Depletion of hsp90beta induces multiple defects in B cell receptor signaling

Hsp90 participates in many distinct aspects of cellular functions and accomplishes these roles by interacting with multiple client proteins. To gain insight into the interactions between Hsp90 and its clients, here we have reduced the protein level of Hsp90 in avian cells by gene targeting in an attempt to elicit the otherwise undetectable (because of the vast amount of cellular Hsp90) Hsp90-interacting proteins. Hsp90beta-deficient cells can grow, albeit more slowly than wild-type cells. B cell antigen receptor signaling is multiply impaired in these mutant cells; in particular, the amount of immunoglobulin M heavy chain protein is markedly reduced. Furthermore, serum activation does not promote ERK phosphorylation in Hsp90beta-deficient cells. These multifaceted depressive effects seem to be provoked independently of each other and possibly recapitulate the proteome-wide in vivo functions of Hsp90. Reintroduction of the Hsp90beta gene efficiently restores all of the defects. Unexpectedly, however, introducing the Hsp90alpha gene is also effective in restoration; thus, these defects might be caused by a reduction in the total expression of Hsp90 rather than by loss of Hsp90beta-specific function.     

Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC

Several methods have been described to enhance antibody-dependent cellular cytotoxicity (ADCC) using different host cells that produce antibody with reduced levels of fucose on their carbohydrates. We compared the suitability of these methods for the serum-free fed-batch production of antibody for clinical trials and commercial uses. Recombinant anti-human CD20 chimeric IgG1-producing clones were established from host-cells that have been shown to produce more than 90% fucose-negative antibody. The cell lines were a FUT8 (alpha-1,6-fucosyltransferase) knockout Chinese hamster ovary (CHO) cell line, Ms704, and two Lens culinaris agglutinin (LCA)-resistant cell lines, one derived from a variant CHO line, Lec13 and the other from a rat hybridoma cell line, YB2/0. The amount of fucose-negative antibody produced by Lec13 and YB2/0 significantly decreased with the culture. The increase in fucosylation was due to remaining synthesis of GDP-fucose via de novo pathway for the CHO line and the elevation of FUT8 expression by the YB2/0 cells. In contrast, Ms704 cells stably produced fucose-negative antibody with a consistent carbohydrate structure until the end of the culture. The productivity of the Ms704 cells reached 1.76 g/L with a specific production rate (SPR) of 29 pg/cell/day for 17 days in serum-free fed-batch culture using a 1 L spinner bioreactor. Our results demonstrate that FUT8 knockout has the essential characteristics of host cells for robust manufacture of fucose-negative therapeutic antibodies with enhanced ADCC.     

Annexin A4 is a possible biomarker for cisplatin susceptibility of malignant mesothelioma cells

Mesothelioma is a highly malignant tumor with a poor prognosis and limited treatment options. Although cisplatin (CDDP) is an effective anticancer drug, its response rate is only 20%. Therefore, discovery of biomarkers is desirable to distinguish the CDDP-susceptible versus resistant cases. To this end, differential proteome analysis was performed to distinguish between mesothelioma cells of different CDDP susceptibilities, and this revealed that expression of annexin A4 (ANXA4) protein was higher in CDDP-resistant cells than in CDDP-susceptible cells. Furthermore, ANXA4 expression levels were higher in human clinical malignant mesothelioma tissues than in benign mesothelioma and normal mesothelial tissues. Finally, increased susceptibility was observed following gene knockdown of ANXA4 in mesothelioma cells, whereas the opposite effect was observed following transfection of an ANXA4 plasmid. These results suggest that ANXA4 has a regulatory function related to the cisplatin susceptibility of mesothelioma cells and that it could be a biomarker for CDDP susceptibility in pathological diagnoses.     

Rational design of DNA sequence-specific zinc fingers

We developed a rational scheme for designing DNA binding proteins. The scheme was applied for a zinc finger protein and the designed sequences were experimentally characterized with high DNA sequence specificity. Starting with the backbone of a known finger structure, we initially calculated amino acid sequences compatible with the expected structure and the secondary structures of the designed fingers were then experimentally confirmed. The DNA-binding function was added to the designed finger by reconsidering a section of the amino acid sequence and computationally selecting amino acids to have the lowest protein-DNA interaction energy for the target DNA sequences. Among the designed proteins, one had a gap between the lowest and second lowest protein-DNA interaction energies that was sufficient to give DNA sequence-specificity.     

Convenient and rapid removal of detergent from glycolipids in detergent-resistant membrane microdomains

Although detergents are often essential in protocols, they are usually incompatible with further biochemical analysis. There are several methods for detergent removal, but the procedures are complicated or suffer from sample loss. Here, we describe a convenient and rapid method for detergent removal from sialic acid-containing glycosphingolipids (gangliosides) and neutral glycolipids in detergent-resistant membrane (DRM) microdomain. It is based on selective detergent extraction, in which the sample is dried on a glass tube, followed by washing with organic solvent. We investigated 18 organic solvents and used high performance thin-layer chromatography (HPTLC) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) to confirm that dichloroethane (DCE) was the most suitable solvent and completely removed the nonionic detergent Triton X-100. Furthermore, DCE extraction effectively removed interference caused by other nonionic, zwitterionic, or ionic detergents in MALDI-QIT-TOF MS analysis.     

Brain insulin resistance accelerates Aβ fibrillogenesis by inducing GM1 ganglioside clustering in the presynaptic membranes

Type 2 diabetes mellitus is thought to be a significant risk factor for Alzheimer's disease. Insulin resistance also affects the central nervous system by regulating key processes, such as neuronal survival and longevity, learning and memory. However, the mechanisms underlying these effects remain uncertain. To investigate whether insulin resistance is associated with the assembly of amyloid β-protein (Aβ) at the cell surface of neurons, we inhibited insulin-signalling pathways of primary neurons. The treatments of insulin receptor (IR)-knockdown and a phosphatidylinositol 3-kinase inhibitor (LY294002), but not an extracellular signal-regulated kinase inhibitor, induced an increase in GM1 ganglioside (GM1) levels in detergent-resistant membrane microdomains of the neurons. The aged db/db mouse brain exhibited reduction in IR expression and phosphorylation of Akt, which later induced an increase in the high-density GM1-clusters on synaptosomes. Neurons treated with IR knockdown or LY294002, and synaptosomes of the aged db/db mouse brains markedly accelerated an assembly of Aβs. These results suggest that ageing and peripheral insulin resistance induce brain insulin resistance, which accelerates the assembly of Aβs by increasing and clustering of GM1 in detergent-resistant membrane microdomains of neuronal membranes.     

Construction of a long hairpin RNA expression library using Cre recombinase

A large number of genome-wide screens using RNA interference (RNAi) libraries have been utilized to determine the function of individual gene products involved in a variety of biological processes. In this study, we describe a new method to enzymatically generate a long hairpin RNA (lhRNA) expression library from a cDNA plasmid library using a nicking endonuclease, BcaBEST DNA polymerase, and Cre recombinase without excising the inserted DNA fragment from the plasmid vector. This method involves 5 steps: (1) conversion of an inserted DNA fragment in a plasmid into a direct repeat (DR); (2) purification of the plasmid containing the DR; (3) subcloning a lox71 cassette into the plasmid; (4) conversion of the DR in the plasmid into an inverted repeat (IR) using Cre recombinase; and (5) purification of the plasmid containing the IR. We also established an efficient method for inserting DNase I-digested DNA fragments into expression plasmids to enable construction of a cDNA plasmid library suitable as source materials to construct the lhRNA expression library. We confirmed that each of the lhRNA expression plasmids constructed using this method induced strong RNAi in a silkworm cell line, NIAS-Bm-oyanagi2.     

The Dr-nanos gene is essential for germ cell specification in the planarian Dugesia ryukyuensis

Homologs of nanos are required for the formation and maintenance of germline stem cell (GSC) systems and for gametogenesis in many metazoans. Planarians can change their reproductive mode seasonally, alternating between asexual and sexual reproduction; they develop and maintain their somatic stem cells (SSCs) and GCSs from pluripotent stem cells known as neoblasts. We isolated a nanos homolog, Dr-nanos, from the expressed sequence tags (ESTs) of the sexualized form of Dugesia ryukyuensis. We examined the expression of Dr-nanos in asexual and sexualized planarians by in situ hybridization and analyzed its function using RNA interference (RNAi) together with a planarian sexualization assay. A nanos homolog, Dr-nanos, was identified in the planarian D. ryukyuensis. Dr-nanos expression was observed in the ovarian primordial cells of the asexual worms. This expression increased in proportion to sexualization and was localized in the early germline cells of the ovaries and testes. In X-ray-irradiated worms, the expression of Dr-nanos decreased to a large extent, indicating that Dr-nanos is expressed in some subpopulations of stem cells, especially in GSCs. During the sexualization process, worms in which Dr-nanos was knocked down by RNAi exhibited decreased numbers of oogonia in the ovaries and failed to develop testes, whereas the somatic sexual organs were not affected. We conclude that Dr-nanos is essential for the development of germ cells in the ovaries and testes and may have a function in the early stages of germ cell specification, but not in the development of somatic sexual organs.     

Self-assembly of amylose-grafted carboxymethyl cellulose

In this study, we performed the self-assembly of the amylose-grafted carboxymethyl cellulose sodium salt (NaCMC) for the formation of nanofiber films under aqueous conditions. The introduction of amylose graft chains was conducted by the chemoenzymatic approach including phosphorylase-catalyzed enzymatic polymerization. The product had the rigid NaCMC main chain, which further assembled leading to nanofibers by the formation of double helix between the long amylose graft chains in the intermolecular NaCMC chains of the products. The lengths of the fibers were depended on degrees of polymerization of amylose chains. The nanofiber films were constructed by drying the alkaline solutions of the amylose-grafted NaCMC. The lengths of the nanofibers strongly affected their arrangements in the films. The nanofibers were merged further by washing out alkali to produce the robust nanofiber films.