Characteristics of the mitochondrial genome of Rana omeimontis and related species in Ranidae: Gene rearrangements and phylogenetic relationships.

The Omei wood frog (Rana omeimontis), endemic to central China, belongs to the family Ranidae. In this study, we achieved detail knowledge about the mitogenome of the species. The length of the genome is 20,120 bp, including 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and a noncoding control region. Similar to other amphibians, we found that only nine genes (ND6 and eight tRNA genes) are encoded on the light strand (L) and other genes on the heavy strand (H). Totally, The base composition of the mitochondrial genome included 27.29% A, 28.85% T, 28.87% C, and 15.00% G, respectively. The control regions among the Rana species were found to exhibit rich genetic variability and A + T content. R. omeimontis was clustered together with R. chaochiaoensis in phylogenetic tree. Compared to R. amurensis and R. kunyuensi, it was more closely related to R. chaochiaoensis, and a new way of gene rearrangement (ND6-trnE-Cytb-D-loop-trnL2 (CUN)-ND5-D-loop) was also found in the mitogenome of R. amurensis and R. kunyuensi. Our results about the mitochondrial genome of R. omeimontis will contribute to the future studies on phylogenetic relationship and the taxonomic status of Rana and related Ranidae species.

The Therapeutic Effects of Magnolia Officinalis Extraction on an Antibiotics-Induced Intestinal Dysbacteriosis in Mice.

Magnolia officinalis bark is a traditional Chinese medicine for gastrointestinal tract disorders. In this study, we explored the effects of M. officinalis extraction on intestinal flora to reveal its mechanism. Thirty SPF mice were divided into five groups: C (control), M (M. officinalis), A (antibiotics: cefradine and gentamicin sulfate), A&M (antibiotics + M. officinalis) and A&N (antibiotics + natural recovery). Faecal samples of all groups were collected and the taxonomic composition and diversity of bacteria was characterized using the 16S rRNA gene (16S). Alpha diversity showed gut bacteria diversity significantly decreased in the A group of mice but increased markedly after administration of M. officinalis extract. Beta diversity indicated that C, M and A&M shared similar bacterial community structure while A and A&N exhibited a different bacterial community. Furthermore, RDA combined with spearman correlation heatmap suggested the five physiological indicators (weight, fur, activity and feces) were highly correlated with bacterial community structure and diversity. Finally, functional categorization of the assigned OTUs was performed using the PICRUSt tool. The changes in PICRUSt inferred that function profile and metabolic pathways were observed in A and A&M, therefore the M. officinalis extract improved the intestinal flora of A&M and normalized its metabolic pathways gradually, improving mouse weight, fur quality, activity and feces qualities.

Complete mitochondrial genome sequence of the Himalayan Griffon, Gyps himalayensis (Accipitriformes: Accipitridae): Sequence, structure, and phylogenetic analyses.

This is the first study to describe the mitochondrial genome of the Himalayan Griffon, Gyps himalayensis, which is an Old World vulture belonging to the family Accipitridae and occurring along the Himalayas and the adjoining Tibetan Plateau. Its mitogenome is a closed circular molecule 17,381 bp in size containing 13 protein-coding genes, 22 tRNA coding genes, two rRNA-coding genes, a control region (CR), and an extra pseudo-control region (CCR) that are conserved in most Accipitridae mitogenomes. The overall base composition of the G. himalayensis mitogenome is 24.55% A, 29.49% T, 31.59% C, and 14.37% G, which is typical for bird mitochondrial genomes. The alignment of the Accipitridae species control regions showed high levels of genetic variation and abundant AT content. At the 5' end of the domain I region, a long continuous poly-C sequence was found. Two tandem repeats were found in the pseudo-control regions. Phylogenetic analysis with Bayesian inference and maximum likelihood based on 13 protein-coding genes indicated that the relationships at the family level were (Falconidae + (Cathartidae + (Sagittariidae + (Accipitridae + Pandionidae))). In the Accipitridae clade, G. himalayensis is more closely related to Aegypius monachus than to Spilornis cheela. The complete mitogenome of G. himalayensis provides a potentially useful resource for further exploration of the taxonomic status and phylogenetic history of Gyps species.

The first complete mitochondrial genome sequence of Nanorana parkeri and Nanorana ventripunctata (Amphibia: Anura: Dicroglossidae), with related phylogenetic analyses.

Members of the Nanorana genus (family Dicroglossidae) are often referred to as excellent model species with which to study amphibian adaptations to extreme environments and also as excellent keystone taxa for providing insights into the evolution of the Dicroglossidae. However, a complete mitochondrial genome is currently only available for Nanorana pleskei. Thus, we analyzed the complete mitochondrial genomes of Nanorana parkeri and Nanorana ventripunctata to investigate their evolutionary relationships within Nanorana and their phylogenetic position in the family Dicroglossidae. Our results showed that the genomes of N. parkeri (17,837 bp) and N. ventripunctata (18,373 bp) encode 13 protein-coding genes (PCGs), two ribosomal RNA genes, 23 transfer RNA (tRNA) genes, and a noncoding control region. Overall sequences and genome structure of the two species showed high degree of similarity with N. pleskei, although the motif structures and repeat sequences of the putative control region showed clear differences among these three Nanorana species. In addition, a tandem repeat of the tRNA-Met gene was found located between the tRNA-Gln and ND2 genes. On both the 5' and 3'-sides, the control region possessed distinct repeat regions; however, the CSB-2 motif was not found in N. pleskei. Based on the nucleotide sequences of 13 PCGs, our phylogenetic analyses, using Bayesian inference and maximum-likelihood methods, illustrate the taxonomic status of Nanorana with robust support showing that N. ventripunctata and N. pleskei are more closely related than they are to N. parkeri. In conclusion, our analyses provide a more robust and reliable perspective on the evolutionary history of Dicroglossidae than earlier analyses, which used only a single species (N. pleskei).

The complete mitochondrial genome sequence of the Sichuan Digging Frog, Kaloula rugifera (Anura: Microhylidae) and its phylogenetic implications.

The Sichuan Digging Frog (Kaloula rugifera) belongs to the family Dicroglossidae, which is endemic to northeastern Sichuan and southernmost Gansu provinces, in southwestern China. In this study, the complete mitochondrial genome of K. rugifera was sequenced. The mitogenome was 17,074bp in length, consisting of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a non-coding control region. As in other vertebrates, most mitochondrial genes are encoded on the heavy strand, except for ND6 and eight tRNA genes which are encoded on the light strand. The overall base composition of the K. rugifera is 30.32% A, 25.76% C, 29.72% T, and 14.20% G, which is consistent with the lowest frequency for G content in typical amphibian animals' mitochondrial genomes. The alignment of the Kaloula species control regions exhibited high genetic variability and rich A+T content. Besides, 3 types of tandem repeat units were also identified in the control region. Phylogenetic tree demonstrated that K. rugifera was clustered together with K. borealis and K. verrucosa and they had a close relationship with each other. The complete mitogenome of K. rugifera can provide an important data for the studies on phylogenetic relationship to further explore the taxonomic status of Kaloula species.

Complete mitochondrial genome sequence of the Asiatic Toad Bufo Gargarizans (Amphibia, Anura, Bufonidae).

The Asiatic toad Bufo gargarizans belongs to Bufonidae. This species is known from the Russian Far East, central, northern and north-eastern China, the Democratic People's Republic of Korea and Japan. In this study, the complete mitochondrial genome of B. gargarizans was sequenced. The mitogenome was 17,407 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a non-coding control region. As in other vertebrates, most mitochondrial genes are encoded on the heavy strand, except for ND6 and eight tRNA genes which are encoded on the light strand. The overall base composition of the B. gargarizans is 28.9% A, 28.2% T, 27.5% C, and 15.3% G. Phylogenetic analysis showed B. gargarizans was closely related to B. bankorensis and B. tibetanus. The complete mitogenome of B. gargarizans can provide an important data for the studies on phylogenetic relationship and population genetics to further explore the taxonomic status of this species.

The complete mitochondrial genome sequence of the Dark-spotted frog Pelophylax nigromaculatus (Amphibia, Anura, Ranidae).

The dark-spotted frog (Pelophylax nigromaculatus) belongs to Ranidae. This species is known from the Russian Far East, central, northern and north-eastern China, the Democratic People's Republic of Korea, the Republic of Korea, and Japan. In this study, the complete mitochondrial genome of P. nigromaculatus was sequenced. The mitogenome was 17 567 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a non-coding control region. As in other vertebrates, most mitochondrial genes are encoded on the heavy strand, except for ND6 and eight tRNA genes which are encoded on the light strand. The overall base composition of the P. nigromaculatus is 29.2% A, 27.4% T, 28.4% C, and 15.0% G. Phylogenetic analysis showed P. nigromaculatus was closely related to P. plancyi and P. chosenicus. The complete mitogenome of P. nigromaculatus can provide important data for the studies on phylogenetic relationship and population genetics to further explore the taxonomic status of this species.

Complete mitochondrial genome sequence of the Sichuan digging frog Kaloula rugifera (Anura: Microhylidae).

The Sichuan Digging Frog (Kaloula rugifera) belongs to the family Dicroglossidae, which is endemic to northeastern Sichuan and southernmost Gansu provinces, in southwestern China. In this study, the complete mitochondrial genome of K. rugifera was sequenced. The mitogenome was 17 074 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a non-coding control region. As in other vertebrates, most mitochondrial genes are encoded on the heavy strand, except for ND6 and eight tRNA genes which are encoded on the light strand. The overall base composition of the K. rugifera is 29.7% A, 30.3% T, 25.8% C, and 14.2% G. The alignment of the Kaloula species control regions exhibited high genetic variability and rich A + T content. Phylogenetic tree demonstrated that K. rugifera was clustered together with K. borealis and K. verrucosa and they had a close relationship with each other. The complete mitogenome of K. rugifera can provide an important data for the studies on phylogenetic relationship to further explore the taxonomic status of Kaloula species.

The complete mitochondrial genome sequence of the Xizang Plateau frog, Nanorana parkeri (Anura: Dicroglossidae).

The Xizang Plateau frog (Nanorana parkeri) belongs to the family Dicroglossidae, which distributes in southern and eastern Xizang, southern-most Qinghai in China, high elevations of north-central Nepal, Himalayan Bhutan, northeastern Kashmir and India. In this study, the complete mitochondrial genome of N. parkeri was sequenced. The mitogenome was 17,837 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA genes, and a non-coding control region (CR). As in other vertebrates, most mitochondrial genes are encoded on the heavy strand, except for ND6 and eight tRNA genes, which are encoded on the light strand. The overall base composition of the N. parkeri is A: 27.7 % A, T: 30.1 % T, C: 26.6% and G: 15.6%. The alignment of the Nanorana species CRs exhibited high genetic variability and rich A + T content. In comparison with the mtDNA sequences typical of vertebrates, a tandem duplication of the tRNA(Met) gene and a rearrangement of the tRNA(Thr), tRNA(Pro) and tRNA(Leu) (CUN) genes were found. The complete mitogenome of N. parkeri can provided an important data for the studies on phylogenetic relationship and population genetics to further explore the taxonomic status of this species.

Biodegradation of phenol by using free and immobilized cells of Acinetobacter sp. BS8Y.

Strain BS8Y with high biodegradation activity and high tolerance of phenol was isolated from activated sludge in an insulating material plant of China. This strain was capable of removing 99.2% of the initial 600 mg/l phenol in liquid minimal medium within 24 h and tolerating phenol at concentrations of up to 1,200 mg/ml. DNA sequencing and homologous analysis of the 16S rRNA gene identified that the strain BS8Y belonged to an Acinetobacter species. Polyvinyl alcohol was used as gel matrix to immobilize the strain BS8Y. The factors affecting the phenol degradation by immobilized cells and the phenol removal efficiency of free and immobilized cells were investigated; the stability of the immobilized cells is also reported. The results show that the immobilized cells could tolerate a higher phenol level and protected the bacteria much more effectively against changes in temperature and pH. The phenol degradation efficiency was high at up to 96% within 30 h, with an initial concentration of 800 mg/l phenol, and the immobilized cells showed better performance than the suspended cells. Reusability tests revealed that the immobilized cells were stable enough even after reuse for ten times or storing at 4°C for 35 d. These results demonstrate that immobilized Acinetobacter sp. BS8Y possesses a good application potential in the treatment of phenol-containing wastewater.

Cloning, expression and characterization of the putative nuclear transport factor 2 (NTF2) gene from moss Conocephalum conicum(L.) Dum.

Biomacromolecules import into the nucleus is a complex progress which requires the participation of several cytosolic factors, and nuclear transport factor 2 (NTF2) is one of essential components in nuclear trafficking. Its main role is to transport RanGDP from cytoplasm to nucleus by interacting with FxFG nucleoporin repeats. In the study a putative new gene, designated as CcNTF2, was obtained from the moss (Conocephalum conicum) cDNA library we have constructed. The full-length cDNA sequence is 913 bp in size contains a 372 bp open reading frame (ORF) flanked by a 195 bp 5'-untranslated sequence and a long 346 bp 3'-non-coding region, encoding 123 amino acids of 13,575.3 Da. Part of the genomic sequence was also cloned and sequenced, which is 1,602 bp long and possesses two exons and one intron. Alignment analysis showed that the CcNTF2 protein is high conserved among plant NTF2 and shares 81% similarity with the ones from Arabidopsis thaliana and Brassica rapa. The expression of wild-type CcNTF2 was detected by immunoblotting of extraction of C. conicum and it indicated the putative protein is integral. Through functional expression of CcNTF2-green fluorescent protein (GFP) in tobacco, it was demonstrated that CcNTF2 can accumulate at the nuclear rim. Site-directed mutagenesis analysis confirmed CcNTF2 P71K has influence on the protein import into nucleus. In addition, overexpression of CcNTF2 P71K was observed to be deleterious for the plant cell. It is the first illumination of NTF2 in moss, and our study established the primary foundation for further research on moss NTF2.

Enhanced secretion of adhesive recognition sequence containing hirudin III mutein in E. coli.

It has been previously shown that Escherichia coli L-asparaginase II (L-ASP) signal peptide is capable of being utilized to direct extracellular secretion of hirudin III (HV3) in shake flask. In this study HV3 muteins R33G34D35(S36)-HV3 were generated by introduction of adhesive recognition sequence RGD(S) into the non-functional region of HV3. The resultant recombinants were cultivated on 30 l bioreactor scale using L-ASP signal peptide expression system and the optimized fed-batch cultivation was well established. After cultivation for approximately 11 h the secreted product accumulated up to approximately 1 g l(-1), which means 17-fold increase in productivity compared to initial expression in shake flask. N-terminal analysis, pI measurement, and MALDI mass spectral analysis on mutein R33G34D35S36-HV3 confirmed the authenticity of the product. Compared to wild-type HV3 and R33G34D35HV3, the mutein R33G34D35S36-HV3 exhibits the improved pharmacological activity. Collectively, a novel secretion strategy using L-ASP signal peptide for the rapid, efficient and cost-effective production of HV3 mutein possessing improved pharmacological activity on bioreactor scale has been well established. Using this expression system downstream processing becomes very simple because secreted product is mature, soluble, active, and without N-terminal extension of Met, which is quite critical for most therapeutic protein to reduce the side effect in clinic use. Thus, it provides a promising alternative for extracellular production of other difficult-to-express protein for biopharmaceutical use.

[Protective effects of shark hepatic stimulator substance against acute hepatic injury induced by acetaminophen in mice].

AIM: To investigate the protective effects of shark hepatic stimulator substance (sHSS) against acute hepatic injury induced by acetaminophen (AAP) in mice. METHODS: Acute hepatic injury model of Balb/c mice was induced by a single intraperitoneal injection of AAP (200 mg.kg-1, i.p.). Serum ALT and AST activities were analyzed. The changes of microstructure and ultrastructure of hepatocyte were observed under optical and electronic microscope. The hepatocyte apoptosis was analyzed by flow cytometer and the expression level of Fas mRNA was determined by RT-PCR. RESULTS: The activities of serum ALT and AST were significantly decreased and both necrosis and inflammatory infiltration were improved in the mice treated with sHSS 3.0 and 1.5 mg.kg-1. sHSS (3.0 mg.kg-1) prevented the ultrastructural changes of hepatocytes caused by AAP, decreased the percentage of apoptotic cells, and downregulated the expression level of Fas mRNA. CONCLUSION: sHSS protected hepatocytes from AAP-induced injury, which might be associated with its protection of the mitochondria and inhibition of apoptosis and expression of Fas mRNA in hepatocytes.

[Purification and characterization of a uridine phosphorylase from Enterobacter aerogenes EAM-Z1].

A uridine phosphorylase(UPase) was isolation from Enterobacter aerogenes EAM-Z1 and purified by means of ammonium sulfate precipitation, DEAE-cellulose, Phenyl-Sepharose, DEAE-Sepharose, FPLC ion exchange, and Sephacryl S-200 column chromatography. The purified UPase showed homogeneity on the native polacrylamide gel electrophoresis. The UPase is a trimer of 43 kD subunits. Fifteen residues from the amino terminal end of UPase were identified as MRMVDLIATKRDGGE. The isoelectric point was pH 4.46. Michaelis constant for uridine was 0.29 mmol/L. The UPase has a maximal activity at a pH value of 7.8 and 50 degrees C. The UPase could catalyses the phosphorolysis of uridine, thymidine, 5-Fluorouridine, 5-Fluoro-2'-deoxyuridine, uracil-beta-D-arbinofuranoside, and could also catalyse the synthesis of 5-Fluorouridine, a better prodrug form of the anticancer drug 5-fluorouracil, from 5-fluorouracil and uridine, and 47% uridine was converted to 5-Fluoro-uridine.

[Studies on biotransformation of 5-FUR].

Conditions for biotransformation and purification of FUR were investigated. The result showed that when the cell concentration of E. aerogenes was 10% (w/v), the temperature and pH were 7.8 and 60 degrees C respectively, 59.7% UR was converted to FUR. It also demonstrated that the ideal procedure for the purification of FUR is silica gel column chromatography by two elution systems (S1:CHCl3:CH3OH:H2O = 61:13:1, and S2; CH3COOCH2CH3:CH3OH:CH3COOH:H2O = 12:3:3:2) with the purity and yield of 98.0% and 86.0% respectively.