies producing a decolorized zone were isolated and they were grouped using RAPD‐PCR. 16S rRNA gene sequencing analysis revealed the isolates were Bacteroides (B.) thetaiotaomicron, B. ovatus, B. vulgatus, B. dorei, B. xylanisolvens, B. uniformis, and Veillonella (V.) rogosae. Thin layer chromatography analysis showed that the dextranases exhibit mainly endo‐type activity and produce various oligosaccharides including isomaltose and isomaltotriose. Zymogram analysis demonstrated that enzymes localized mainly in the cell membrane fraction and the molecular weight was 50–70 kDa. When cultured in a dextran‐containing medium, all strains isolated in this study produced short‐chain fatty acids, with butyric acid as the major compound. This is the first study to report that human intestinal B. xylanisolvens, B. dorei, and V. rogosae metabolize dextran utilizing dextranolytic enzymes. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 321–327, 2015.     

A Novel Application of Furazolidone: Anti-Leukemic Activity in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common malignant myeloid disorder of progenitor cells in myeloid hematopoiesis and exemplifies a genetically heterogeneous disease. The patients with AML also show a heterogeneous response to therapy. Although all-trans retinoic acid (ATRA) has been successfully introduced to treat acute promyelocytic leukemia (APL), it is rather ineffective in non-APL AML. In our present study, 1200 off-patent marketed drugs and natural compounds that have been approved by the Food and Drug Administration (FDA) were screened for anti-leukemia activity using the retrovirus transduction/transformation assay (RTTA). Furazolidone (FZD) was shown to inhibit bone marrow transformation mediated by several leukemia fusion proteins, including AML1-ETO. Furazolidone has been used in the treatment of certain bacterial and protozoan infections in human and animals for more than sixty years. We investigated the anti-leukemic activity of FZD in a series of AML cells. FZD displayed potent antiproliferative properties at submicromolar concentrations and induced apoptosis in AML cell lines. Importantly, FZD treatment of certain AML cells induced myeloid cell differentiation by morphology and flow cytometry for CD11b expression. Furthermore, FZD treatment resulted in increased stability of tumor suppressor p53 protein in AML cells. Our in vitro results suggest furazolidone as a novel therapeutic strategy in AML patients.