Formation of tau inclusions in knock-in mice with familial Alzheimer disease (FAD) mutation of presenilin 1 (PS1)

Mutations in the presenilin 1 (PS1) gene are responsible for the early onset of familial Alzheimer disease (FAD). Accumulating evidence shows that PS1 is involved in gamma-secretase activity and that FAD-associated mutations of PS1 commonly accelerate Abeta(1-42) production, which causes Alzheimer disease (AD). Recent studies suggest, however, that PS1 is involved not only in Abeta production but also in other processes that lead to neurodegeneration. To better understand the causes of neurodegeneration linked to the PS1 mutation, we analyzed the development of tau pathology, another key feature of AD, in PS1 knock-in mice. Hippocampal samples taken from FAD mutant (I213T) PS1 knock-in mice contained hyperphosphorylated tau that reacted with various phosphodependent tau antibodies and with Alz50, which recognizes the conformational change of PHF tau. Some neurons exhibited Congo red birefringence and Thioflavin T reactivity, both of which are histological criteria for neurofibrillary tangles (NFTs). Biochemical analysis of the samples revealed SDS-insoluble tau, which under electron microscopy examination, resembled tau fibrils. These results indicate that our mutant PS1 knock-in mice exhibited NFT-like tau pathology in the absence of Abeta deposition, suggesting that PS1 mutations contribute to the onset of AD not only by enhancing Abeta(1-42) production but by also accelerating the formation and accumulation of filamentous tau.     

Cu,Zn-superoxide dismutase is an intracellular catalyst for the H(2)O(2)-dependent oxidation of dichlorodihydrofluorescein

Dichlorodihydrofluorescein (DCFH(2)) is a widely used probe for intracellular H(2)O(2). However, H(2)O(2) can oxidize DCFH(2) only in the presence of a catalyst, whose identity in cells has not been clearly defined. We compared the peroxidase activity of Cu,Zn-superoxide dismutase (CuZnSOD), cytochrome c, horseradish peroxidase (HRP), Cu(2+), and Fe(3+) under various condi-tions to identify an intracellular catalyst. Enormous increase by bicarbonate in the rate of DCFH(2) oxidation distinguished CuZnSOD from cytochrome c and HRP. Cyanide inhibited the reaction catalyzed by CuZnSOD but accelerated that by Cu(2+) and Fe(3+). Oxidation of DCFH(2) by H(2)O(2) in the presence of a cell lys-ate was also enhanced by bicarbonate and inhibited by cyanide. Confocal microscopy of H(2)O(2)-treated cells showed enhanced DCF fluorescence in the presence of bicarbonate and attenuated fluorescence for the cells pre-incubated with KCN. Moreover, DCF fluorescence was intensified in CuZnSOD-transfected HaCaT and RAW 264.7 cells. We propose that CuZnSOD is a potential intracellular catalyst for the H(2)O(2)-dependent oxidation of DCFH(2).     

Isolation of mutations that bypass the requirement of the septation initiation network for septum formation and conidiation in Aspergillus nidulans

The kinase cascade of the septation initiation network (SIN), first revealed in fission yeast, activates the contraction of the actomyosin ring, and plays an essential role in fungal septation. Mob1p, an evolutionarily conserved SIN protein, is associated with the most downstream kinase of this cascade in fission yeast. In this study, the mobA gene encoding a homologous protein was isolated from the filamentous fungus Aspergillus nidulans, whose mycelium is made of multinucleate cells. The MOBA protein was required for septation and conidiation, but was not essential for hyphal extension and colony formation. To identify genes that act antagonistically against the SIN, UV mutagenesis was carried out to isolate suppressor (smo) mutations that restored conidiation when MOBA was not expressed. Microscopic examination indicated that the restored conidiation was concomitant with restored septation in the absence of the MOBA protein. Eight recessive smo mutations in five complementation groups also bypassed the requirement of the SIN kinases SEPH and SIDB for septum formation and conidiation. However, none of these smo mutations affected the localization of MOBA. Among smo mutations, smoA and smoB mutations caused reduced hyphal growth and colony formation. They also rendered hypersensitivity to low doses of the microtubule-depolymerizing agent benomyl for conidiation. Therefore, in A. nidulans, proteins encoded by the smo genes likely have an antagonistic interaction against the SIN pathway to regulate septation and conidiation.     

Cyclosporin A does not increase the oxidative susceptibility of low density lipoprotein in vitro

Accelerated atherosclerosis is the leading cause of morbidity in renal transplant recipients. The pathogenic mechanisms responsible for the progression of atherosclerosis in renal transplant recipients have not been elucidated. Cyclosporin A (CsA) is an immunosuppressive agent used post-transplant and may contribute to increased oxidative susceptibility of low density lipoprotein (LDL). There is a paucity of data testing the effect of CsA on LDL oxidation. Hence, the aim of this study was to test the effect of in vitro enrichment of LDL with CsA on LDL oxidation. LDL oxidation in presence of different concentrations of CsA was tested using metal-dependent (copper), metal-independent (AAPH) and cell-mediated (macrophages) oxidation systems. In all 3 systems, CsA had no significant effect on LDL oxidation. Also, pre-incubation of LDL with CsA did not affect LDL oxidation and LDL alpha tocopherol levels. Thus, the results of our studies with CsA indicate that it is not a direct pro-oxidant.     

Syntheses and biological evaluation of 1-heteroaryl-2-aryl-1H-benzimidazole derivatives as c-Jun N-terminal kinase inhibitors with neuroprotective effects

1-Heteroaryl-2-aryl-1H-benzimidazole derivatives were synthesized as inhibitors of c-Jun N-terminal kinases, JNK3. Their activities were evaluated through measurement of K_d using SPR, JNK3 kinase assay, and cell-viability of human neuroblastoma cells. Most tested compounds showed high affinity (10 μM–46 nM) to JNK3. Among them, compound 16f exhibited potent activities (K_d = 46 nM). Especially, 16f was also found to present a potent cell protective effect (IC₅₀ = 1.09 μM) against toxicity induced by anisomycin, showing a possibility as protective therapeutics in neuronal cell apoptosis.     

Plasmodium vivax: collaborative roles for plasmepsin 4 and vivapains in hemoglobin hydrolysis

Plasmepsins, a family of aspartic proteases of Plasmodium species, are known to participate in a wide variety of cellular processes essential for parasite survival. Therefore, the plasmepsins of malaria parasites have been recognized as attractive antimalarial drug targets. Although the plasmepsins of P. falciparum have been extensively characterized, the plasmepsins of P. vivax are currently not well known. To expand our understanding of the plasmepsins of P. vivax, we characterized plasmepsin 4 of P. vivax (PvPM4). The bacterially expressed recombinant PvPM4 was insoluble, but it was easily refolded into a soluble protein. The processing of PvPM4 into a mature enzyme occurred through autocatalytic activity under acidic conditions in a pepstatin A-sensitive manner, in which process a portion of prodomain was essential for correct folding. PvPM4 could hydrolyze native human hemoglobin at acidic pHs, but preferred denatured hemoglobin as a substrate. PvPM4 acted synergistically with vivapain-2 and vivapain-3, cysteine proteases of P. vivax, in the hydrolysis of hemoglobin. The vivapains also mediated processing of PvPM4 into a mature enzyme. These results collectively suggest that PvPM4 is an active hemoglobinase of P. vivax that works collaboratively with vivapains to enhance the parasite’s ability to hydrolyze hemoglobin.     

Structures of iron-dependent alcohol dehydrogenase 2 from Zymomonas mobilis ZM4 with and without NAD+ cofactor

The ethanologenic bacterium Zymomonas mobilis ZM4 is of special interest because it has a high ethanol yield. This is made possible by the two alcohol dehydrogenases (ADHs) present in Z. mobilis ZM4 (zmADHs), which shift the equilibrium of the reaction toward the synthesis of ethanol. They are metal-dependent enzymes: zinc for zmADH1 and iron for zmADH2. However, zmADH2 is inactivated by oxygen, thus implicating zmADH2 as the component of the cytosolic respiratory system in Z. mobilis. Here, we show crystal structures of zmADH2 in the form of an apo-enzyme and an NAD⁺-cofactor complex. The overall folding of the monomeric structure is very similar to those of other functionally related ADHs with structural variations around the probable substrate and NAD⁺ cofactor binding region. A dimeric structure is formed by the limited interactions between the two subunits with the bound NAD⁺ at the cleft formed along the domain interface. The catalytic iron ion binds near to the nicotinamide ring of NAD⁺, which is likely to restrict and locate the ethanol to the active site together with the oxidized Cys residue and several nonpolar bulky residues. The structures of the zmADH2 from the proficient ethanologenic bacterium Z. mobilis, with and without NAD⁺ cofactor, and modeling ethanol in the active site imply that there is a typical metal-dependent catalytic mechanism.     

Overall Prevalence and Distribution of Knockdown Resistance (kdr) Mutations in Aedes aegypti from Mandalay Region,Myanmar

Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) of mosquitoes confer resistance to insecticides. Although insecticide resistance has been suspected to be widespread in the natural population of Aedes aegypti in Myanmar, only limited information is currently available. The overall prevalence and distribution of kdr mutations was analyzed in Ae. aegypti from Mandalay areas, Myanmar. Sequence analysis of the VGSC in Ae. aegypti from Myanmar revealed amino acid mutations at 13 and 11 positions in domains II and III of VGSC, respectively. High frequencies of S989P (68.6%), V1016G (73.5%), and F1534C (40.1%) were found in domains II and III. T1520I was also found, but the frequency was low (8.1%). The frequency of S989P/V1016G was high (55.0%), and the frequencies of V1016G/F1534C and S989P/V1016G/F1534C were also high at 30.1% and 23.5%, respectively. Novel mutations in domain II (L963Q, M976I, V977A, M994T, L995F, V996M/A, D998N, V999A, N1013D, and F1020S) and domain III (K1514R, Y1523H, V1529A, F1534L, F1537S, V1546A, F1551S, G1581D, and K1584R) were also identified. These results collectively suggest that high frequencies of kdr mutations were identified in Myanmar Ae. aegypti, indicating a high level of insecticide resistance.