Hydrogen-deuterium (H/D) exchange mapping of Abeta 1-40 amyloid fibril secondary structure using nuclear magnetic resonance spectroscopy

We describe here details of the hydrogen-deuterium (H/D) exchange behavior of the Alzheimer's peptide Abeta(1)(-)(40), while it is a resident in the amyloid fibril, as determined by high-resolution solution NMR. Kinetics of H/D exchange in Abeta(1)(-)(40) fibrils show that about half the backbone amide protons exchange during the first 25 h, while the other half remain unexchanged because of solvent inaccessibility and/or hydrogen-bonded structure. After such a treatment for 25 h with D(2)O, fibrils of (15)N-enriched Abeta were dissolved in a mixture of 95% dimethyl sulfoxide (DMSO) and 5% dichloroacetic acid (DCA) and successive heteronuclear (1)H-(15)N HSQC spectra were collected to identify the backbone amides that did not exchange in the fibril. These studies showed that the N and C termini of the peptide are accessible to the solvent in the fibril state and the backbone amides of these residues are readily exchanged with bulk deuterium. In contrast, the residues in the middle of the peptide (residues 16-36) are mostly protected, suggesting that that many of the residues in this segment of the peptide are involved in a beta structure in the fibril. Two residues, G25 and S26, exhibit readily exchangeable backbone amide protons and therefore may be located on a turn or a flexible part of the peptide. Overall, the data substantially supports current models for how the Abeta peptide folds when it engages in the amyloid fibril structure, while also addressing some discrepancies between models.     

The solution structure of the N-terminal domain of human vitronectin: proximal sites that regulate fibrinolysis and cell migration

The three-dimensional structure of an N-terminal fragment comprising the first 51 amino acids from human plasma vitronectin, the somatomedin B (SMB) domain, has been determined by two-dimensional NMR approaches. An average structure was calculated, representing the overall fold from a set of 20 minimized structures. The core residues (18-41) overlay with a root mean square deviation of 2.29 +/- 0.62 A. The N- and C-terminal segments exhibit higher root mean square deviations, reflecting more flexibility in solution and/or fewer long-range NOEs for these regions. Residues 26-30 form a unique single-turn alpha-helix, the locus where plasminogen activator inhibitor type-1 (PAI-1) is bound. This structure of this helix is highly homologous with that of a recombinant SMB domain solved in a co-crystal with PAI-1 (Zhou, A., Huntington, J. A., Pannu, N. S., Carrell, R. W., and Read, R. J. (2003) Nat. Struct. Biol. 10, 541-544), although the remainder of the structure differs. Significantly, the pattern of disulfide cross-links observed in this material isolated from human plasma is altogether different from the disulfides proposed for recombinant forms. The NMR structure reveals the relative orientation of binding sites for cell surface receptors, including an integrin-binding site at residues 45-47, which was disordered and did not diffract in the co-crystal, and a site for the urokinase receptor, which overlaps with the PAI-1-binding site.     

Beneficial role of aminoguanidine on acute cardiomyopathy related to doxorubicin-treatment

Doxorubicin (DOX) is a broad-spectrum anthracycline antibiotic that has cardiotoxicity as a major side effect. One mechanism of this toxicity is believed to involve the reactive oxygen radical species (ROS); these agents likely account for the pathophysiology of DOX-induced cardiomyopathy. Aminoguanidine (AG) is an effective antioxidant and free radical scavenger which has long been known to protect against ROS formation. We investigated the effects of AG on DOX-induced changes in thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) content. The rats were divided into four groups:1) Control; 2) DOX group; injected intraperitoneally (i.p.) with DOX 20 mg/kg in a single dose 3) AG-treated group; injected i.p. in single dose of 20 mg/kg DOX plus 100 mg/kg AG 1 h before the DOX for 3 days, 4) AG group; injected i.p. with AG 100 mg/kg for 3 days. DOX administration to control rats increased TBARS and decreased GSH levels. AG administration before DOX injection caused significant decrease in TBARS and increase in GSH levels in the heart tissue when compared with DOX only. Morphological changes, including severe myocardial fibrosis and inflammatory cell infiltration were clearly observed in the DOX-treated heart. AG reversed the DOX-induced heart damage. Therefore AG could protect the heart tissue against free radical injury. The application of AG during cancer chemotherapy may attenuate tissue damage and improve the therapeutic index of DOX.     

Molecular determinants of affinity for aminoglycoside binding to the aminoglycoside nucleotidyltransferase(2')-Ia

One of the most commonly occurring aminoglycoside resistance enzymes is aminoglycoside 2'-O-nucleotidyltransferase [ANT(2')]. In the present study molecular determinants of affinity and specificity for aminoglycoside binding to this enzyme are investigated using isothermal titration calorimetry (ITC). Binding of aminoglycosides is enthalpically driven accompanied by negative entropy changes. The presence of metal-nucleotide increases the affinity for all but one of the aminoglycosides studied but has no effect on specificity. The substituents at positions 1, 2', and 6' are important determinants of substrate specificity. An amino group at these positions leads to greater affinity. No correlation is observed between the change in affinity and enthalpy. At the 2' position greater affinity results from a more negative enthalpy for an aminoglycoside containing an amino rather than a hydroxyl at that position. At the 6' position the greater affinity for an aminoglycoside containing an amino substituent results from a less disfavorable entropic contribution. The thermodynamic basis for the change in affinity at position 1 could not be determined because of the weak binding of one of the aminoglycoside substrates, amikacin. The effect of increasing osmotic stress on affinity was used to determine that a net release of approximately four water molecules occurs when tobramycin binds to ANT(2'). No measurable net change in the number of bound water molecules is observed when neomycin binds the enzyme. Data acquired in this work provide the rationale for the ability of ANT(2') to confer resistance against kanamycins but not neomycins.     

Not4 enhances JAK/STAT pathway-dependent gene expression in Drosophila and in human cells

The JAK/STAT pathway is essential for organogenesis, innate immunity, and stress responses in Drosophila melanogaster. The JAK/STAT pathway and its associated regulators have been highly conserved in evolution from flies to humans. We have used a genome-wide RNAi screen in Drosophila S2 cells to identify regulators of the JAK/STAT pathway, and here we report the characterization of Not4 as a positive regulator of the JAK/STAT pathway. Overexpression of Not4 enhanced Stat92E-mediated gene responses in vitro and in vivo in Drosophila. Specifically, Not4 increased Stat92E-mediated reporter gene activation in S2 cells; and in flies, Not4 overexpression resulted in an 8-fold increase in Turandot M (TotM) and in a 4-fold increase in Turandot A (TotA) stress gene activation when compared to wild-type flies. Drosophila Not4 is structurally related to human CNOT4, which was found to regulate interferon-γ- and interleukin-4-induced STAT-mediated gene responses in human HeLa cells. Not4 was found to coimmunoprecipitate with Stat92E but not to affect tyrosine phosphorylation of Stat92E in Drosophila cells. However, Not4 is required for binding of Stat92E to its DNA recognition sequence in the TotM gene promoter. In summary, Not4/CNOT4 is a novel positive regulator of the JAK/STAT pathway in Drosophila and in humans.     

Association between ABCB1 gene polymorphisms and fentanyl's adverse effects in Turkish patients undergoing spinal anesthesia

The ATP-binding cassette transporter (ABCB1) gene product, P-glycoprotein plays an important role in the prevention of intracellular accumulation of potentially toxic substances and metabolites in various tissues. Single nucleotide polymorphisms in this gene are claimed to be correlated with changes in the function of P-glycoprotein. There is evidence that fentanyl, may be a substrate for P-glycoprotein. The aim of the study was to assess whether an association exists between ABCB1 gene polymorphism and early respiratory and sedative adverse effects of intravenous fentanyl in Turkish patients who underwent spinal anesthesiaIn all 83 unrelated Turkish patients were enrolled in this study. In this study, spinal anesthesia was provided and a single dose of intravenous fentanyl (2.5 μg.kg⁻¹) at the beginning of surgery was used as a sedative agent. Bispectral index, respiration rate and peripheral oxygen saturation were measured continuously and recorded throughout the study.The allele and genotype frequencies were similar to previous data from Turkish population.Respiratory rate (RR) and SpO₂ parameters of the patients did not show any significant difference according to the genotype distribution for C1236T and C3435T SNPs. Fentanyl-induced decrease in respiration rate was most remarkable at 15 min (23%) in CC genotype of C1236T, whereas in TT genotype of C3435T (18%) polymorphism. SpO₂ parameters in allele distribution were also not significant among the groups (p = 0.374, p = 0.985, respectively). For the C1236T polymorphism, patients carrying T allele showed a significant decrease in pH, and a significant increase in pCO₂ (p < 0.001).ABCB1 polymorphisms did not seem to have a significant effect on sedation and respiratory depression caused by intravenous fentanyl in spinal anesthesia in Turkish patients.     

Efficient iPS cell production with the MyoD transactivation domain in serum-free culture

A major difficulty of producing induced pluripotent stem cells (iPSCs) has been the low efficiency of reprogramming differentiated cells into pluripotent cells. We previously showed that 5% of mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs when they were transduced with a fusion gene composed of Oct4 and the transactivation domain of MyoD (called M(3)O), along with Sox2, Klf4 and c-Myc (SKM). In addition, M(3)O facilitated chromatin remodeling of pluripotency genes in the majority of transduced MEFs, including cells that did not become iPSCs. These observations suggested the possibility that more than 5% of cells had acquired the ability to become iPSCs given more favorable culture conditions. Here, we raised the efficiency of making mouse iPSCs with M(3)O-SKM to 26% by culturing transduced cells at low density in serum-free culture medium. In contrast, the efficiency increased from 0.1% to only 2% with the combination of wild-type Oct4 and SKM (OSKM) under the same culture condition. For human iPSCs, M(3)O-SKM achieved 7% efficiency under a similar serum-free culture condition, in comparison to 1% efficiency with OSKM. This study highlights the power of combining the transactivation domain of MyoD with a favorable culture environment.     

The cytotoxic effect of diphtheria toxin on the actin cytoskeleton

Diphtheria toxin (DT) and its N-terminal fragment A (FA) catalyse the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) into a covalent linkage with eukaryotic elongation factor 2 (eEF2). DT-induced cytotoxicity is versatile, and it includes DNA cleavage and the depolymerisation of actin filaments. The inhibition of the ADP-ribosyltransferase (ADPrT) activity of FA did not affect the deoxyribonuclease activity of FA or its interaction with actin. The toxin entry rate into cells (HUVEC) was determined by measuring the ADP-ribosyltransferase activity. DT uptake was nearly 80% after 30 min. The efficiency was determined as K_m = 2.2 nM; V_max = 0.25 pmol.min⁻¹. The nuclease activity was tested with hyperchromicity experiments, and it was concluded that G-actin has an inhibitory effect on DT nuclease activity. In thepresence of DT and mutant of diphtheria toxin (CRM197), F-actin depolymerisation was determined with gel filtration, WB and fluorescence techniques. In the presence of DT and CRM197, 60–65% F-actin depolymerisation was observed. An in vitro FA-actin interaction and F-actin depolymerisation were reported in our previous paper. The present study thus confirms the depolymerisation of actin cytoskeleton in vivo.     

Evidence for the co-circulation of dengue virus type 3 genotypes III and V in the Northern region of Brazil during the 2002-2004 epidemics

The reintroduction of dengue virus type 3 (DENV-3) in Brazil in 2000 and its subsequent spread throughout the country was associated with genotype III viruses, the only DENV-3 genotype isolated in Brazil prior to 2002. We report here the co-circulation of two different DENV-3 genotypes in patients living in the Northern region of Brazil during the 2002-2004 epidemics. Complete genomic sequences of viral RNA were determined from these epidemics, and viruses belonging to genotypes V (Southeast Asia/South Pacific) and III were identified. This recent co-circulation of different DENV-3 genotypes in South America may have implications for pathological and epidemiological dynamics.     

Determination of the growth and solubilization capabilities of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> T1

The growth capability of Trichoderma harzianum Rifaii Tl was tested on Malt Extract and Czapeks Dox agar containing different concentrations of Cu²⁺, Zn²⁺, Mn²⁺, Fe²⁺ and Ca²⁺. The T. harzianum Tl isolate was observed to produce mycelia and spores in various mineral-containing media. It showed the lowest tolerance to Ca²⁺ and the highest tolerance to Fe²⁺. Solubilization capability of T. harzianum Tl for some insoluble minerals via acidification of medium has been tested on MnO₂, CuO, Fe₂O₃ and metallic Zn. T. harzianum Tl was able to solubilize MnO₂ and metallic Zn in a liquid medium.