Characterization of a murine gene encoding an acidic-basic dipeptide repeat that interacts with GADD34

GADD34 is one of a subset of proteins induced after DNA damage or cell growth arrest. To examine the function of GADD34, we used the yeast two-hybrid system to clone the protein that interacts with murine GADD34. We utilized as bait the partial product of GADD34 cDNA including the PEST region and the gamma(1)34.5. One cDNA clone was almost the same as MuRED, which encodes an acidic-basic dipeptide repeat; we named it G34BP. The interaction between GADD34 and G34BP was also confirmed in the NIH3T3 cells by in vivo two-hybrid analysis. For the binding of two proteins, the PEST region was important, and the C-terminal of G34BP was necessary. G34BP was detected in all the mouse tissues examined. Although GADD34 was significantly elevated with methyl methanesulfonate treatment, G34BP expression was not induced. Overexpression of G34BP in the NIH3T3 cells inhibited the cell growth analyzed by WST1 assay.     

Label-free quantitative proteomics using large peptide data sets generated by nanoflow liquid chromatography and mass spectrometry

We developed an integrated platform consisting of machinery and software modules that can apply vast amounts of data generated by nanoflow LC-MS to differential protein expression analyses. Unlabeled protein samples were completely digested with modified trypsin and separated by low speed (200 nl/min) one-dimensional HPLC. Mass spectra were obtained every 1 s by using the survey mode of a hybrid Q-TOF mass spectrometer and displayed in a two-dimensional plane with m/z values along the x axis, and retention time was displayed along the y axis. The time jitter of nano-LC was adjusted using newly developed software based on a dynamic programming algorithm. The comprehensiveness (60,000-160,000 peaks above the predetermined threshold detectable in 60-microg cell protein samples), reproducibility (average coefficient of variance of 0.35-0.39 and correlation coefficient of over 0.92 between duplicates), and accurate quantification with a wide dynamic range (over 10(3)) of our platform warrant its application to various types of experimental and translational proteomics.     

Production of anti-self-H-2 antibodies by C3D2F1 mice hyperimmune to L cell/L1210 hybrids and L1210 leukemia cells

During the course of immunization of (C3H × DBA/2)F₁ mice (genotype H-2^k/b) with L cell (H-2^k/k)/L1210 leukemia cell (H-2^d/d) hybrids and L1210 leukemia cells, some of them produced a good titer of anti-self-H-2 (H-2^d) antibodies. Antigens recognized by this anti-self-H-2 antiserum were shown to be controlled by the H-2K-IA-IB-IJ-IE subregions of the H-2^d but not H-2^k nor H-2^b haplotypes of parental as well as F₁ origins and to have a tissue distribution identical to that of class 1 H-2 (H-2K/D) antigens.     

Glucose-dependent insulinotropic polypeptide receptor null mice exhibit compensatory changes in the enteroinsular axis

The incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are gut hormones that act via the enteroinsular axis to potentiate insulin secretion from the pancreas in a glucose-dependent manner. Both GLP-1 receptor and GIP receptor knockout mice (GLP-1R(-/-) and GIPR(-/-), respectively) have been generated to investigate the physiological importance of this axis. Although reduced GIP action is a component of type 2 diabetes, GIPR-deficient mice exhibit only moderately impaired glucose tolerance. The present study was directed at investigating possible compensatory mechanisms that take place within the enteroinsular axis in the absence of GIP action. Although serum total GLP-1 levels in GIPR knockout mice were unaltered, insulin responses to GLP-1 from pancreas perfusions and static islet incubations were significantly greater (40-60%) in GIPR(-/-) than in wild-type (GIPR(+/+)) mice. Furthermore, GLP-1-induced cAMP production was also elevated twofold in the islets of the knockout animals. Pancreatic insulin content and gene expression were reduced in GIPR(-/-) mice compared with GIPR(+/+) mice. Paradoxically, immunocytochemical studies showed a significant increase in beta-cell area in the GIPR-null mice but with less intense staining for insulin. In conclusion, GIPR(-/-) mice exhibit altered islet structure and topography and increased islet sensitivity to GLP-1 despite a decrease in pancreatic insulin content and gene expression.     

Design, synthesis, and biological activity of non-basic compounds as factor Xa inhibitors: SAR study of S1 and aryl binding sites

Compound 7 was identified as the active metabolite of 6 by HPLC and mass spectral analysis. Modification of lead compound 7 by transformation of its N-oxide 6-6 biaryl ring system and fused aromatics produced a series of non-basic fXa inhibitors with excellent potency in anti-fXa and anticoagulant assays. The optimized compounds 73b and 75b showed sub to one digit micromolar anticoagulant activity (PTCT2). Particularly, anti-fXa activity was detected in plasma of rats orally administered with 1mg/kg of compound 75b.     

Physiological characterization of anaerobic ammonium oxidizing bacterium ‘Candidatus Jettenia caeni’

To date, six candidate genera of anaerobic ammonium‐oxidizing (anammox) bacteria have been identified, and numerous studies have been conducted to understand their ecophysiology. In this study, we examined the physiological characteristics of an anammox bacterium in the genus ‘Candidatus Jettenia’. Planctomycete KSU‐1 was found to be a mesophilic (20–42.5°C) and neutrophilic (pH 6.5–8.5) bacterium with a maximum growth rate of 0.0020 h^?1. Planctomycete KSU‐1 cells showed typical physiological and structural features of anammox bacteria; i.e. ²⁹N₂ gas production by coupling of ¹⁵NH₄⁺ and ¹⁴NO₂^?, accumulation of hydrazine with the consumption of hydroxylamine and the presence of anammoxosome. In addition, the cells were capable of respiratory ammonification with oxidation of acetate. Notably, the cells contained menaquinone‐7 as a dominant respiratory quinone. Proteomic analysis was performed to examine underlying core metabolisms, and high expressions of hydrazine synthase, hydrazine dehydrogenase, hydroxylamine dehydrogenase, nitrite/nitrate oxidoreductase and carbon monoxide dehydrogenase/acetyl‐CoA synthase were detected. These proteins require iron or copper as a metal cofactor, and both were dominant in planctomycete KSU‐1 cells. On the basis of these experimental results, we proposed the name ‘Ca. Jettenia caeni’ sp. nov. for the bacterial clade of the planctomycete KSU‐1.     

Cloning and characterisation of a peroxiredoxin from the swine roundworm Ascaris suum

Antioxidant enzymes in parasites play an important role in protection against the oxygen radicals by generating during aerobic metabolism, as well as in defence against host immune cell assault. Here we report the cloning and characterisation of a cDNA encoding peroxiredoxin from Ascaris suum (AsPrx). AsPrx is 776bp long and contains the nematode 22bp splice leader sequence at the 5' end and polyadenylation signal followed by poly(A) tail at the 3' end. AsPrx codes a full-length protein with a predicted molecular mass of 22. 6kDa, and possesses two cysteine residues at amino acid 49 and 168 that are conserved among Prx proteins. GenBank() analysis showed that the deduced amino acid sequence had significant similarity to parasite and mammalian Prx at the amino acid level. DNA nicking revealed that Escherichia coli-expressed recombinant AsPrx (rAsPrx) is enzymatically inhibited to form oxidative-nicking of supercoiled plasmid DNA. Two-dimensional immunoblot analysis with mouse anti-rAsPrx serum reacted two major constituent protein spots in extracts of adult female worms, suggesting that the native AsPrx might be function as a major antioxidant enzyme in Ascaris suum.     

Tyrosinase‐catalyzed oxidation of resveratrol produces a highly reactive ortho‐quinone: Implications for melanocyte toxicity

trans‐Resveratrol (3,5,4′‐trihydroxy‐trans‐stilbene, RES), a naturally occurring polyphenol, has recently attracted increased interest as a health‐beneficial agent. However, based on its p‐substituted phenol structure, RES is expected to be a substrate for tyrosinase and to produce a toxic o‐quinone metabolite. The results of this study demonstrate that the oxidation of RES by tyrosinase produces 4‐(3′,5′‐dihydroxy‐trans‐styrenyl)‐1,2‐benzoquinone (RES‐quinone), which decays rapidly to an oligomeric product (RES‐oligomer). RES‐quinone was identified after reduction to its corresponding catechol, known as piceatannol. RES‐quinone reacts with N‐acetylcysteine, a small thiol, to form a diadduct and a triadduct, which were identified by NMR and MS analyses. The production of a triadduct is not common for o‐quinones, suggesting a high reactivity of RES‐quinone. RES‐quinone also binds to bovine serum albumin through its cysteine residue. RES‐oligomer can oxidize GSH to GSSG, indicating its pro‐oxidant activity. These results suggest that RES could be cytotoxic to melanocytes due to the binding of RES‐quinone to thiol proteins.