Complete mitochondrial genome of a Chinese scorpion Mesobuthus martensii (Chelicerata, Scorpiones, Buthidae).

The complete mitochondrial genome (15,034 bp) of a Chinese scorpion Mesobuthus martensii (Buthidae) was sequenced and characterized in detail. The genome contains 13 protein-coding genes, 21 transfer RNA genes, two ribosomal RNA genes and a large non-coding region ( = CR). Its gene arrangement pattern is identical to that of Limulus polyphemus (Chelicerata, Xiphosura), with the exceptions of the tRNA(Glu)-tRNA(Ile)-tRNA(Met) (Q-I-M) arrangement and tRNA(Asp)-loss. Additional interesting features are found and discussed: high frequency of Leu(UUG) codon use, low A+T content of the genome (66.75%), and six repeat units (five 60-nt-long and one 58-nt-long repeats) in the 998-nt CR. Bayesian analysis based on amino acid sequences of the 12 proteincoding genes (excluding ATP8) reveals that the family Buthidae (Order Scorpiones) and the class Arachnida form strong monophyletic groups within Chelicerata, respectively. It indicated that the scorpions are the most ancestral arachnids.     

Regulation of ANP secretion by cardiac Na+/Ca2+ exchanger using a new controlled atrial model

The myocardial interstitium is important in regulating cardiac function. Between the atrial lumen and the pericardial space are transmural pathways, and movement of interstitial fluid (ISF) through these pathways is one of the main driving forces regulating translocation of substances from the interstitium into the blood. To define how ISF translocation from the interstitial space into the luminal space is regulated by each component of atrial hemodynamics, we devised a new rabbit atrial model in which each physical parameter could be controlled independently. Using this system, we also defined the physiological role of the cardiac Na(+)/Ca(2+) exchanger on secretion of atrial natriuretic peptide (ANP) by depletion of extracellular Na(+) ([Na(+)](o)). Increases in stroke volume and atrial end-systolic volume increased ISF translocation and ANP secretion. However, an increase in atrial rate did not influence ISF translocation but, rather, increased ANP secretion. Gradual depletion of [Na(+)](o) caused gradual increases in ANP secretion and intracellular Ca(2+) ([Ca(2+)](i)), which were blocked in the presence of Ca(2+)-free buffer and Ni(2+), but not in the presence of KB-R7943, diltiazem, mibefradil, caffeine, or monensin. Amiloride and its analog blocked an increase in ANP secretion but not an increase in [Ca(2+)](i) by [Na(+)](o) depletion. Therefore, we suggest that ANP secretion and ISF translocation may be differently controlled by each physical factor. These results also suggest that the increase in ANP secretion in response to [Na(+)](o) depletion may involve inhibition of Na(+)/Ca(2+) and Na(+)/H(+) exchangers but not an increase in [Ca(2+)](i).     

Immunohistochemical study on the distribution of neuronal nitric oxide synthase-immunoreactive neurons in the spinal cord of aged rat

Summary Despite in vivo studies suggesting an important function for nitric oxide (NO) in the spinal cord in the transmission of pain signals, sympathetic nerve activity and presumably other spinal functions, changes of neuronal NO synthase (nNOS)-containing neurons with aging in the spinal cord has not been investigated. In the present study, we demonstrated for the first time that the number of nNOS-immunoreactive neurons was significantly decreased in the central autonomic nucleus and the superficial dorsal horn of spinal cord in aged rats. Morphologically, the number and length of dendritic branches also seemed to be decreased. Combined with our previous studies, age-related decreases in the number of nNOS-immunoreactive neurons in the central autonomic nucleus and the superficial dorsal horn might be associated with the abnormality of micturition function or pain perception encountered in the elderly. However, the mechanisms underlying the decreased immunoreactivity for nNOS, and the functional implications require elucidation.     

Neuroprotective effects of naturally occurring biflavonoids

We examined neuroprotective effects of naturally occurring biflavonoids on oxidative stress-induced and amyloid beta peptide-induced cell death in neuronal cells. Among the nine biflavonoids tested, amentoflavone, ginkgetin, and isoginkgetin exhibited strong neuroprotection against cytotoxic insults induced by oxidative stress and amyloid beta, suggesting their therapeutic potential against neurodegenerative diseases, including ischemic stroke and Alzheimer's disease.     

Induction of thioredoxin is required for nodule development to reduce reactive oxygen species levels in soybean roots

Nodules are formed on legume roots as a result of signaling between symbiotic partners and in response to the activities of numerous genes. We cloned fragments of differentially expressed genes in spot-inoculated soybean (Glycine max) roots. Many of the induced clones were similar to known genes related to oxidative stress, such as thioredoxin and beta-carotene hydroxylase. The deduced amino acid sequences of full-length soybean cDNAs for thioredoxin and beta-carotene hydroxylase were similar to those in other species. In situ RNA hybridization revealed that the thioredoxin gene is expressed on the pericycle of 2-d-old nodules and in the infected cells of mature nodules, suggesting that thioredoxin is involved in nodule development. The thioredoxin promoter was found to contain a sequence resembling an antioxidant responsive element. When a thioredoxin mutant of yeast was transformed with the soybean thioredoxin gene it became hydrogen peroxide tolerant. These observations prompted us to measure reactive oxygen species levels. These were decreased by 3- to 5-fold in 7-d-old and 27-d-old nodules, coincident with increases in the expression of thioredoxin and beta-carotene hydroxylase genes. Hydrogen peroxide-producing regions identified with cerium chloride were found in uninoculated roots and 2-d-old nodules, but not in 7-d-old and 27-d-old nodules. RNA interference-mediated repression of the thioredoxin gene severely impaired nodule development. These data indicate that antioxidants such as thioredoxin are essential to lower reactive oxygen species levels during nodule development.     

Generation of a cell culture-adapted hepatitis C virus with longer half life at physiological temperature

Background

We previously reported infectious HCV clones that contain the convenient reporters, green fluorescent protein (GFP) and Renilla luciferase (Rluc), in the NS5a-coding sequence. Although these viruses were useful in monitoring viral proliferation and screening of anti-HCV drugs, the infectivity and yield of the viruses were low.

Methodology/Principal Findings

In order to obtain a highly efficient HCV cultivation system, we transfected Huh7.5.1 cells [1] with JFH 5a-GFP RNA and then cultivated cells for 20 days. We found a highly infectious HCV clone containing two cell culture-adapted mutations. Two cell culture-adapted mutations which were responsible for the increased viral infectivity were located in E2 and p7 protein coding regions. The viral titer of the variant was ∼100-fold higher than that of the parental virus. The mutation in the E2 protein increased the viability of virus at 37°C by acquiring prolonged interaction capability with a HCV receptor CD81. The wild-type and p7-mutated virus had a half-life of ∼2.5 to 3 hours at 37°C. In contrast, the half-life of viruses, which contained E2 mutation singly and combination with the p7 mutation, was 5 to 6 hours at 37°C. The mutation in the p7 protein, either singly or in combination with the E2 mutation, enhanced infectious virus production about 10–50-fold by facilitating an early step of virion production.

Conclusion/Significance

The mutation in the E2 protein generated by the culture system increases virion viability at 37°C. The adaptive mutation in the p7 protein facilitates an earlier stage of virus production, such as virus assembly and/or morphogenesis. These reporter-containing HCV viruses harboring adaptive mutations are useful in investigations of the viral life cycle and for developing anti-viral agents against HCV.     

Mutational biosynthesis of tacrolimus analogues by fkbO deletion mutant of Streptomyces sp. KCTC 11604BP

Tacrolimus (FK506) is an important macrocyclic polyketide showing antifungal and immunosuppressive activities, as well as neuroregenerative properties. Tacrolimus biosynthetic machinery should incorporate the shikimate-derived 4,5-dihydroxycyclohex-1-enecarboxylic acid (DHCHC) as a biosynthetic starter unit into the biosynthetic line of tacrolimus. fkbO is a homologue of rapK encoding chorismatase related to the biosynthesis of starter unit DHCHC from chorismate in the rapamycin biosynthetic gene cluster. FkbO and RapK are good targets for mutational biosynthesis to produce novel analogues of tacrolimus, ascomycin, and rapamycin, which could be important drugs for clinical application in the treatment of cancer and immune and neurodegenerative diseases. To make novel tacrolimus analogues, we prepared an fkbO in-frame deletion mutant, Streptomyces sp. GT110507, from a tacrolimus high producer. We scrutinized the cyclic carboxylic acids that were possibly incorporated instead of DHCHC by precursor-directed mutasynthesis using Streptomyces sp. GT110507 to lead tacrolimus analogues. Among them, trans-4-hydroxycyclohexanecarboxylic acid and 3-hydroxybenzoic acid were successfully incorporated into the tacrolimus backbone, which led to the production of 31-desmethoxytacrolimus and TC-225, respectively. Especially, adding of trans-4-hydroxycyclohexanecarboxylic acid produced a high amount (55 mg/L) of 31-desmethoxytacrolimus. Interestingly, in the rapK mutant, it has been reported that the incorporation of cyclohexanecarboxylic acid (CHC) led to 39-desmethoxy rapamycin. However, in Streptomyces sp. GT110507, CHC is not successfully incorporated. This discrepancy should reflect the differences in the DHCHC biosynthesis mechanism and/or substrate specificity of starter unit loading machineries (FkbP and RapP) of tacrolimus and rapamycin.     

The estimation of absorbed dose rates for non-human biota: an extended intercomparison

An exercise to compare 10 approaches for the calculation of unweighted whole-body absorbed dose rates was conducted for 74 radionuclides and five of the ICRP's Reference Animals and Plants, or RAPs (duck, frog, flatfish egg, rat and elongated earthworm), selected for this exercise to cover a range of body sizes, dimensions and exposure scenarios. Results were analysed using a non-parametric method requiring no specific hypotheses about the statistical distribution of data. The obtained unweighted absorbed dose rates for internal exposure compare well between the different approaches, with 70% of the results falling within a range of variation of?±20%. The variation is greater for external exposure, although 90% of the estimates are within an order of magnitude of one another. There are some discernible patterns where specific models over- or under-predicted. These are explained based on the methodological differences including number of daughter products included in the calculation of dose rate for a parent nuclide; source-target geometry; databases for discrete energy and yield of radionuclides; rounding errors in integration algorithms; and intrinsic differences in calculation methods. For certain radionuclides, these factors combine to generate systematic variations between approaches. Overall, the technique chosen to interpret the data enabled methodological differences in dosimetry calculations to be quantified and compared, allowing the identification of common issues between different approaches and providing greater assurance on the fundamental dose conversion coefficient approaches used in available models for assessing radiological effects to biota.     

An economical and highly productive cell-free protein synthesis system utilizing fructose-1,6-bisphosphate as an energy source

In this study, we describe the development of a cost effective and highly productive cell-free protein synthesis system derived from Escherichia coli. Through the use of an optimal energy source and cell extract, approximately 1.3 mg/mL of protein was generated from a single batch reaction at greatly reduced reagent costs. Compared to previously reported systems, the described method yields approximately 14-fold higher productivity per unit reagent cost making this cell-free synthesis technique a promising alternative for more efficient protein production.     

Improved expression of a soluble single chain antibody fusion protein containing tumor necrosis factor in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An epoxide hydrolase gene of about 0.8 kb was cloned from Rhodococcus opacus ML-0004, and the open reading frame (ORF) sequence predicted a protein of 253 amino acids with a molecular mass of about 28 kDa. An expression plasmid carrying the gene under the control of the tac promotor was introduced into Escherichia coli, and the epoxide hydrolase gene was successfully expressed in the recombinant strains. Some characteristics of purified recombinant epoxide hydrolase were also studied. Epoxide hydrolase showed a high stereospecificity for l(+)-tartaric acid, but not for d(+)-tartaric acid. The epoxide hydrolase activity could be assayed at the pH ranging from 3.5 to 10.0, and its maximum activity was obtained between pH 7.0 and 7.5. The enzyme was sensitive to heat, decreasing slowly between 30°C and 40°C, and significantly at 45°C. The enzyme activity was activated by Ca²⁺ and Fe²⁺, while strongly inhibited by Ag⁺ and Hg⁺, and slightly inhibited by Cu²⁺, Zn²⁺, Ba²⁺, Ni⁺, EDTA–Na₂ and fumarate.