TFR1 expression in induced sputum is associated with asthma severity.

Background: Asthma is characterized as a chronic inflammatory airway disease. Iron accumulation is related to asthma pathogenesis. Transferrin receptor 1(TFR1) expression is associated with intracellular iron overload in macrophages. In our study, we explored the association among TFR1 expression, the inflammatory macrophage phenotype, and asthma severity. Methods: Induced sputum was collected from 50 asthma patients. Real-time PCR was used to evaluate mRNA expression. The status of inflammatory macrophage phenotype was assessed using flow cytometry. Results: TFR1 levels were inversely correlated with forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) and FEV1/vital capacity (VC). Among inflammatory cytokines, TFR1 expression was positively correlated with IL-1ß, TNF-α, IL-6, IFN-γ, and IL-17A mRNA expression in induced sputum. Moreover, TFR1 expression was positively correlated with the number of proinflammatory M1 macrophages and iNOS expression in induced sputum. Neutrophil counts in induced sputum were significantly and positively related to TFR1 expression. Furthermore, TFR1 expression showed an increasing trend in asthma patients with no family history. Our findings indicated that TFR1 expression was consistent with the asthma severity index, especially the proinflammatory M1 macrophage phenotype. TFR1 expression may be a good marker to indicate asthma severity.

Studies revealing bioremediation potential of the strain Burkholderia sp. GB-01 for abamectin contaminated soils.

Burkholderia sp. GB-01 strain was used to study different factors affecting its growth for inoculum production and then evaluated for abamectin degradation in soil for optimization under various conditions. The efficiency of abamectin degradation in soil by strain GB-01 was seen to be dependent on soil pH, temperature, initial abamectin concentration, and inoculum size along with inoculation frequency. Induction studies showed that abamectin depletion was faster when degrading cells were induced by pre-exposure to abamectin. Experiments performed with varying concentrations (2-160 mg Kg(-1)) of abamectin-spiked soils showed that strain GB-01 could effectively degrade abamectin over the range of 2-40 mg Kg(-1). The doses used were higher than the recommended dose for an agricultural application of abamectin, taking in account the over-use or spill situations. A cell density of approximately 10(8) viable cells g(-1) dry weight of soil was found to be suitable for bioremediation over a temperature range of 30-35°C and soil pH 7.5-8.5. This is the first report on bacterial degradation of abamectin in soil by a Burkholderia species, and our results indicated that this bacterium may be useful for efficient removal of abamectin from contaminated soils.

[Study on the atrazine-degrading genes in Arthrobacter sp. AG1].

Atrazine could be used as the sole carbon, nitrogen and energy sources for growth by strain Arthrobacter sp. AG1, and the atrazine-degrading genes of AG1 were found to be the combination of trzN, atzB and atzC. The atrazine chloride hydrolysase gene trzN was cloned by PCR amplification,whose sequence shared 99% identity with that of Norcardioides sp. C190. Two large plasmids were found in AG1,and trzN and atzB were confirmed to be localized on the larger plasmid pAG1 by the method of southern hybridization. Subculture of AG1 in liquid LB for three generations, 34% of the subsequent cells were found to lose degrading activity, however, neither plasmid was lost. PCR amplification results showed that the mutants had only lost the trzN gene instead of atzB and atzC. It was deduced that mutation might be due to the trzN gene deletion from the plasmid. This study provided new evidence that atrazine metabolic genotypes were resulted from horizontal gene transfer between different bacteria under environmental selective pressure.

[Isolation and characterization of an atrazine-degrading bacterium strain SA1].

Atrazine (AT), a kind of herbicide for the pre and post-emergence control of annual and broad leaved weeds and perennial grasses, had been widely used in the world. However, the extensive use of atrazine had led to widespread environmental pollution. A bacterium strain SA1, which could degrade AT completely, was isolated from an atrazine-degrading consortium by long-time repeated alternative cultivation and plate striking. Combining cultural and physiobiochemical characteristics with 16S rDNA sequence analysis, SA1 was identified as Pseudomonas sp.. SAl could use atrazine as the sole carbon, nitrogen and energy sources for growth, and the main product of AT biodegradation was cyanuric acid. AT degrading activity of SA1 was not affected by the addition of nitrogen resources. However, cyanuric acid could be degraded quickly to an undetectable level when glucose was added. The optimal temperature and pH value for SAl growth was 37 degrees C and pH7, respectively. Atrazine could be degraded efficiently by the resting cells of SAl under the conditions of 10 degrees C - 40 degrees C or pH value 4-11, and SA1 had a wide range of temperature and pH value for AT degradation when compared with ADP. atzABCD and conserved sequence of tnpA gene of IS1071 could be amplified from SA1, and these genes could be lost during subculture.

[Isolation of a monocrotophos-degrading bacterial strain and characterization of enzymatic degradation].

A monocrotophos [dimethyl (E)-1-2-methylcarbamoylvinylphosphate or MCP] -degrading strain named as M-1 was isolated from sludge collected from the wastewater treatment pool of a pesticide factory and identified as Paracoccus sp. according to its morphology and biochemical properties and 16S rDNA sequence analysis. Using MCP as a sole carbon source, M-1 was able to degrade MCP(100 mg x L(-1)) by 92.47% in 24 h. The key enzyme(s) involved in the initial biodegradation of monocrotophos in M-1 was shown to be constitutively expressed cytosolic proteins and showed the greatest activity at pH 8.0 and 25 degrees C, with its Michaelis-Mentn's constant (K(m)) and maximum degradation rate (V(max)) of 0.29 micromol x mL(-1) and 682.12 micromol (min x mg)(-1) respectively. This degrading enzyme(s) was sensitive to high temperature, but kept high activity under alkaline conditions.

[Construction of a versatile degrading bacteria Pseudomonas putida KT2440-DOP and its degrading characteristics].

Triazophos is a kind of organophosphorous pesticide which was widely used by farmers all over the world in 1990's. It is effective in controlling pesticide but harmful to human beings. Bioremediation is an effective and economic method to treat environment that has been polluted by hazardous organic compounds, so researchers paid much attention in this area. Most of which focused on isolating functional bacteria, studying its degrading mechanism, and cloning degradation-related genes. MP-4 was isolated from soil polluted by Triazophos for a long time and identified as Ochrobactrum sp.. The triazophos hydrolase (tpd) gene was cloned by the method of shotgun cloning, and the sequences were determined and analyzed. In the former tests it was found that there was only 18 base pairs different in tpd gene from mpd gene, which was isolated from methyl parathion degrading strain Pseudomonas putida DLL-1. Enzyme TPD can hydrolyze triazophos and methyl parathion while MPD cannot hydrolyze triazophos. Pseudomonas putida KT2440 is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes. This bacterium is known for its diverse metabolism and potential for development of biopesticides and plant growth promoters because of its ability to colonize rhizosphere of crop plants. Tpd gene was isolated from the genomic DNA of Ochrobactrum sp. MP-4 by PCR amplification. Recombinant plasmids pTPD was constructed by ligating tpd gene into broad host vector pBBRMCS-5. With the help of plasmid pRK2013, pTPD was transferred into Pseudomonas putida KT2440 to construct KT2440-DOP. KT2440-DOP can degrade many organophosphate pesticides and aromatics compounds. The specific activity of organophosphate hydrolase of KT2440-DOP was approximately 2 times of MP-4. Later, parameters affecting bioremediation of Organophosphate pesticide in soil using KT2440-DOP will be studied.

[Cloning of the ectoine biosynthesis gene ectABC from Halomonas sp. BYS-1 and salt stressed expression in Escherichia coli].

Ectoine was the main compatible solute of moderately halophilic bacteria. In order to clone the ectABC gene which involved in the ectoine biosynthesis pathway from total DNA of moderately halophilic bacteria Halomonas sp. BYS-1, firstly a 750bp fragment of ectABC gene was amplified by PCR using combinations of forward primers and reverse primers designed according to the ectABC genes of Halomonas elongata 2851T and Halomonas elongata DSM3043. Then the upstream and downstream sequences of the 750bp fragment were amplified by SEFA PCR (SElf-Formed Adaptor PCR), a new PCR method amplified relatively long flanking sequences from tagged sequences in a simple way without enzyme excision and ligation. The 3532bp fragment include 2423bp ectABC, 980bp upstream sequences and 129bp downstream sequences were cloned from Halomonas sp. BYS-1 using a pair of conserved primers designed according to acquired sequences by SEFA PCR. The GenBank accession number of the 3532bp fragment is DQ017757. ORF analysis revealed that ectA, ectB, ectC cluster to an operon, the size of ectA, ectB, and ectC were 573bp, 1251bp and 387bp respectively. The predicted molecular masses of the encoded proteins were 21.1kDa (191 amino acids, EctA), 45.7 kDa (417 amino acids, EctB), and 14.5 kDa (129 amino acids, EctC) respectively. The 3532bp fragment was ligated to the MCS site of vector pUC19 and transformed E. coli DH5alpha to construct E. coli (pUC19ECT). Transformant E. coli (pUC19ECT) could synthesis ectoine under salt stress, the intracellular ectoine level were 7.1, 19.4 and 32.3 micromol/(g x dry x wt) when the salinities of the mediums were 0, 0.4 and 0.8mol/L sodium chloride respectively. But the accumulation of ectoine could not promote the growth of E. coli (pUC19ECT)under high salinity.

[Horizontal transfer of environmental pollutant-degrading gene and application in bioremediation].

Horizontal gene transfer, unlike vertical gene transfer, is a means of genetic communication in bacteria. In the special polluted environment, horizontal transfer of polluted-degrading gene has significant functions. Study on horizontal transfer of degrading gene in polluted environment may deepen our understanding of the mechanism of bacterial adaptation to the organic-polluted environment. In the practical application in bioremediation, horizontal transfer of degrading gene can be regulated to promote degrading ability of microorganisms. In this article, we will review the advances in the study on mechanisms of genetic interactions among bacteria, the effect of degrading gene transfer in contaminated environment on microorganisms'adaptation to contaminated environment and the degradation of the pollutants.

[Construction of multifunctional genetically engineered pesticides-degrading bacteria by homologous recombination].

Construction of multifunctional pesticides-degrading genetically engineered microorganisms (GEMs) is increasing important in the bioremediation of various pesticides contaminants in environment. However, construction of genetically stable GEMs without any exogenous antibiotic resistance is thought to be one of the bottlenecks in GEMs construction. In this article, homologous recombination vectors with the recipient's 16S rDNA as homologous recombination directing sequence (HRDS) and sacB gene as double crossover recombinants positive selective marker were firstly constructed. The methyl parathion hydroalse gene (mpd) was inserted into the 16S rDNA site of the carbofuran degrading strain Sphingomonas sp. CDS-1 by homologous recombination single crossover in the level of about 3.7 x 10-(7) - 6.8 x 10(-7). Multifunctional pesticides-degrading GEMs with one or two mpd genes inserted into the chromosome without any antibiotic marker were successfully constructed. The homologous recombination events were confirmed by PCR and southern blot methods. The obtained GEMs were genetically stable and could degrade methyl parathion and carbofuran simultaneously. The insertion of mpd gene into rrn site did not have any significant effect on recipient' s physiological and original degrading characteristics. The methyl parathion hydrolase (MPH) was expressed at a relatively high level in the recombinants and the recombinant MPH specific activity in cell lysate was higher than that of original bacterium (DLL-1) in every growth phase tested. The highest recombinant MPH specific activity was 6.22 mu/tg. In this article, we describe a first attempt to use rRNA-encoding regions of Sphingomonas strains as target site for expression of exogenous MPH, and constructed multifunctional pesticides degrading GEMs, which are genetically stable and promising for developing bioremediation strategies for the decontamination of pesticides polluted soils.

[Isolation and characteristic of a moderately halophilic bacterium accumulated ectoine as main compatible solute].

A moderately halophilic bacterium(designated strain I15) was isolated from lawn soil. Based on the analysis of 16S rDNA (GenBank accession number DQ010162), morphology, physiological and biochemical characteristics, strain I15 was identified as Virgibacillus marismortuii. This strain was capable of growing under 0% approximately 25% NaCl, and exhibited an optimum NaCl concentration of 10% and an optimum temperature of 30 degrees C and an optimum pH of 7.5 - 8.0 for its growth, respectively. Under hyperosmotic stress, strain 115 accumulated ectoine as the main compatible solute. Under 15% NaCl conditions the intracellar ectoine can reach to 1.608 mmol/(g x cdw), accounted for 89.6% of the total compatible solutes. The biosynthesis of ectoine was under the control of osmotic, and the accumulated ectoine synthesized intraceilularly can released under hypoosmotic shocks and resynthesis under hyperosmotic shock rapidly.