[Effects of Nitrogen Fertilizer and Straw Returning Methods on N2O Emissions in Wheat-Maize Rotational Soils].

In order to explore the impacts of nitrogen fertilizer and straw returning methods on N2O emissions, a two-factor split-zone design was adopted for experimentation under the winter wheat-summer maize rotation model in the Guanzhong area of Shanxi, China. The main areas of interest were conventional nitrogen (G) and reduced nitrogen (70% G); the sub-areas were straw no return (N), straw return (S), and straw return + biochar (SB); we analyzed their impacts on N2O emissions and crop yield, and the relationships with related impact factors. The results showed that the N2O emissions peaks appeared in the wheat season and maize season treatments within 5-16 days after fertilization, and also appeared after rainfall. The N2O flux was significantly and positively correlated with soil temperature and NH4+-N content. Regardless of the wheat season, maize season, or annual total N2O emissions, the 70% GSB treatment was the lowest and the GS treatment was the highest. At the same level of nitrogen application, S treatment increased N2O emissions, SB treatment could reduce N2O emissions, both S and SB treatments could significantly increase crop yields, and SB production increased more; 70%G-level annual N2O emissions, when compared with the G level, had been reduced by 40% to 48%, while the yield has not decreased significantly. Through comprehensive consideration, a reduction of nitrogen by 30% was achieved through the combination of straw + biochar on the basis of conventional nitrogen application, while ensuring high crop yields and the best N2O emissions reduction.

MicroRNA expression profiles of head and neck squamous cell carcinoma with docetaxel-induced multidrug resistance.

BACKGROUND: The purpose of this study was to investigate the potential role of variable microRNA (miRNA) expression in the development of multidrug resistance (MDR) in head and neck cancer. METHODS: Head and neck squamous cell carcinoma cell lines UMSCC-1 and SQ20B were treated with docetaxel at increasing concentrations to develop resistant cell lines. Parental and resistant cells were treated with cisplatin, 5-fluorouracil, paclitaxel, methotrexate, and doxorubicin to confirm cross-resistance. The miRNA pattern of resistant cells was then compared with their parental cells. RESULTS: Docetaxel treatment successfully induced resistance primarily and induced multidrug cross-resistance. Resistant cells showed significant downregulation of miR-100, miR-130a, and miR-197 and upregulation in miR-101, miR-181b, miR-181d, and miR-195 expression when compared with their parent cells (p < .01). Real-time polymerase chain reaction (PCR) analysis confirmed statistically significant downregulation in miR-100 and miR-130a and upregulation in miR-181d expression (p < .001). CONCLUSION: Alterations in miRNA expression has direct relationship to MDR in head and neck cancer and may serve as biomolecular targets for reversal of MDR.

Dietary rice protein isolate attenuates atherosclerosis in apoE-deficient mice by upregulating antioxidant enzymes.

Rice-based diets may have been reported to protect against the development of atherosclerosis; however, the underlying mechanism(s) for this protection remains unknown. In this report, the mechanism(s) contributing to the atheroprotective effects of rice-based diet was addressed using the apolipoprotein E knockout (apoE-/-) mice fed rice protein isolate (RPI) or casein (CAS). Reduced atherosclerotic lesions were observed in aortic sinus and enface analyses of the descending aorta in RPI-fed apoE-/- mice compared with CAS-fed mice. Plasma total- and HDL-cholesterol levels were not different amongst the two groups, suggesting alternative mechanism(s) could have contributed to the atheroprotective effect of rice-based diets. Plasma oxLDL and anti-oxLDL IgG levels were significantly decreased in RPI-fed compared to CAS-fed animals. Plasma and aortic tissue GSH levels and GSH:GSSG ratio were higher in RPI-fed mice compared to CAS-fed group. Interestingly, RPI feeding increased mRNA and protein expression of superoxide dismutase, and mRNA expression of catalase, glutathione peroxidase and glutathione reductase, key antioxidant enzymes implicated inhibiting oxidative stress leading to atherosclerosis. In conclusion, these findings suggest that the reduction in atherosclerotic lesions observed in mice fed the rice-based diet is mediated in part by inhibiting oxidative stress and subsequent oxLDL generation that could result in reduced foam cell formation, an early event during atherogenesis.

Progressive tumor features accompany epithelial-mesenchymal transition induced in mitochondrial DNA-depleted cells.

The growth of LNCaP, a human prostate adenocarcinoma cell line, and MCF-7, a human breast adenocarcinoma cell line, is initially hormone dependent. We previously demonstrated that LNrho0-8 and MCFrho0, derived from LNCaP and MCF-7 by depleting mitochondrial DNA (mtDNA), exhibited hormone-independent growth that led to progressed phenotypes. Here, we demonstrate that LNrho0-8 and MCFrho0 have invasive characters as evaluated by the ability of invasion through the extracellular matrix (ECM) in vitro. In addition, the induction of vimentin and the repression of E-cadherin expression in rho0 cells indicate that they are mesenchymal cells. Since LNrho0-8 and MCFrho0 were derived from epithelial cancer cell lines, LNCaP and MCF-7 must have lost epithelial features and gained the mesenchymal phenotype by epithelial-mesenchymal transition (EMT) during the mtDNA depletion. In the rho0 cell lines, the Raf/MAPK signaling cascade was highly activated together with the expressions of transforming growth factor-beta (TGF-beta) and type I TGF-beta receptor (TGF-betaRI). EMT requires cooperation of TGF-beta signaling with activation of the Raf/MAPK cascade, suggesting that EMT was induced in mtDNA depleted cells resulting in the acquisition of progressive tumor features, such as higher invasiveness and loss of hormone dependent growth. Our results indicate that decreasing mtDNA content induces EMT, enabling the progressive phenotypes observed in cancer.

Induction of acquired resistance to antiestrogen by reversible mitochondrial DNA depletion in breast cancer cell line.

Although the net benefits of tamoxifen in adjuvant breast cancer therapy have been proven, the recurrence of the cancer in an aggressive and hormone independent form has been highly problematic. We previously demonstrated the important role mitochondrial DNA (mtDNA) plays in hormone-independence in prostate cancer. Here, the role of mtDNA in breast cancer progression was investigated. We established hydroxytamoxifen (4-OHT) resistant HTRMCF by growing MCF-7, human breast adenocarcinoma cells, in the presence of 4-OHT. HTRMCF was cross-resistant to 4-OHT and ICI182,780 concurrent with the depletion of mtDNA. To further investigate the role of mtDNA depletion, MCF-7 was depleted of mtDNA by treatment with ethidium bromide. MCF Rho 0 was resistant to both 4-OHT and ICI182,780. Furthermore, cybrid (MCFcyb) prepared by fusion MCF Rho 0 with platelet to transfer mtDNA showed susceptibility to antiestrogen. Surprisingly, after withdrawal of 4-OHT for 8 weeks, HTRMCF and their clones became susceptible to both drugs concurrent with a recovery of mtDNA. Herein, our results substantiated the first evidence that the depletion of mtDNA induced by hormone therapy triggers a shift to acquired resistance to hormone therapy in breast cancer. In addition, we showed that mtDNA depletion can be reversed, rendering the cancer cells susceptible to antiestrogen. The fact that the hormone independent phenotype can be reversed should be a step toward more effective treatments for estrogen-responsive breast cancer.

Mitochondrial regulation of cancer associated nuclear DNA methylation.

The onset and progression of cancer is associated with the methylation-dependent silencing of specific genes, however, the mechanism and its regulation have not been established. We previously demonstrated that reduction of mitochondrial DNA content induces cancer progression. Here we found that mitochondrial DNA-deficient LNrho0-8 activates the hypermethylation of the nuclear DNA promoters including the promoter CpG islands of the endothelin B receptor, O6-methylguanine-DNA methyltransferase, and E-cadherin. These are unmethylated and the corresponding gene products are expressed in the parental LNCaP containing mitochondrial DNA. The absence of mitochondrial DNA induced DNA methyltransferase 1 expression which was responsible for the methylation patterns observed. Inhibition of DNA methyltransferase eliminated hypermethylation and expressed gene products in LNrho0-8. These studies demonstrate loss or reduction of mitochondrial DNA resulted in the induction of DNA methyltransferase 1, hypermethylation of the promoters of endothelin B receptor, O6-methylguanine-DNA methyltransferase, and E-cadherin, and reduction of the corresponding gene products.

Paenibacillus terrigena sp. nov., isolated from soil.

A novel Gram-positive bacterium, strain A35(T), was isolated from coastal soil at Chiba, Japan, and was identified as a member of the genus Paenibacillus on the basis of phenotypic and phylogenetic analyses. The bacterium was found to be a facultatively anaerobic and endospore-forming rod. The predominant menaquinone was MK-7, the major cellular fatty acid was anteiso-C(15 : 0) and the DNA G+C content was 48.1 mol%. The levels of 16S rRNA gene sequence similarity between strain A35(T) and Paenibacillus species with validly published names were less than 94 %. Strain A35(T) was clearly distinguishable from reference species for the genus Paenibacillus. Therefore, on the basis of these data, a novel species of the genus Paenibacillus, Paenibacillus terrigena sp. nov., is proposed. The type strain is A35(T) (=IAM 15291(T)=CCTCC AB206026(T)).

Reclassification of [Flavobacterium] ferrugineum as Terrimonas ferruginea gen. nov., comb. nov., and description of Terrimonas lutea sp. nov., isolated from soil.

Strain DY(T), which was isolated from garden soil in Japan, was subjected to a polyphasic taxonomic study. Sequence analysis of the 16S rRNA gene and the GyrB protein revealed that the closest relative of strain DYT was [Flavobacterium] ferrugineum Sickles and Shaw 1934, with 94.8 and 90.1 % similarity, respectively. The two strains had similar chemotaxonomic characteristics, with menaquinone 7 as the major quinone system, 47.2-48.9 mol% DNA G+C content and 15 : 0 iso, 15 : 1 iso, 17 : 0 iso 3-OH and summed feature 3 as the major fatty acids. Based on genotypic and phenotypic characteristics, [Flavobacterium] ferrugineum IAM 15098T could be clearly differentiated from other members of the genus Flavobacterium. Strain DYT and [Flavobacterium] ferrugineum IAM 15098T could be easily distinguished from neighbouring taxa by morphological features (non-motile, non-gliding and non-filamentous single cells). Therefore, it is proposed that [Flavobacterium] ferrugineum IAM 15098T and strain DYT represent two separate species of a new genus, Terrimonas gen. nov., with the names Terrimonas ferruginea comb. nov. (type species; type strain IAM 15098T=ATCC 13524T) and Terrimonas lutea sp. nov. (type strain DYT=IAM 15284T=CCTCC AB205006T), respectively.

Sphingomonas azotifigens sp. nov., a nitrogen-fixing bacterium isolated from the roots of Oryza sativa.

Three yellow-pigmented strains associated with rice plants were characterized by using a polyphasic approach. The nitrogen-fixing abilities of these strains were confirmed by acetylene reduction assay and nifH gene detection. The three strains were found to be very closely related, with 99.9 % 16S rRNA gene sequence similarity and greater than 70 % DNA-DNA hybridization values, suggesting that the three strains represent a single species. 16S rRNA gene sequence analysis indicated that the strains were closely related to Sphingomonas trueperi, with 99.5 % similarity. The chemotaxonomic characteristics (G+C content of the DNA of 68.0 mol%, ubiquinone Q-10 system, 2-OH as the only hydroxy fatty acid and homospermidine as the sole polyamine) were similar to those of members of the genus Sphingomonas. Based on DNA-DNA hybridization values and physiological characteristics, the three novel strains could be differentiated from other recognized species of the genus Sphingomonas. The name Sphingomonas azotifigens sp. nov. is proposed to accommodate these bacterial strains; the type strain is Y39T (=NBRC 15497T = IAM 15283T = CCTCC AB205007T).

Zoogloea oryzae sp. nov., a nitrogen-fixing bacterium isolated from rice paddy soil, and reclassification of the strain ATCC 19623 as Crabtreella saccharophila gen. nov., sp. nov.

Two strains of free-living diazotrophs isolated from soil from a rice paddy field were characterized by using a polyphasic approach. The novel strains, A-7T and A-4, were found to be very closely related, with 99.9% 16S rRNA gene sequence similarity and a DNA-DNA hybridization value of 89.5%, suggesting that they represent a single species. 16S rRNA gene sequence analyses indicated that the two strains fell within the Zoogloea lineage, with less than 96.7 % sequence similarity to other Zoogloea species. Chemotaxonomic characteristics of the novel strains, including DNA G + C content (65.1 mol%), the major quinone system (Q-8), predominant fatty acids (16:1omega7c and 16:0) and major hydroxy fatty acids (3-OH 10:0 and 3-OH 12:0), are similar to those of the genus Zoogloea. The novel strains showed positive results for floc formation which is accepted as confirmatory for species of the genus Zoogloea. However, the novel strains can be distinguished from the other species of Zoogloea by physiological characteristics. The name Zoogloea oryzae sp. nov. is therefore proposed for the novel strains with strain A-7T (= IAM 15218T = CCTCC AB 2052005T) as the type strain. Phylogenetic and chemotaxonomic analyses indicate that strain ATCC 19623, designated as a reference strain of Zoogloea ramigera, does not belong to the genus Zoogloea but to a new genus of Alphaproteobacteria. The name Crabtreella saccharophila gen. nov., sp. nov. is proposed for strain ATCC 19623T (= IAM 12669T).

Reclassification of Alcaligenes latus strains IAM 12599T and IAM 12664 and Pseudomonas saccharophila as Azohydromonas lata gen. nov., comb. nov., Azohydromonas australica sp. nov. and Pelomonas saccharophila gen. nov., comb. nov., respectively.

The aim of this study was to clarify the taxonomic position of the nitrogen-fixing and hydrogen-oxidizing bacteria Alcaligenes latus strains IAM 12599T, IAM 12664 and IAM 12665 and Pseudomonas saccharophila IAM 14368T. It was found that the type strain of Alcaligenes latus, IAM 12599T, showed 99 x 9 and 96 x 1 % 16S rRNA gene sequence similarity to strains IAM 12665 and IAM 12664, respectively. A comparison using DNA-DNA hybridization suggested that strains IAM 12599T and IAM 12665 belong to a single species (89 x 7 %) and that strain IAM 12664 (35 x 1 %) forms a separate species. The phenotypic characteristics also support the conclusion that these bacteria should be identified as two species of a new genus: Azohydromonas lata gen. nov., comb. nov. (type strain IAM 12599T=DSM 1122T=LMG 3321T=ATCC 29712T; reference strain IAM 12665=DSM 1123=LMG 3325=ATCC 29714) and Azohydromonas australica sp. nov. (type strain IAM 12664T=DSM 1124T=LMG 3324T=ATCC 29713T). Pseudomonas saccharophila IAM 14368T was found to be closely related to the phototrophic bacterium Roseateles depolymerans, with 96 x 8 % 16S rRNA gene sequence similarity, but the two bacteria are quite different with respect to their metabolism and some significant phenotypic characteristics, suggesting that they cannot be included in a single genus. Further studies on their nifH gene sequences, G+C content of the DNA and cellular fatty acid composition confirm that Pseudomonas saccharophila should be reclassified: the name Pelomonas saccharophila gen. nov., comb. nov. is proposed, with the type strain IAM 14368T (=LMG 2256T=ATCC 15946T).

Azospirillum oryzae sp. nov., a nitrogen-fixing bacterium isolated from the roots of the rice plant Oryza sativa.

The taxonomic position of the free-living diazotrophic strain COC8(T) isolated from rice was investigated based on phylogenetic analyses. 16S rRNA gene sequence analyses indicated that strain COC8(T) was closely related to the genus Azospirillum (96% similarity). Chemotaxonomic characteristics (G+C content of the DNA 66.8 mol%, Q-10 quinone system, 18:1omega7c as the major fatty acid and 14:0 3-OH and 16:0 3-OH as the major hydroxy fatty acids) were also similar to those of the genus Azospirillum. Based on its physiological characteristics, strain COC8(T) can clearly be differentiated from recognized species of Azospirillum. The name Azospirillum oryzae sp. nov. is proposed to accommodate this bacterial strain; the type strain is COC8(T) (=IAM 15130(T)=CCTCC AB204051(T)).

Pleomorphomonas oryzae gen. nov., sp. nov., a nitrogen-fixing bacterium isolated from paddy soil of Oryza sativa.

Five strains of free-living diazotrophs isolated from rice were characterized by using a polyphasic approach. The strains were found to be very closely related, with 99-100 % 16S rRNA gene sequence similarity and DNA-DNA hybridization values greater than 70 %, suggesting that they represent a single species. When compared with other recognized species, they showed not more than 93 and 89 % similarity for the 16S rRNA and nifH gene sequences, respectively. Phylogenetic distances showed that these isolates were distinct from other taxa within the alpha-Proteobacteria. Chemotaxonomic characteristics of these isolates included the DNA G + C content (62.1-63.1 mol%), the major quinone system (Q-10), predominant fatty acids (18 : 1omega7c, cyclo 19 : 0omega8c and 16 : 0) and major hydroxy fatty acids (14 : 0 3-OH, 18 : 0 3-OH and 16 : 0 3-OH). Based on phylogenetic and phenotypic analyses, these isolates are considered to represent a novel genus and species, for which the name Pleomorphomonas oryzae gen. nov., sp. nov. is proposed. The type strain is F-7(T) (=IAM 15079(T) = ATCC BAA-940(T) = DSM 16300(T)).

Phylogenetic analysis of Alysiella and related genera of Neisseriaceae: proposal of Alysiella crassa comb. nov., Conchiformibium steedae gen. nov., comb. nov., Conchiformibium kuhniae sp. nov. and Bergeriella denitrificans gen. nov., comb. nov.

A phylogenetic analysis based on 16S rRNA gene sequences reveals that Alysiella filiformis belongs to the family Neisseriaceae. The genus Simonsiella is phylogenetically separated by the genera Kingella and Neisseria. The species Simonsiella crassa and A. filiformis show a close phylogenetic relationship, with the 16S rDNA sequence similarity and the DNA-DNA hybridization representing 98.7% and 35%, respectively. Therefore, S. crassa should be transferred from the genus Simonsiella to the genus Alysiella as Alysiella crassa comb. nov. Simonsiella steedae and Simonsiella sp. of cat origin show strong genetic affinities and are distantly related with the type species of Simonsiella, S. mulleri. Thus, a new genus, Conchiformibium is proposed; Conchiformibium steedae comb. nov. and Conchiformibium kuhniae sp. nov. are accommodated in this new genus. On the basis of the phylogenetic, phenotypic and chemotaxonomic distinction from the genus Neisseria, N. denitrificans should be reclassified, for which a new genus and new combination Bergeriella denitrificans are proposed.

Dyella japonica gen. nov., sp. nov., a gamma-proteobacterium isolated from soil.

Three strains isolated from the soil of a garden in Tokyo, Japan, were characterized physiologically, biochemically and in terms of fatty acid profile, DNA-DNA relatedness and 16S rRNA gene sequence. The isolates were Gram-negative, aerobic, rod-shaped cells with polar flagellation. According to DNA-DNA similarity, the strains belonged to the same species. The bacteria grew at temperatures from 10 to 37 degrees C, with an optimum around 25-30 degrees C. Growth was observed at pH values from 5.6 to 8.0. The DNA G+C content ranged from 63.4 to 64.0 mol%. Phylogenetic analyses of 16S rRNA gene sequences revealed a clear affiliation with members of the family 'Xanthomonadaceae'. The closest relationship was seen with Fulvimonas soli and Frateuria aurantia, but, in terms of physiology and fatty acid profile, the bacteria described were rather distant from Fulvimonas and Frateuria. On the basis of phenotypic and phylogenetic distinctness, it is proposed that the isolates represent a novel species in a novel genus, namely Dyella japonica gen. nov., sp. nov. The type strain is XD53(T) (=IAM 15069(T)=DSM 16301(T)=ATCC BAA-939(T)).

Transfer of the misnamed [Alysiella] sp. IAM 14971 (=ATCC 29468) to the genus Moraxella as Moraxella oblonga sp. nov.

Phylogenetic analysis based on 16S rRNA gene sequences revealed that [Alysiella] sp. IAM 14971 (=ATCC 29468) is closely related to the genus Moraxella of the gamma-Proteobacteria (96-97 % similarity). The newly obtained phenotypic data also indicate that [Alysiella] sp. IAM 14971 is distinct from the genus Alysiella and similar to the genus Moraxella. On the basis of these results, the strain should be classified in the genus Moraxella, as Moraxella oblonga sp. nov. The type strain is IAM 14971T (=ATCC 29468T).

Transfer of Hyphomicrobium indicum to the genus Photobacterium as Photobacterium indicum comb. nov.

Hyphomicrobium indicum Johnson and Weisrock 1969 lacks true budding and hyphal branching, and some phenotypic characteristics are in contrast to other true hyphomicrobia. The major quinone system (ubiquinone Q-8), the G+C content of the DNA (40 mol%) and the cellular fatty acid composition (16 : 0, 16 : 1 and 18 : 1 as the major components, and 12 : 0 3-OH and 14 : 0 3-OH as the hydroxy fatty acids) of H. indicum are different from the genus Hyphomicrobium, but similar to the genus Photobacterium. Like the marine bacteria Photobacterium, H. indicum can be tolerant of sea water, while Hyphomicrobium cannot. Phylogenetic analyses of 16S rRNA and gyrB gene sequences revealed that H. indicum is most closely related to the genus Photobacterium of the gamma-Proteobacteria. Based on the phylogenetic, phenotypic and chemotaxonomic evidence, the results indicate that H. indicum should be transferred to the genus Photobacterium, and the name Photobacterium indicum comb. nov. (type strain, NBRC 14233(T)=ATCC 19614(T)) is proposed.

Phylogenetic analyses of the nitrogen-fixing genus Derxia.

Phylogenetic analyses of the 16S rRNA gene sequence indicate that the genus Derxia forms a distinct lineage in the beta-Proteobacteria. On the NJ tree Derxia has a low bootstrap value (30.9%) with Alcaligeneceae, and on the ML tree it shows an independent cluster separated from other families. Moreover, there is below 93.4% 16S rDNA sequence similarity between genus Derxia and the genera of the beta-Proteobacteria. These facts reveal that Derxia is not grouped with any known family of beta-Proteobacteria and should be placed as a separate genus of beta-Proteobacteria. The data on high G+C content (71 mol%), the cellular fatty acid composition, and the physiological characteristics of facultative hydrogen autotrophy and nitrogen fixation are unique for Derxia. The nifH gene sequence was found in this genus and phylogenetically compared among nitrogen-fixing bacteria to indicate that Derxia is clustered with the diazotrophs of beta-Proteobacteria.