Conserved Acidic Amino Acid Residues in a Second RNA Recognition Motif Regulate Assembly and Function of TDP-43

Accumulating evidence suggests that pathogenic TAR DNA-binding protein (TDP)-43 fragments contain a partial RNA-recognition motif domain 2 (RRM2) in amyotrophic lateral sclerosis (ALS)/frontotemporal lobar degeneration. However, the molecular basis for how this domain links to the conformation and function of TDP-43 is unclear. Previous crystal analyses have documented that the RRM2-DNA complex dimerizes under acidic and high salt conditions, mediated by the intermolecular hydrogen bonds of Glu246-Ile249 and Asp247-Asp247. The aims of this study were to investigate the roles of Glu246 and Asp247 in the molecular assembly of RRM2 under physiological conditions, and to evaluate their potential use as markers for TDP-43 misfolding due to the aberrantly exposed dimer interface. Unexpectedly, gel filtration analyses showed that, regardless of DNA interaction, the RRM2 domain remained as a stable monomer in phosphate-buffered saline. Studies using substitution mutants revealed that Glu246 and, especially, Asp247 played a crucial role in preserving the functional RRM2 monomers. Substitution to glycine at Glu246 or Asp247 induced the formation of fibrillar oligomers of RRM2 accompanied by the loss of DNA-binding affinity, which also affected the conformation and the RNA splicing function of full-length TDP-43. A novel monoclonal antibody against peptides containing Asp247 was found to react with TDP-43 inclusions of ALS patients and mislocalized cytosolic TDP-43 in cultured cells, but not with nuclear wild-type TDP-43. Our findings indicate that Glu246 and Asp247 play pivotal roles in the proper conformation and function of TDP-43. In particular, Asp247 should be studied as a molecular target with an aberrant conformation related to TDP-43 proteinopathy.     

Hybrid Transverse Magneto-Thermoelectric Cooling in Artificially Tilted Multilayers

In artificially tilted multilayers comprising two different conductors that are alternately and obliquely stacked, transverse thermoelectric conversion occurs, in which charge and heat currents are interconverted in the orthogonal direction. Although transverse thermoelectric conversion also occurs in homogeneous materials as an intrinsic transport phenomenon owing to the effects of magnetic fields, magnetization, and spins on conduction carriers, such magneto-thermoelectric effects are investigated independently of thermoelectrics for artificially tilted multilayers. Here, this study shows that the synergy of these different principles improves the performance of transverse thermoelectric conversion. Using lock-in thermography techniques, transverse thermoelectric conversion processes are visualized in artificially tilted multilayers and the experiments clarify how nonuniform charge currents are converted into orthogonal heat currents. Through the measurements of temperature change under magnetic fields, the contributions of the magneto-thermoelectric effects are quantified in the artificially tilted multilayers and magnetically enhanced hybrid transverse thermoelectric cooling is demonstrated. By replacing one of the conductors in the multilayer with permanent magnets, the same functionality is obtained even in the absence of magnetic fields, paving the way for the creation of “thermoelectric permanent magnets” that exhibit efficient transverse thermoelectric conversion together with spontaneous magnetization. This study provides a new material design guideline for transverse thermoelectrics.     

Mutated C-terminal fragments of Clostridium perfringens enterotoxin have increased affinity to claudin-4 and reversibly modulate tight junctions in vitro

Passage across epithelial cell sheets is the first step in drug absorption. Tight junctions (TJs) are located between adjacent epithelial cells and seal the intercellular space preventing leakage of solutes. Claudin, a tetra-transmembrane protein family, is a pivotal functional and structural component of the TJ barrier. Modulation of the claudin-based TJ seal is a strategy for mucosal drug absorption. We previously found that a claudin-4 binder, a C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE194), was a modulator of the TJ seal and a potent mucosal absorption enhancer. In the present study, we attempted to improve claudin-4 binders by modification of C-CPE194. Substitution of Asn at position 309 and Ser at position 313 with Ala increased the affinity to claudin-4 by 9.9-fold as compared to C-CPE194. Deletion of 10 amino acids in the N-terminal domain of the double-alanine-substituted mutant increased affinity to claudin-4 by 23.9-fold as compared to C-CPE194. These C-CPE194 mutants reversibly modulated the TJ seal in human intestinal epithelial cell sheets. The N-terminal-truncated mutant was the most potent modulator of the TJ seal. These findings indicate that the C-CPE mutant may be a promising lead for the development of a clinical TJ modulator.     

Antimicrobial susceptibility and genetic relatedness of bovine Stenotrophomonas maltophilia isolates from a mastitis outbreak

Aims: To evaluate the antimicrobial susceptibility and genetic relatedness of 11 Stenotrophomonas maltophilia isolates from an outbreak of bovine clinical mastitis in one herd and two isolates from two separate mastitis cases in two other herds. Methods and Results: Thirteen S. maltophilia isolates were obtained from milk samples from 11 cows from three dairy herds in Japan during 2008. We tested their susceptibility to 14 antimicrobials by broth microdilution and identified their genotypes by enterobacterial repetitive intergenic consensus 2 (ERIC2)‐PCR. Every cow had acute mild mastitis (slightly watery foremilk with flakes) without systemic symptoms and all resolved within 3–5 weeks of diagnosis. Eleven of the 13 isolates derived from nine cows in one herd over a 7‐month period exhibited a closely related ERIC2 type (A). The remaining two isolates derived from two cows from two other herds exhibited two distinct ERIC2 types (B and C). Most of the 13 isolates exhibited susceptibility to trimethoprim‐sulfamethoxazole, chloramphenicol, minocycline and levofloxacin; however, they were resistant to four β‐lactams, kanamycin, gentamicin and oxytetracycline. They were intermediate to enrofloxacin. Conclusions: Eleven closely related S. maltophilia isolates were involved in a herd outbreak of mastitis to some extent. Bovine S. maltophilia isolates exhibited resistance to many classes of antimicrobials. Significance and impact of study: This is a rare report of a herd outbreak of bovine mastitis involving closely related S. maltophilia isolates.     

Structural basis of protein-bound endogenous aldehydes. Chemical and immunochemical characterizations of configurational isomers of a 4-hydroxy-2-nonenal-histidine adduct

4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centers in the cyclic hemiacetal structure. In the present study, we characterized configurational isomers of a HNE-N(alpha)-acetylhistidine adduct by NMR spectroscopy and by molecular orbital calculations. In addition, we raised monoclonal antibodies against (R)-HNE-histidine and (S)-HNE-histidine adducts, characterized their specificities, and examined in vivo localizations of each adduct under oxidative stress. To facilitate structural characterization of the configurational isomers of an HNE-histidine adduct, we prepared the (R)-HNE-histidine and (S)-HNE-histidine adducts by incubating N(alpha)-acetylhistidine with each HNE enantiomer, both of which provided two peaks (Ra and Rb from (R)-HNE-histidine and Sa and Sb from (S)-HNE-histidine adducts) in reversed-phase high-performance liquid chromatography. The NMR analysis showed that each peak was a mixture of two diastereomers. In addition, the analysis of the nuclear Overhauser effect enabled the determination of configurations of the eight isomers. The relative amounts of these isomers in the NMR analysis correlated with the relative energies calculated by molecular orbital methods. On the other hand, using (R)-HNE-modified and (S)-HNE-modified keyhole limpet hemocyanins as the antigens, we raised the monoclonal antibodies, mAbR310 and mAbS412, which enantioselectively recognized the (R)-HNE-histidine and (S)-HNE-histidine adducts, respectively. Among the mixtures (Ra, Rb, Sa, and Sb) of diastereomers, mAbR310 showed the highest immunoreactivity to Rb (the mixture of 2R,4S,5R and 2S,4S,5R isomers), whereas mAbS412 preferentially recognized Sa (the mixture of 2R,4S,5S and 2S,4S,5S isomers). The presence of (R)-HNE and (S)-HNE epitopes in vivo was immunohistochemically examined in the kidney of rats exposed to the renal carcinogen, ferric nitrilotriacetate, by which nuclear and cytosolic stainings with mAbR310 and mAbS412, respectively, were detected.     

A Novel Method for the Synthesis of ddA and F-ddA Via Regioselective 2′-O-Deacetylation of 9-(2,5-DI-O-Acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)adenine

Abstract

Regioselective 2′-O-deacetylation of 9-(2,5-di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)adenine (1) is achieved by treatment of 1 with β-cyclodextrin (β-CyD) / aq. NaHCO₃ or N₂H₄·H₂O / EtOH. The 9-(5-O-Acetyl-3-bromo-3-deoxy-β-D-xylo-furanosyl)adenine (2) obtained is a common intermediate for the synthesis of 2′,3′-dideoxy-adenosine (ddA) (7) and 9-(2-fluoro-2,3-dideoxy-β-D-threo-pentofuranosyl)-adenine (F-ddA) (9).     

Immunohistochemical Localization of Manserin,a Novel Neuropeptide Derived from Secretogranin II,in Rat Adrenal Gland,and its Upregulation by Physical Stress

We recently identified a novel 40-amino acid neuropeptide designated manserin from the rat brain (Yajima in NeuroReport 15: 1755–1759, 2004). Manserin is highly expressed in pituitary and hypothalamic nuclei, which suggests that it plays a role in the endocrine system. In this study, we employed immunohistochemical methods to investigate the presence of manserin in rat adrenal glands, as well as its regulation by physical stress. Immunohistochemical analysis using anti-manserin antibody showed that manserin is present in the rat adrenal medulla but not in the cortex. When the colocalization of manserin and phenylethanolamine N-methyltransferase (PNMT), an epinephrine-synthesizing enzyme, was examined, virtually all PNMT-positive cells expressed manserin. Interestingly, the immunoreactivity of manserin was significantly increased when the rats were exposed to water-immersion restraint stress. These results demonstrate for the first time that adrenal manserin, a novel neuropeptide, may have a potential physiological role under stress-inducing conditions.