Side-necked turtle (Pleurodira, Chelonia, reptilia) hemoglobin: cDNA-derived primary structures and X-ray crystal structures of Hb A

Red blood cells of yellow-spotted river turtles (Podocnemis unifilis, Pleurodira, Chelonia, REPTILIA) have two hemoglobin (Hb) components, Hb A and Hb D. We purified the hemoglobin component homologous to amniote (reptiles, birds, and mammals) adult Hb A which comprises two identical α(A) -globin polypeptides and two identical β-globin polypeptides. To establish the crystal structure of Podocnemis Hb A, we first determined the globin primary structures using cDNA nucleotide sequencing with the assistance of protein sequencing. The purified Podocnemis Hb A produced a different form of crystal for each of the two different buffer systems used: form A, tetragonal crystals (space group, P4?2?2), produced under neutral pH (pH 7-8) conditions; and form B, hexagonal crystals (space group, P6?22), produced under high alkaline pH (pH 11-13) conditions. Single crystals of the two forms were examined by Raman microscopy with an excitation of 532 nm, indicating their structural differences. The crystal structures of the two forms were constructed by X-ray crystallographic diffraction at a resolution of 2.20 ? for form A and 2.35 ? for form B. The differences of the tertiary and quaternary structures of the two forms were marginal; however, one clear difference was found in helix structure. When comparing Podocnemis Hb A with Hb A from specimens in other taxa, such as Anser indicus (birds) and Homo sapiens (mammals) by SHELXPRO, the root mean square deviation (RMSD) between the corresponding Cα atoms of the two globins does not exceed 2.0 ?. These low values indicate the crystal structures resemble each other. Our data on X-ray crystal structures and Raman spectra not only reveal the first findings on the two crystal forms of Podocnemis unifilis Hb A but also provide the first refined models for reptilian adult Hb A.     

Origin of agouti-melanistic polymorphism in Wild Black Rats (Rattus rattus) inferred from Mc1r gene sequences

We examined nucleotide changes that underlie coat color variation in Black Rats (the Rattus rattus species complex), which show polymorphism in dorsal fur color, including either grayish brown (agouti) or black (melanistic) forms. We examined the full coding sequence of a gene known to produce melanism in other vertebrates-melanocortin-1-receptor gene Mc1r (954 bp) -using samples of both R. rattus (with 2n = 38) and its close relative Asian Black Rat (R. tanezumi; 2n = 42). We used 61 specimens from Japan with karyotype-known individuals and four samples from Pakistan. We found 11 allele sequences and constructed a network tree that shows two distinct clusters, with allelic segregation according to karyotype and by inference, representing the two species. We found that a nucleotide substitution from G to A at site 280, producing an amino acid change from glutamic acid to lysine, was associated with the dominant trait of the melanistic form of the coat color in R. rattus. Notably, the derived SNP 280A was found in a single allele, with the ancestral SNP 280G present in seven alleles. By contrast, all three alleles for R. tanezumi retain the ancestral SNP 280G. These results suggest a possible recent origin of melanism in R. rattus.     

How does the QB site influence propagate to the QA site in photosystem II?

The redox potential of the primary quinone Q(A) [E(m)(Q(A))] in photosystem II (PSII) is lowered by replacement of the native plastoquinone (PQ) with bromoxynil (BR) at the secondary quinone Q(B) binding site. Using the BR-bound PSII structure presented in the previous Fourier transform infrared and docking calculation studies, we calculated E(m)(Q(A)) considering both the protein environment in atomic detail and the protonation pattern of the titratable residues. The calculated E(m)(Q(A)) shift in response to the replacement of PQ with deprotonated BR at the Q(B) binding site [ΔE(m)(Q(A))(PQ→BR)] was -55 mV when the three regions, Q(A), the non-heme iron complex, and Q(B) (Q(B) = PQ or BR), were treated as a conjugated supramolecule (Q(A)-Fe-Q(B)). The negative charge of BR apparently contributes to the downshift in ΔE(m)(Q(A))(PQ→BR). This downshift, however, is mostly offset by the influence of the residues near Q(B). The charge delocalization over the Q(A)-Fe-Q(B) complex and the resulting H-bond strength change between Q(A) and D2-His214 are crucial factors that yield a ΔE(m)(Q(A))(PQ→BR) of -55 mV by (i) altering the electrostatic influence of the H-bond donor D2-His214 on E(m)(Q(A)) and (ii) suppressing the proton uptake events of the titratable residues that could otherwise upshift ΔE(m)(Q(A))(PQ→BR) during replacement of PQ with BR at the Q(B) site.     

Geographical variation in the early regeneration process of Siebold's Beech (Fagus crenata BLUME) in Japan

The causes and timing of seed death in early regeneration process of Siebold's beech (Fagus crenata Blume) was studied at 15 sites along a snowfall gradient in Japan, in order to clarify why the seedling density of the species has geographic difference remarkably. Seed production did not significantly differ along the snowfall gradient. Pre-dispersal seed mortality by insect damage was higher at sites with light snowfall than at sites with heavy snowfall, but this only seemed to be a minor factor influencing the population. A large proportion of the viable nuts that fall in autumn ware killed in winter before germination. Winter mortality was much higher at sites with thin snow cover than that at sites with thick snow cover, and this factor was strongly correlated with the geographic variation of seedling regeneration probability. There was little seed mortality by winter desiccation. The main factor contributing to the geographic difference seemed to be a seed predation by rodents in winter. Deep snow cover may reduce the success of rodents finding seeds in winter. Thus the observed relationship between snowpack depth and early mortality may be due to an indirect effect through the process of seed predation.p>     

Methotrexate chronotherapy is effective against rheumatoid arthritis

Methotrexate (MTX) is the most important drug for treating rheumatoid arthritis (RA). It has been stated that cytokines play an important role in the pathogenesis of RA, and that cytokine levels increase and show 24-h rhythms in RA patients. Previously, we found that arthritis was relieved after the administration of MTX at specific times in synchronization with the 24-h rhythm of tumor necrosis factor (TNF)-α in collagen-induced arthritis (CIA) animals. Based on our findings in an earlier study of the dosing time-dependent effects of MTX in MRL/lpr mice, which develop autoimmune disorders that share similarities with human RA, we examined here the utility of MTX chronotherapy in Japanese RA patients. In an initial animal modeling study, we collected blood from MRL/lpr mice at different times (2, 6, 10, 14, 18, or 22 hours after the light was turned on [HALO]), and we measured TNF-α mRNA expression in leukocytes. MTX was administered to the mice at two different dosing times (6 or 18 HALO), and various blood parameters were measured to estimate arthritis activity. TNF-α mRNA levels showed a clear 24-h rhythm with a peak at 22 HALO and a trough at 18 HALO after RA had developed. In these MRL/lpr mice, inflammation and TNF-α were markedly reduced when the MTX dosing time was matched to the time (18 HALO) when the TNF-α level began to increase. We then applied these findings to Japanese RA patients by switching them from the standard MTX three times/wk (day 1: after breakfast and supper; day 2: after breakfast schedule), to chronotherapy, in which the dose and number of doses/wk were not changed but MTX was administered once-a-day at bedtime. Disease Activity Score (DAS)28, modified health assessment questionnaire (MHAQ), and adverse effects were assessed. With MTX chronotherapy, DAS28, which is commonly used to quantitatively assess RA symptoms, was significantly improved at all follow-up clinical visit times compared with the baseline (vs. 1 mo: p?=?.0197, 2 mos: p?=?.0107, 3 mos: p?=?.0087). Significant symptom recovery was observed in 41.2% of patients, and 23.5% of patients achieved clinical remission during the 3 mos of follow-up. Functional capacity of RA patients, as indicated by the MHAQ, was markedly improved by chronotherapy. There were no severe adverse effects. Thus, we demonstrated (i) inflammation and plasma TNF-α concentrations were significantly reduced in MRL/lpr mice treated with MTX at 18 HALO, the time when TNF-α mRNA level began to increase; and (ii) MTX bedtime chronotherapy was safe, markedly reduced disease activity, and improved the functional capacity of RA patients. The findings on RA patients show that bedtime MTX chronotherapy can improve RA symptoms compared to the current standard dosing methods.     

High performance liquid chromatographic methods for the determination of aripiprazole with ultraviolet detection in rat plasma and brain: application to the pharmacokinetic study

High performance liquid chromatographic (HPLC) methods were validated for the determination of aripiprazole (OPC-14597, Abilify) in rat plasma and brain. Separation was by Nova-pak phenyl column; flow rate, 1.0 ml/min; mobile phase, acetonitrile-methanol-20 mM sodium sulfate-acetic acid (27:25:48:1, v/v/v/v); UV detection at 254 nm. Reproducibility in plasma and brain showed excellent precision (within 7.8 and 10.6%) and accuracy (96.0-102.4% and 99.0-108.7%) with calibration curve ranges 10.0-2000 ng/ml and 30.0-6000 ng/g, respectively. Validated HPLC methods were successfully applied to pharmacokinetic study of aripiprazole in rats, demonstrating brain concentrations after oral administration five times higher than plasma concentrations.     

Application of ultrafiltration method to measurement of catecholamines in plasma of human and rodents by high-performance liquid chromatography

In order to develop a reliable, simple and routine method using small sample volume to determine norepinephrine (NE) and epinephrine (E) concentrations in plasma of humans and rodents, we utilize the ultrafiltration (UF) method by Ultrafree-MC filter device and a high-performance liquid chromatography equipped with electrochemical detector (HPLC-ECD) to detect NE and E. Optimum UF and HPLC conditions were as follows: the filter nominal molecular weight limit size is 30,000, the pH of added phosphate buffer to each plasma sample for UF is 3.0, and the mobile phase is 0.1M phosphate buffer (pH 3)/acetonitrile (98:2) containing 0.05% sodium disulfite and 0.001% EDTA 2Na. The plasma samples and 1.0M phosphate buffer (pH 3) containing 3,4-dihydroxybenzylamine (DHBA), as an internal standard, was mixed and poured into the UF units. After the centrifugation for 60 min at 13,000 x g at 4 degrees C, the filtrate was directly injected into HPLC. The calibration curve of NE and E was linear for the concentrations studied (20-400 pg) with a correlation coefficient of >0.999. Intra-assay coefficients of variation for NE and E using this method were less than 3%. The method also correlated well with the well-established alumina method (r=0.954). The present findings suggest that a newly-developed UF method with HPLC-ECD would apply successfully to measure plasma NE and E concentrations in humans and rodents.     

Genetic structure of masu salmon (Oncorhynchus masou) populations in Hokkaido, northernmost Japan, inferred from mitochondrial DNA variation

Genetic structure of masu salmon Oncorhynchus masou populations in Hokkaido was examined by analysing mtDNA NADH dehydrogenase subunit 5 gene (561 bp) of 382 individuals collected from 12 rivers, in which there were no records of artificial release. Analysis of molecular variance showed that between groups level and between populations within-group level explained each c. 10% of genetic variance. In neighbour-joining tree, four populations located in southern Hokkaido were clustered into a single group; however, other populations did not form any clear clusters. Fu's F _S, Tajima's D and a mismatch distribution test indicated a sudden expansion of population in the entire population of Hokkaido and the northernmost population of Chiraibetsu, which was genetically close to the southern Hokkaido group. The Sea of Japan and southern rivers, including those of southern Hokkaido, seem to have served as refugia for masu salmon during glacial periods, and their dispersal and straying in interglacial periods affected the genetic structure of masu salmon populations in Hokkaido.     

Characterization of cysteine protease induced by oxidative stress in cells of Chlamydomonas sp. strain W80

Unlike known Chlamydomonas species, Chlamydomonas sp. strain W80, which was isolated from seawater, shows tolerance to salt and cadmium. In this study, we purified and characterized cysteine protease from Chlamydomonas sp. strain W80 cells and also investigated their response to oxidative stress. The protease was purified 2760-fold with a yield of 2.6% by five steps of successive chromatography. This protease had a pH optimum of 8.0 and was specific only for tert -butoxycarbonyl (Boc)-Leu-Arg-Arg-4-methylcoumaryl-7-amide (MCA) (Boc-LRR-MCA) and Boc-Val-Leu-Lys-MCA as substrates among eight fluorogenic peptides tested. The K _m value was estimated to be 44.4 μ M for Boc-LRR-MCA. The molecular weight of the protease was determined to be approximately 102 kDa by Superdex 200 gel filtration and 60 kDa by SDS–PAGE, suggesting that this enzyme is a dimer. This enzyme was inhibited by the cysteine protease inhibitors leupeptin and N-ethylmaleimide but neither inhibited by phenylmethylsulfonyl fluoride or ethylenediaminetetraacetic acid nor activated by metal cations. These findings indicate that this enzyme is likely a cysteine protease. When strain W80 was grown under oxidative stress in the presence of methyl viologen and cadmium chloride, cysteine protease activity was about 30–90% higher than normal, whereas no changes were observed in carbon enrichment or senescence. It is likely that this protease is upregulated in response to oxidative stress and plays a role in the maintenance of cell metabolism under oxidative stress conditions.