Protein purification and cloning of diacylglycerol lipase from rat brain
Diacylglycerol (DG) lipase, which hydrolyses 1-stearoyl-2-arachidonyl-sn-glycerol to produce an endocannabinoid, 2-arachidonoylglycerol, was purified from the soluble fraction of rat brain lysates. DG lipase was purified about 1,200-fold by a sequential column chromatographic procedure. Among proteins identified by mass spectrometry analysis in the partially purified DG lipase sample, only DDHD domain containing two (DDHD2), which was formerly regarded as a phospholipase A1, exhibited significant DG lipase activity. Rat DDHD2 expressed in Chinese hamster ovary cells showed similar enzymatic properties to partially purified...
Source: Journal of Biochemistry - May 30, 2016 Category: Biochemistry Authors: Aso, C., Araki, M., Ohshima, N., Tatei, K., Hirano, T., Obinata, H., Kishi, M., Kishimoto, K., Konishi, A., Goto, F., Sugimoto, H., Izumi, T. Tags: Regular Papers Source Type: research
Comparative lipid analysis in the normal and cancerous organoids of MDCK cells
Epithelial organs are made of a well-polarized monolayer of epithelial cells, and their morphology is maintained strictly for their proper functioning. The roles of lipids are not only to generate the membrane, but also to provide the specific domains for signal transduction, or to transmit signals as second messengers. By using a liquid chromatography-electrospray ionization mass spectrometry (LC-MS)/MS method, we here analyzed sphingolipids in MDCK cysts under various conditions. Our result showed that, compared to the three-dimensional cyst, the two-dimensional MDCK sheet is relatively enriched in sphingolipids. During ...
Source: Journal of Biochemistry - May 30, 2016 Category: Biochemistry Authors: Yoshizaki, H., Ogiso, H., Okazaki, T., Kiyokawa, E. Tags: Regular Papers Source Type: research
A/T gap tolerance in the core sequence and flanking sequence requirements of non-canonical p53 response elements
In this study, we investigated the possible number of A/T bases used by p53 and showed that a six-base A/T gap CATATATG core sequence was the maximum A/T gap in the p53 response element that could be upregulated by p53 and p63. Canonical and non-canonical p53 response elements also have three-base flanking sequences. A/T bases could be substituted by G/C bases, including CACACG and CGTGTG, but not CGCGCG. We found that the SV40 promoter with functional six- and two-base A/T gap core sequences could be activated by TAp63 and that TAp63 could upregulate SV40 small and large T antigens expression in COS7 cells. We also found ...
Source: Journal of Biochemistry - May 30, 2016 Category: Biochemistry Authors: Cai, B.-H., Chao, C.-F., Lin, H.-C., Huang, H.-Y., Kannagi, R., Chen, J.-Y. Tags: Regular Papers Source Type: research
Statistical analyses in trials for the comprehensive understanding of organogenesis and histogenesis in humans and mice
Statistical analyses based on the quantitative data from real multicellular organisms are useful as inductive-type studies to analyse complex morphogenetic events in addition to deductive-type analyses using mathematical models. Here, we introduce several of our trials for the statistical analysis of organogenesis and histogenesis of human and mouse embryos and foetuses. Multidimensional scaling has been applied to prove the existence and examine the mode of interkinetic nuclear migration, a regulatory mechanism of stem cell proliferation/differentiation in epithelial tubular tissues. Several statistical methods were used ...
Source: Journal of Biochemistry - May 30, 2016 Category: Biochemistry Authors: Otani, H., Udagawa, J., Naito, K. Tags: JB Review Source Type: research
The N-terminal motif of PMP70 suppresses cotranslational targeting to the endoplasmic reticulum
Many membrane proteins possessing hydrophobic transmembrane (TM) segments are cotranslationally integrated into the endoplasmic reticulum (ER) membrane. Various peroxisomal and mitochondrial membrane proteins escape the ER-targeting mechanism and are targeted to their destinations. Here, we discovered a short segment in the 70-kDa peroxisomal membrane protein (PMP70) that suppresses ER targeting. The first TM segment has an intrinsic signal function that targets the nascent chain to the ER. The ER targeting was suppressed by a short N-terminal sequence of nine residues that is 80 residues upstream of the TM segment. Among ...
Source: Journal of Biochemistry - April 25, 2016 Category: Biochemistry Authors: Sakaue, H., Iwashita, S., Yamashita, Y., Kida, Y., Sakaguchi, M. Tags: Regular Papers Source Type: research
Redox-coupled structural changes in nitrite reductase revealed by serial femtosecond and microfocus crystallography
Serial femtosecond crystallography (SFX) has enabled the damage-free structural determination of metalloenzymes and filled the gaps of our knowledge between crystallographic and spectroscopic data. Crystallographers, however, scarcely know whether the rising technique provides truly new structural insights into mechanisms of metalloenzymes partly because of limited resolutions. Copper nitrite reductase (CuNiR), which converts nitrite to nitric oxide in denitrification, has been extensively studied by synchrotron radiation crystallography (SRX). Although catalytic Cu (Type 2 copper (T2Cu)) of CuNiR had been suspected to tol...
Source: Journal of Biochemistry - April 25, 2016 Category: Biochemistry Authors: Fukuda, Y., Tse, K. M., Suzuki, M., Diederichs, K., Hirata, K., Nakane, T., Sugahara, M., Nango, E., Tono, K., Joti, Y., Kameshima, T., Song, C., Hatsui, T., Yabashi, M., Nureki, O., Matsumura, H., Inoue, T., Iwata, S., Mizohata, E. Tags: Regular Papers Source Type: research
PURE mRNA display for in vitro selection of single-chain antibodies
mRNA display is a method to form a covalent linkage between a cell-free synthesized protein (phenotype) and its encoding mRNA (genotype) through puromycin for in vitro selection of proteins. Although a wheat germ cell-free translation system has been previously used in our mRNA display system, a protein synthesis using recombinant elements (PURE) system is a more attractive approach because it contains no endogenous nucleases and proteases and is optimized for folding of antibodies with disulphide bonds. However, when we used the PURE system for mRNA display of single-chain Fv (scFv) antibodies, the formation efficiency of...
Source: Journal of Biochemistry - April 25, 2016 Category: Biochemistry Authors: Nagumo, Y., Fujiwara, K., Horisawa, K., Yanagawa, H., Doi, N. Tags: Regular Papers Source Type: research
Effects of polyamines from Thermus thermophilus, an extreme-thermophilic eubacterium, on tRNA methylation by tRNA (Gm18) methyltransferase (TrmH)
Thermus thermophilus is an extreme-thermophilic eubacterium, which grows at a wide range of temperatures (50–83°C). This thermophile produces various polyamines including long and branched polyamines. In tRNAs from T. thermophilus, three distinct modifications, 2’-O-methylguanosine at position 18 (Gm18), 5-methyl-2-thiouridine at position 54 and N1-methyladenosine at position 58, are assembled at the elbow region to stabilize the L-shaped tRNA structure. However, the structures of unmodified tRNA precursors are disrupted at high temperatures. We hypothesize that polyamine(s) might have a positive effect on ...
Source: Journal of Biochemistry - April 25, 2016 Category: Biochemistry Authors: Hori, H., Terui, Y., Nakamoto, C., Iwashita, C., Ochi, A., Watanabe, K., Oshima, T. Tags: Regular Papers Source Type: research
Capsid protease domain as a tool for assessing protein-domain folding during organelle import of nascent polypeptides in living cells
Various proteins synthesized by ribosomes are imported into specific organelles. To elucidate the behavior of protein domains during import, we developed a folding probe, in which the capsid protease (CP) domain of the Semliki Forest virus was connected to enhanced green fluorescent protein (EGFP). The probe was fused to appropriate N-terminal organelle-targeting signal sequences and expressed in cultured cells. When the entire CP-domain was present in the cytosol, it became folded and cleaved off the following EGFP-domain. Once cleaved, EGFP stability was not affected by upstream sequences. Based on EGFP localization, we ...
Source: Journal of Biochemistry - April 25, 2016 Category: Biochemistry Authors: Kang, K., Takahara, M., Sakaue, H., Sakaguchi, M. Tags: Regular Papers Source Type: research
Microglia in central nervous system repair after injury
Accumulating evidence suggests that immune cells perform crucial inflammation-related functions including clearing dead tissue and promoting wound healing. Thus, they provide a conducive environment for better neuronal regeneration and functional recovery after adult mammalian central nervous system (CNS) injury. However, activated immune cells can also induce secondary damage of intact tissue and inhibit post-injury CNS repair. The inflammation response is due to the microglial production of cytokines and chemokines for the recruitment of peripheral immune cell populations, such as monocytes, neutrophils, dendritic cells ...
Source: Journal of Biochemistry - April 25, 2016 Category: Biochemistry Authors: Jin, X., Yamashita, T. Tags: JB Reviews Source Type: research
Basigin (CD147), a multifunctional transmembrane glycoprotein with various binding partners
Basigin, also called CD147 or EMMPRIN, is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily. Basigin has isoforms; the common form (basigin or basigin-2) has two immunoglobulin domains, and the extended form (basigin-1) has three. Basigin is the receptor for cyclophilins, S100A9 and platelet glycoprotein VI, whereas basigin-1 serves as the receptor for the rod-derived cone viability factor. Basigin tightly associates with monocarboxylate transporters and is essential for their cell surface translocation and activities. In the same membrane plane, basigin also associates with other proteins includi...
Source: Journal of Biochemistry - April 25, 2016 Category: Biochemistry Authors: Muramatsu, T. Tags: JB Reviews Source Type: research
Effects of the N terminus of mouse DNA polymerase {kappa} on the bypass of a guanine-benzo[a]pyrenyl adduct
DNA polymerase (Pol), one of the typical member of the Y-family DNA polymerases, has been demonstrated to bypass the 10S (+)-trans-anti-benzo[a]pyrene diol epoxide-N2-deoxyguanine adducts (BPDE-dG) efficiently and accurately. A large structural gap between the core and little finger as well as an N-clasp domain are essential to its unique translesion capability. However, whether the extreme N-terminus of Pol is required for its activity is unclear. In this work, we constructed two mouse Pol deletions, which have either a catalytic core (mPol1-516) or a core without the first 21-residues (mPol22-516), and tested their acti...
Source: Journal of Biochemistry - March 20, 2016 Category: Biochemistry Authors: Liu, Y., Ma, X., Guo, C. Tags: Regular Papers Source Type: research
Heterologous expression of peptidyl-Lys metallopeptidase of Armillaria mellea and mutagenic analysis of the recombinant peptidase
A method to express, purify and modify the Peptidyl-Lys metallopeptidase (LysN) of Armillaria mellea in Pichia pastoris was developed to enable functional studies of the protease. Based on prior work, we propose a mechanism of action of LysN. Catalytic residues were investigated by site-directed mutagenesis. As anticipated, these mutations resulted in significantly reduced catalytic rates. Additionally, based on molecular modelling eleven mutants were designed to have altered substrate specificity. The S1' binding pocket of LysN is quite narrow and lined with negative charge to specifically accommodate lysine. To allow for...
Source: Journal of Biochemistry - March 20, 2016 Category: Biochemistry Authors: Odum, A. S. R., Ostergaard, S., Norby, I., Meldal, M., Olesen, K. Tags: Regular Papers Source Type: research
Purification and molecular cloning of aspartic proteinases from the stomach of adult Japanese fire belly newts, Cynops pyrrhogaster
Six aspartic proteinase precursors, a pro-cathepsin E (ProCatE) and five pepsinogens (Pgs), were purified from the stomach of adult newts (Cynops pyrrhogaster). On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the molecular weights of the Pgs and active enzymes were 37–38 kDa and 31–34 kDa, respectively. The purified ProCatE was a dimer whose subunits were connected by a disulphide bond. cDNA cloning by polymerase chain reaction and subsequent phylogenetic analysis revealed that three of the purified Pgs were classified as PgA and the remaining two were classified as PgBC belonging to C-type Pg. Our...
Source: Journal of Biochemistry - March 20, 2016 Category: Biochemistry Authors: Nagasawa, T., Sano, K., Kawaguchi, M., Kobayashi, K.-i., Yasumasu, S., Inokuchi, T. Tags: Regular Papers Source Type: research
Characterization of the 5-flanking region of the human TP53 gene and its response to the natural compound, Resveratrol
In this study, we constructed a Luciferase expression vector, pGL4-TP53-551, containing 551 bp of the 5’-upstream region of the human TP53 gene, which was then transfected into HeLa S3 cells. A Luciferase assay showed that Rsv treatment increased the promoter activity of the TP53 gene in comparison to that of PIF1. Detailed deletion and mutation analyses revealed that Nkx-2.5 and E2F-binding elements are required in addition to duplicated GGAA (TTCC), for the regulation of TP53 promoter activity. In this study, it is suggested that the transient induction of TP53 gene expression by Rsv treatment might be partly invol...
Source: Journal of Biochemistry - March 20, 2016 Category: Biochemistry Authors: Uchiumi, F., Shoji, K., Sasaki, Y., Sasaki, M., Sasaki, Y., Oyama, T., Sugisawa, K., Tanuma, S.-I. Tags: Regular Papers Source Type: research
