Peripheral region for core cross-beta plays important role in amyloidogenicity

The role of the peripheral sequence neighboring the core cross-beta region was investigated using a peptide library constructed with all possible combinations of Lys, Glu, Ser, and Leu at three residue positions (X1-X3) forming the N-terminal region linked to the amyloid core sequence of the barnase-derived segment (A4-K22). By means of CD spectra and thioflavin T binding assay for 64 peptides, not only the composition but also the sequence in the peripheral region were found to be responsible for amyloid formation. The preferences of amino acid residues in the peripheral region of the amyloid-forming peptides were in the order of Leu approximately SerGlu>Lys. A balance of positive and negative charges was found to be essential for amyloid formation, suggesting that the electrostatic interaction at the surface of the amyloid fibrils is relevant to their stability. On the basis of the maximum fluorescence wavelength of fibril-bound thioflavin T, the highly amyloidogenic peptides were classified into two classes, which exhibited the sequence preferences of (Leu, Ser/Glu, and Leu) and (Glu, Leu, and Ser) for the peripheral sequence (X1, X2, and X3). The former class can be rationally assigned to the structural model with deep grooves along the fibril axis. Thus, the peripheral sequence regulates the manner of molecular packing in the fibrils as well as the amyloidogenicity. In addition, the chains of the peripheral sequence are most likely to form thioflavin T binding sites.     

Oxygen- and carbon monoxide-binding kinetic parameters for liposomal heme under semi-physiological condition

Binding and dissociation rate constants of oxygen and carbon monoxide with the meso-tetra(α,α,α,α-(o-pivalamidophenyl))porphinato iron-mono(1-lauryl-2-methylimidazole) complex incorporated into the liposomes of dimyristoylphosphatidylcholine (liposomal heme) were measured with flash-photolysis method at pH 7.0 and 20 °C. Extra large quantum yield was observed for the photo-dissociation of the oxygen adduct, while that for the CO adduct was relatively small. Rate constants for the binding and the dissociation were comparable to those of hemoglobin except for the oxygen-binding rate constant.     

FIA functions as an early signal component of abscisic acid signal cascade in Vicia faba guard cells

An abscisic acid (ABA)-insensitive Vicia faba mutant, fia (fava bean impaired in ABA-induced stomatal closure) had previously been isolated. In this study, it was investigated how FIA functions in ABA signalling in guard cells of Vicia faba. Unlike ABA, methyl jasmonate (MeJA), H(2)O(2), and nitric oxide (NO) induced stomatal closure in the fia mutant. ABA did not induce production of either reactive oxygen species or NO in the mutant. Moreover, ABA did not suppress inward-rectifying K(+) (K(in)) currents or activate ABA-activated protein kinase (AAPK) in mutant guard cells. These results suggest that FIA functions as an early signal component upstream of AAPK activation in ABA signalling but does not function in MeJA signalling in guard cells of Vicia faba.     

Toxic and nontoxic components of botulinum neurotoxin complex are evolved from a common ancestral zinc protein

Zinc atoms play an essential role in a number of enzymes. Botulinum neurotoxin (BoNT), the most potent toxin known in nature, is a zinc-dependent endopeptidase. Here we identify the nontoxic nonhemagglutinin (NTNHA), one of the BoNT-complex constituents, as a zinc-binding protein, along with BoNT. A protein structure classification database search indicated that BoNT and NTNHA share a similar domain architecture, comprising a zinc-dependent metalloproteinase-like, BoNT coiled-coil motif and concanavalin A-like domains. Inductively coupled plasma-mass spectrometry analysis demonstrated that every single NTNHA molecule contains a single zinc atom. This is the first demonstration of a zinc atom in this protein, as far as we know. However, the NTNHA molecule does not possess any known zinc-coordinating motif, whereas all BoNT serotypes possess the classical HEXXH motif. Homology modeling of the NTNHA structure implied that a consensus K-C-L-I-K-X(35)-D sequence common among all NTNHA serotype molecules appears to coordinate a single zinc atom. These findings lead us to propose that NTNHA and BoNT may have evolved distinct functional specializations following their branching out from a common ancestral zinc protein.     

<i>Acetobacter okinawensis</i> sp. nov., <i>Acetobacter papayae</i> sp. nov., and <i>Acetobacter persicus</i> sp. nov.; novel acetic acid bacteria isolated from stems of sugarcane, fruits, and a flower in Japan

Eleven strains of acetic acid bacteria were isolated from stems of sugarcane, fruits, and a flower in Japan. The isolates were separated into three groups, Groups I, II, and III, in the genus Acetobacter according to phylogenetic analysis based on 16S rRNA sequences. The isolates had sequence similarities of 99.8-100% within the Group, 99.3-99.6% to those of the type strains of each related Acetobacter species, and less than 98.4% to those of the type strains of other Acetobacter species. Genomic DNA G+C contents of Groups I, II, and III were 59.2-59.4, 60.5-60.7, and 58.7-58.9 mol%, respectively. The isolates in the Group showed high values of DNA-DNA relatedness to each other, but low values less than 46% to the type strains of related Acetobacter species. A good correlation was found between the three Groups and groups based on DNA G+C contents and DNA-DNA relatedness. All the strains had Q-9 as the main component, and Q-8 and Q-10 as minor components. The isolates in the three Groups did not completely match with any Acetobacter species on catalase reaction, the production of ketogluconic acids from D-glucose, growth on ammoniac nitrogen with ethanol (Hoyer-Frateur medium and Frateur modified Hoyer medium), growth on 30% (w/v) D-glucose, growth in 10% (v/v) ethanol, or DNA G+C contents. On the basis of phylogenetic relationships in the genus Acetobacter and chemosystematic and phenotypic characteristics, the three Groups were regarded as novel species in the genus Acetobacter. Acetobacter okinawensis sp. nov. is proposed for Group I, Acetobacter papayae sp. nov. for Group II, and Acetobacter persicus sp. nov. for Group III.     

Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair

Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome-assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2-knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome-assembly activity, it significantly stimulated FANCD2-mediated nucleosome assembly. These observations suggest that FANCD2-FANCI may regulate chromatin dynamics during DNA repair.     

Effects of exogenous proline and glycinebetaine on the salt tolerance of rice cultivars

Salinity significantly increased trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS) uptake and decreased the K(+)/Na(+) ratio in salt-sensitive rice (Nipponbare) but did not markedly in salt-tolerant rice (Pokkali). Proline and glycinebetaine (betaine) suppressed the increase in PTS uptake and the decrease in the K(+)/Na(+) ratio in Nipponbare, but did not affect PTS uptake or the K(+)/Na(+) ratio in Pokkali.     

Higher serum bone alkaline phosphatase as a predictor of mortality in male hemodialysis patients

AimsHigher serum alkaline phosphatase predicts lower mortality in chronic kidney disease and hemodialysis patients without liver dysfunction because it reflects high bone turnover. The purpose of our study was to compare the significance of serum bone alkaline phosphatase (BAP) with that of other bone markers in prediction of all-cause mortality(ACM) in male hemodialysis patients.Main methodsThe study was performed for 5 years. Serum BAP, intact osteocalcin (iOC), ß-CrossLaps (CTX), and intact parathyroid hormone (iPTH) were measured in 196 male hemodialysis patients without radiographic fracture. Their day-to-day variation during 5 consecutive days and diurnal variation were determined in 13 healthy males.Key findingsThe patients were divided into higher and lower groups based on serum levels of bone markers(mean ± SD: iPTH 218.6 ± 214.5 pg/ml, BAP 23.6 ± 12.2U/L, iOC 42.8 ± 45.2 ng/ml, CTX 1.71 ± 1.23 nmol/L BCE). In Kaplan–Meier analysis, the higher BAP group had significantly higher ACM than the lower BAP group (P = 0.013), whereas mortality did not differ between the higher and lower groups in other markers. Cox regression hazard analysis identified higher log BAP as a significant independent predictor [hazard ratio(HR) 8.32(95%CI:1.18–58.98)] for ACM after adjustment for various factors including pre-existing cardiovasucular disease, presence of DM. The significant association of mortality with serum BAP alone, in contrast with other markers including CTX [HR0.64 (95%CI:0.16–2.47)], iOC [HR0.97(95%CI:0.36–2.64)], iPTH [HR0.84(95%CI:0.44–1.60)],it may be due to the narrower day-to-day variation and the absence of diurnal variation in serum BAP compared to other markers.SignificanceHigher serum BAP may be a predictor of ACM in male hemodialysis patients.