Mutagenesis, heterogeneous gene expression, and purification and amino acid substitution analyses of plant peroxidase, PrxA3a

We introduced mutations into prxA3a, a peroxidase gene of hybrid aspen, Populus kitakamiensis, to substitute the amino acid residues at the surface of the protein, and analyzed substrate specificities. PrxA3a and mutated enzymes heterogeneously gene expressed in Saccharomyces cerevisiae were purified by Ni affinity chromatography, hydrolysis of sugar chain (Endoglycosidase H_f) and gel filtration. The substrate specificities were altered by substituted amino acid residues. PrxA3a F77Y A165W acquired the substrate specificity to m-chlorophenol. PrxA3a F77Y and PrxA3a F77YA165W could polymerize sinapyl alcohol. In addition, PrxA3a A165W, F77Y, and F77YA165W improved cytochrome c oxidizing activity. These substituted amino acid residues should function as a catalytic site outside of the heme pocket.     

Bacterial community incorporating carbon derived from plant residue in an anoxic non-rhizosphere soil estimated by DNA-SIP analysis

Purpose

Plant residues are one of the main sources of soil organic matter in paddy fields, and elucidation of the bacterial communities decomposing plant residues was important to understand their function and roles, as the microbial decomposition of plant residues is linked to soil fertility. We conducted a DNA stable isotope probing (SIP) experiment to elucidate the bacterial community assimilating 13-carbon (¹³C) derived from plant residue under an anoxic soil condition. In addition, we compared the bacterial community with that under the oxic soil condition, which was elucidated in our previous study (Lee et al. in Soil Biol Biochem 43:814–822, 2011).

Materials and methods

We used the ¹³C-labeled dried rice callus cells as a model of rice plant residue. A paddy field soil was incubated with unlabeled and ¹³C-labeled callus cells. DNA extracted from the soils was subjected to buoyant density gradient centrifugation to fractionate ¹³C-enriched DNA. Then, polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis of bacterial 16S rDNA band patterns and band sequencing method were used to evaluate bacterial community.

Results and discussion

DGGE analysis showed that the band patterns in the ¹³C-enriched fractions were distinctly changed over time, while the changes in the community structure before fractionation were minor. Sequencing of the ¹³C-labeled DGGE bands revealed that Clostridia were a major group in the bacterial communities incorporating the callus-derived carbon although Gram-negative bacteria, and Actinobacteria also participated in the carbon flow from the callus under the anoxic condition. The proportion of Gram-negative bacteria and Actinobacteria increased on 14 days after the onset of incubation, suggesting that the callus was decomposed by diverse bacterial members on this phase. When the bacterial groups incorporating the ¹³C were compared between under anoxic and oxic soil conditions, the composition was largely different under the two opposite conditions. However, some members of Gram-negative bacteria were commonly found under the anoxic and oxic soil conditions.

Conclusions

The majority of bacterial members assimilating the callus carbon was Clostridia in the soil under anoxic conditions. However, several Gram-negative bacterial members, such as Acidobacteria, Bacteroidetes, and Proteobacteria, also participated in the decomposition of callus under anoxic soil conditions. Our study showed that carbon flow into the diverse bacterial members during the callus decomposition and the distinctiveness of the bacterial communities was formed under the anoxic and oxic soil conditions.

     

Epidemiological study on periodontal diseases and some other dental disorders in dogs

The prevalence of dental disorders in dogs was studied by applying index systems for human with some modifications. A total of 251 mongrel dogs including 143 stray dogs kept in the Animal Protection Offices in Tokyo and Hokkaido and 108 pet dogs visiting veterinary clinicians in Chiba Prefecture and Hokkaido were used. Periodontitis was prevalent among these dogs regardless of their sources and its incidence was increased with age. The lesion was more severe and more frequent in the premolar and molar regions than in the maxillary and mandibular incisor regions. Missing of teeth was observed at a high and increasing incidence with age. The tooth most commonly lost was the first premolar, followed by the other premolars and molars, where severe periodontitis was frequently found. Calculus was seen on many teeth, and aging agravated its prevalence and severity. Dental caries was observed in stray dogs, but neither to a serious degree nor at a significant level. These findings emphasize the necessity of dental hygiene, proper dental care and continuous periodical survey for dogs.     

The effect of rhizosphere of various plants on the growth of Rhizobium

As elucidated in the previous paper¹⁾, the rhizosphere of the host plant maintains more abundant root nodule bacteria than non-rhizosphere soil. The success of seed inoculation of the root nodule bacteria is supported by the multiplication in the vicinity of the host plant.     

Histological examination of callogenesis and adventitious embryogenesis in immature ovary culture of grapevine (Vitis vinifera L.)

Summary

Histological examinations of callogenesis and adventitious embryogenesis in immature ovary culture of grapevine (Vitis vinifera L. ‘Neo Mat’) were carried out during different phases of ontogenetic development. Adventitious embryos and/or embryogenic calli were obtained when immature ovary explants were cultured on a callus induction medium (C medium) for two months followed by transfer of callus-forming explants onto an embryogenesis induction medium (E medium). Microscopic observations of serial sections of the explants revealed that the calli formed on C medium were initiated preferentially from receptacle parenchyma cells. No cell division in the embryo sac was observed and most of them degenerated four weeks after the onset of culture. Two to four weeks after transfer of the explants onto E medium, calli characterized by dense cytoplasm, conspicuous nuclei and thick cell walls were newly formed in the initially-formed, receptacle-derived ones. Proembryos simultaneously developed in the newly formed calli, indicating that they were embryogenic calli. Cell division of embryo sacs was never observed even on E medium, and adventitious embryos and embryogenic calli were hence of somatic origin. Adventitious embryos developed asynchronously and passed through globular, heart, torpedo and cotyledonary stages. These adventitious embryos germinated and developed into plantlets following their transfer onto a plant growth regulator-free medium.     

Interspecific variations in age and size at settlement of 8 emperor fishes (Lethrinidae) at the southern Ryukyu Islands,Japan

Molecular and otolith analyses were conducted for 173 settlement-stage larvae of emperor fishes (family Lethrinidae) collected by light traps at Ishigaki Island, southern Japan, in July and August (summer season), to (1) present diagnostic DNA markers for identification of lethrinid species and (2) compare the size and age at settlement of each species. PCR–RFLP and direct nucleotide sequencing analyses identified 8 species. Size (standard length, SL) at settlement differed significantly between species; Lethrinus ornatus (mean SL ± SD, 12.8 ± 1.5 mm), L. obsoletus (14.2 ± 0.8 mm) and L. harak (15.8 ± 1.6 mm) settled at a smaller size than L. atkinsoni (17.0 ± 1.3 mm), L. genivittatus (17.3 ± 1.0 mm), L. olivaceus (18.1 ± 0.6 mm), L. nebulosus (18.6 ± 4.2 mm), and L. sp.2 reported by Lo Galbo et al. (J Mol Evol 54:754–762, 2002) (21.7 ± 1.4 mm). Age at settlement tends to increase with settlement size; L. obsoletus (mean age ± SD, 25.6 ± 1.2 days), L. atkinsoni (26.1 ± 2.1 days) and L. ornatus (26.3 ± 2.9 days) were younger at settlement than L. nebulosus (28.4 ± 2.1 days), L. harak (29.2 ± 1.7 days), L. olivaceus (29.5 ± 1.0 days), L. genivittatus (30.5 ± 1.7 days) and L. sp.2 (31.0 ± 2.0 days). Although our study showed interspecific variation in body size and age at settlement among 8 lethrinid species, further seasonal replication is necessary to clarify the general patterns.     

Detection of Cryptosporidium sp. Oocyst and Giardia sp. cyst in faucet water samples from cattle and goat farms in Taiwan

A survey on the presence of Cryptosporidium oocyst and Giardia cyst in livestock drinking water as well as the urban tap water throughout Taiwan was carried out. Water examination for the presence of the protozoa was conducted by filtering through a PTFE membrane followed by immunomagnetic separation (IMS) and immunostaining the sediment with commercially available monoclonal antibody against Cryptosporidium and Giardia. Of the 55 different water samples from various sources examined, 2 were found to contain both of Cryptosporidium oocyst and Giardia cyst, 1 was found to contain Cryptosporidium oocyst only. These protozoa-positive water samples, originating from underground well and from the mountain spring, were also used as drinking water for livestock. However, no Cryptosporidium oocyst was found in the city tap water. This is the first report of Cryptosporidium oocyst and Giardia cyst being found in the drinking water for livestock.     

Synthesis of morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) by dipeptidyl aminopeptidase IV derived from Aspergillus oryzae

Morphiceptin (Tyr-Pro-Phe-Pro-NH(2)), tetrapeptide, was synthesized using dipeptidyl aminopeptidase IV (DP IV, EC 3.4.14.5) derived from Aspergillus oryzae RIB 915 as a catalyst. Tyr-Pro-OEt was incubated with Phe-Pro-NH(2) in the presence of DP IV under various conditions of temperature, concentrations of ethylene glycol, pH, reaction time, and others. Morphiceptin was obtained at 40% yield under the optimal reaction conditions: substrate, 4 mM Tyr-Pro-OEt.HCl and 20 mM Phe-Pro-NH(2).HCl; enzyme, DP IV, 0.275 nkat; solvent, 60% ethylene glycol containing 20 mM phosphate buffer at pH 7.0; amine, 4.2 mM diisopropylamine at 4 degrees C for 24 h. Amino group protection was unnecessary for synthesis of morphiceptin by DP IV.