A novel oligoalginate lyase from abalone, Haliotis discus hannai, that releases disaccharide from alginate polymer in an exolytic manner

We previously reported the isolation and cDNA cloning of an endolytic alginate lyase, HdAly, from abalone Haliotis discus hannai [Carbohydr. Res.2003, 338, 2841-2852]. Although HdAly preferentially degraded mannuronate-rich substrates, it was incapable of degrading unsaturated oligomannuronates smaller than tetrasaccharide. In the present study, we used conventional chromatographic techniques to isolate a novel unsaturated-trisaccharide-degrading enzyme, named HdAlex, from the digestive fluid of the abalone. The HdAlex showed a molecular weight of 32,000 on SDS-PAGE and could degrade not only unsaturated trisaccharide but also alginate and mannuronate-rich polymers at an optimal pH and temperature of 7.1 and 42 degrees C, respectively. Upon digestion of alginate polymer, HdAlex decreased the viscosity of the alginate at a slower rate than did HdAly, producing only unsaturated disaccharide without any intermediate oligosaccharides. These results indicate that HdAlex degrades the alginate polymer in an exolytic manner. Because HdAlex split saturated trisaccharide producing unsaturated disaccharide, we considered that this enzyme cleaved the alginate at the second glycoside linkage from the reducing terminus. The primary structure of HdAlex was deduced with cDNAs amplified from an abalone hepatopancreas cDNA library by the polymerase chain reaction. The translational region of 822 bp in the total 887-bp sequence of HdAlex cDNA encoded an amino-acid sequence of 273 residues. The N-terminal sequence of 16 residues, excluding the initiation methionine, was regarded as the signal peptide of this enzyme. The amino-acid sequence of the remaining 256 residues shared 62-67% identities with those of the polysaccharide lyase family-14 (PL14) enzymes such as HdAly and turban-shell alginate lyase SP2. To our knowledge, HdAlex is the first exolytic oligoalginate lyase belonging to PL14.     

Radiocesium distribution in the tissues of Japanese black beef heifers fed fallout-contaminated roughage due to the fukushima daiichi nuclear power station accident

This study examined the accumulation and tissue distribution of radioactive cesium nuclides in Japanese Black beef heifers raised on roughage contaminated with radioactive fallout due to the accident at the Fukushima Daiichi Nuclear Power Station on March 2011. Radiocesium feeding increased both (134)Cs and (137)Cs levels in all tissues tested. The kidney had the highest level and subcutaneous adipose had the lowest of radioactive cesium in the tissues. Different radioactive cesium levels were not found among parts of the muscles. These results indicate that radiocesium accumulated highly in the kidney and homogenously in the skeletal muscles in the heifers.     

Impact of normothermic preservation with extracellular type solution containing trehalose on rat kidney grafting from a cardiac death donor

Background

The aim of this study was to investigate factors that may improve the condition of a marginal kidney preserved with a normothermic solution following cardiac death (CD) in a model of rat kidney transplantation (RTx).

Methods

Post-euthanasia, Lewis (LEW) donor rats were left for 1 h in a 23°C room. These critical kidney grafts were preserved in University of Wisconsin (UW), lactate Ringer''s (LR), or extracellular-trehalose-Kyoto (ETK) solution, followed by intracellular-trehalose-Kyoto (ITK) solution at 4, 23, or 37°C for another 1 h, and finally transplanted into bilaterally nephrectomized LEW recipient rats (n = 4–6). Grafts of rats surviving to day 14 after RTx were evaluated by histopathological examination. The energy activity of these marginal rat kidneys was measured by high-performance liquid chromatography (HPLC; n = 4 per group) and fluorescence intensity assay (n = 6 per group) after preservation with UW or ETK solutions at each temperature. Finally, the transplanted kidney was assessed by an in vivo luciferase imaging system (n = 2).

Results

Using the 1-h normothermic preservation of post-CD kidneys, five out of six recipients in the ETK group survived until 14 days, in contrast to zero out of six in the UW group (p<0.01). Preservation with ITK rather than ETK at 23°C tended to have an inferior effect on recipient survival (p = 0.12). Energy activities of the fresh donor kidneys decreased in a temperature-dependent manner, while those of post-CD kidneys remained at the lower level. ETK was superior to UW in protecting against edema of the post-CD kidneys at the higher temperature. Luminescence intensity of successful grafts recovered within 1 h, while the intensity of grafts of deceased recipients did not change at 1 h post-reperfusion.

Conclusions

Normothermic storage with extracellular-type solution containing trehalose might prevent reperfusion injury due to temperature-dependent tissue edema.     

Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli

ABSTRACT: BACKGROUND: Escherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli. RESULTS: Because the redox enzymes can reduce the disulfide that forms on proteins, wefirst tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coli L-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (cysI and cysJ) and the L-cysteine synthase gene (cysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (cysC or cysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coli L-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell . CONCLUSIONS: In this work, we showed that Grx1 and NrdH reduce SSC to L-cysteine, and the generated sulfite is then utilized as the sulfur source to produce additional L-cysteine molecule through the sulfate pathway in E. coli. We also found that co-overexpression of NrdH, CysI, and CysK increases L-cysteine production. Our results propose that the enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from SSC is a novel method for improvement of L-cysteine production.     

Recognition of Non-Harmonic Natural Sounds by Small Mammals Using Competitive Training

Animals recognize biologically relevant sounds, such as the non-harmonic sounds made by some predators, and respond with adaptive behaviors, such as escaping. To clarify which acoustic parameters are used for identifying non-harmonic, noise-like, broadband sounds, guinea pigs were conditioned to a natural target sound by introducing a novel training procedure in which 2 or 3 guinea pigs in a group competed for food. A set of distinct behavioral reactions was reliably induced almost exclusively to the target sound in a 2-week operant training. When fully conditioned, individual animals were separately tested for recognition of a set of target-like sounds that had been modified from the target sound, with spectral ranges eliminated or with fine or coarse temporal structures altered. The results show that guinea pigs are able to identify the noise-like non-harmonic natural sounds by relying on gross spectral compositions and/or fine temporal structures, just as birds are thought to do in the recognition of harmonic birdsongs. These findings are discussed with regard to similarities and dissimilarities to harmonic sound recognition. The results suggest that similar but not identical processing that requires different time scales might be used to recognize harmonic and non-harmonic sounds, at least in small mammals.     

Factors Influencing Collaborative Activities between Non-Professional Disaster Volunteers and Victims of Earthquake Disasters

Background

Assistance from non-professional disaster volunteers (hereinafter, volunteers) is essential for disaster victims to recover physically and rebuild their lives; however, disaster victims in some areas are reluctant to accept assistance from volunteers. This study explored factors that may influence collaborative activities between volunteers and victims of earthquake disasters.

Methods

From July to September 2008, a self-reporting questionnaire survey was conducted with all 302 leaders of neighborhood associations in a city within Niigata Prefecture at the time of the Niigataken Chuetsu-oki Earthquake in 2007. Each factor was determined based on the Health Belief Model. Multiple regression analysis was conducted, using collaborative activities as the objective variable.

Results

From 261 valid responses received (response rate 86.4%), 41.3% of leaders collaborated with volunteers, and 60.2% of associations had residents who collaborated with volunteers. Collaboration with volunteers was significantly and positively related to perceived severity of an earthquake disaster (standardized partial regression coefficient β = 0.224, p<0.001) and neighborhood association activities during the earthquake disaster (β = 0.539, p<0.001). A positive and marginally significant relation was found between such collaboration and sense of coherence within a community (β = 0.137, p = 0.06), social capital (β = 0.119, p = 0.08), and perceived benefits (β = 0.116, p = 0.09).

Conclusion

Collaboration between disaster victims and volunteers during the response to an earthquake may require the preemptive estimation of damage by residents during normal times and the enhancement of neighborhood association activities during a disaster. For residents to have such estimation abilities, public institutions should provide information related to anticipated disaster damage and appropriate disaster prevention training and education. In addition, residents should create a disaster prevention map with other residents. Lastly, promoting neighborhood association activities may require the participation of many residents in disaster drills and education as well as a preemptive discussion of neighborhood activities during a disaster.     

Comparative Genomics of Community-Acquired ST59 Methicillin-Resistant Staphylococcus aureus in Taiwan: Novel Mobile Resistance Structures with IS1216V

Methicillin-resistant Staphylococcus aureus (MRSA) with ST59/SCCmecV and Panton-Valentine leukocidin gene is a major community-acquired MRSA (CA-MRSA) lineage in Taiwan and has been multidrug-resistant since its initial isolation. In this study, we studied the acquisition mechanism of multidrug resistance in an ST59 CA-MRSA strain (PM1) by comparative genomics. PM1’s non-β-lactam resistance was encoded by two unique genetic traits. One was a 21,832-bp composite mobile element structure (MES_PM1), which was flanked by direct repeats of enterococcal IS1216V and was inserted into the chromosomal sasK gene; the target sequence (att) was 8 bp long and was duplicated at both ends of MES_PM1. MES_PM1 consisted of two regions: the 5′-end side 12.4-kb region carrying Tn551 (with ermB) and Tn5405-like (with aph[3′]-IIIa and aadE), similar to an Enterococcus faecalis plasmid, and the 3′-end side 6,587-bp region (MES_cat) that carries cat and is flanked by inverted repeats of IS1216V. MES_cat possessed att duplication at both ends and additional two copies of IS1216V inside. MES_PM1 represents the first enterococcal IS1216V-mediated composite transposon emerged in MRSA. IS1216V-mediated deletion likely occurred in IS1216V-rich MES_PM1, resulting in distinct resistance patterns in PM1-derivative strains. Another structure was a 6,025-bp tet-carrying element (MES_tet) on a 25,961-bp novel mosaic penicillinase plasmid (pPM1); MES_tet was flanked by direct repeats of IS431, but with no target sequence repeats. Moreover, the PM1 genome was deficient in a copy of the restriction and modification genes (hsdM and hsdS), which might have contributed to the acquisition of enterococcal multidrug resistance.     

Muscle RING-finger protein-1 (MuRF1) as a connector of muscle energy metabolism and protein synthesis

During pathophysiological muscle wasting, a family of ubiquitin ligases, including muscle RING-finger protein-1 (MuRF1), has been proposed to trigger muscle protein degradation via ubiquitination. Here, we characterized skeletal muscles from wild-type (WT) and MuRF1 knockout (KO) mice under amino acid (AA) deprivation as a model for physiological protein degradation, where skeletal muscles altruistically waste themselves to provide AAs to other organs. When WT and MuRF1 KO mice were fed a diet lacking AA, MuRF1 KO mice were less susceptible to muscle wasting, for both myocardium and skeletal muscles. Under AA depletion, WT mice had reduced muscle protein synthesis, while MuRF1 KO mice maintained nonphysiologically elevated levels of skeletal muscle protein de novo synthesis. Consistent with a role of MuRF1 for muscle protein turnover during starvation, the concentrations of essential AAs, especially branched-chain AAs, in the blood plasma significantly decreased in MuRF1 KO mice under AA deprivation. To clarify the molecular roles of MuRF1 for muscle metabolism during wasting, we searched for MuRF1-associated proteins using pull-down assays and mass spectrometry. Muscle-type creatine kinase (M-CK), an essential enzyme for energy metabolism, was identified among the interacting proteins. Coexpression studies revealed that M-CK interacts with the central regions of MuRF1 including its B-box domain and that MuRF1 ubiquitinates M-CK, which triggers the degradation of M-CK via proteasomes. Consistent with MuRF1's role of adjusting CK activities in skeletal muscles by regulating its turnover in vivo, we found that CK levels were significantly higher in the MuRF1 KO mice than in WT mice. Glucocorticoid modulatory element binding protein-1 and 3-hydroxyisobutyrate dehydrogenase, previously identified as potential MuRF1-interacting proteins, were also ubiquitinated MuRF1-dependently. Taken together, these data suggest that, in a multifaceted manner, MuRF1 participates in the regulation of AA metabolism, including the control of free AAs and their supply to other organs under catabolic conditions, and in the regulation of ATP synthesis under metabolic-stress conditions where MuRF1 expression is induced.     

X-ray structures of Aerococcus viridans lactate oxidase and its complex with D-lactate at pH 4.5 show an alpha-hydroxyacid oxidation mechanism

l-Lactate oxidase (LOX) belongs to a family of flavin mononucleotide (FMN)-dependent α-hydroxy acid-oxidizing enzymes. Previously, the crystal structure of LOX (pH 8.0) from Aerococcus viridans was solved, revealing that the active site residues are located around the FMN. Here, we solved the crystal structures of the same enzyme at pH 4.5 and its complex with d-lactate at pH 4.5, in an attempt to analyze the intermediate steps. In the complex structure, the d-lactate resides in the substrate-binding site, but interestingly, an active site base, His265, flips far away from the d-lactate, as compared with its conformation in the unbound state at pH 8.0. This movement probably results from the protonation of His265 during the crystallization at pH 4.5, because the same flip is observed in the structure of the unbound state at pH 4.5. Thus, the present structure appears to mimic an intermediate after His265 abstracts a proton from the substrate. The flip of His265 triggers a large structural rearrangement, creating a new hydrogen bonding network between His265-Asp174-Lys221 and, furthermore, brings molecular oxygen in between d-lactate and His265. This mimic of the ternary complex intermediate enzyme-substrate-O₂ could explain the reductive half-reaction mechanism to release pyruvate through hydride transfer. In the mechanism of the subsequent oxidative half-reaction, His265 flips back, pushing molecular oxygen into the substrate-binding site as the second substrate, and the reverse reaction takes place to produce hydrogen peroxide. During the reaction, the flip-flop action of His265 has a dual role as an active base/acid to define the major chemical steps. Our proposed reaction mechanism appears to be a common mechanistic strategy for this family of enzymes.     

Mitochondrial and nuclear genes suggest that stony corals are monophyletic but most families of stony corals are not (Order Scleractinia, Class Anthozoa, Phylum Cnidaria)

Modern hard corals (Class Hexacorallia; Order Scleractinia) are widely studied because of their fundamental role in reef building and their superb fossil record extending back to the Triassic. Nevertheless, interpretations of their evolutionary relationships have been in flux for over a decade. Recent analyses undermine the legitimacy of traditional suborders, families and genera, and suggest that a non-skeletal sister clade (Order Corallimorpharia) might be imbedded within the stony corals. However, these studies either sampled a relatively limited array of taxa or assembled trees from heterogeneous data sets. Here we provide a more comprehensive analysis of Scleractinia (127 species, 75 genera, 17 families) and various outgroups, based on two mitochondrial genes (cytochrome oxidase I, cytochrome b), with analyses of nuclear genes (ss-tubulin, ribosomal DNA) of a subset of taxa to test unexpected relationships. Eleven of 16 families were found to be polyphyletic. Strikingly, over one third of all families as conventionally defined contain representatives from the highly divergent "robust" and "complex" clades. However, the recent suggestion that corallimorpharians are true corals that have lost their skeletons was not upheld. Relationships were supported not only by mitochondrial and nuclear genes, but also often by morphological characters which had been ignored or never noted previously. The concordance of molecular characters and more carefully examined morphological characters suggests a future of greater taxonomic stability, as well as the potential to trace the evolutionary history of this ecologically important group using fossils.