Therapeutic role and potential mechanisms of active Vitamin D in renal interstitial fibrosis

Vitamin D, especially its most active metabolite 1,25-dihydroxyvitamin D₃ or calcitriol, is essential in regulating a wide variety of biologic processes, such as calcium homeostasis, immune modulation, cell proliferation and differentiation. Clinical studies show that the circulating level of calcitriol is substantially reduced in patients with chronic renal insufficiency. Administration of active Vitamin D results in significant amelioration of renal dysfunction and fibrotic lesions in various experimental models of chronic kidney diseases. Active Vitamin D elicits its renal protective activity through multiple mechanisms, such as inhibiting renal inflammation, regulating renin–angiotensin system and blocking mesangial cell activation. Recent studies indicate that calcitriol induces anti-fibrotic hepatocyte growth factor expression, which in turn blocks the myofibroblastic activation and matrix production in interstitial fibroblasts. Furthermore, in vivo and in vitro studies demonstrate that active Vitamin D effectively blocks tubular epithelial to mesenchymal transition (EMT), a phenotypic conversion process that plays a central role in the evolution of renal interstitial fibrosis. Together, it is becoming increasingly clear that a high level of active Vitamin D may be obligatory in the maintenance of normal kidney structure and function. Thus, supplementation of active Vitamin D could be a rational strategy for the therapeutics of chronic kidney diseases.     

Establishing a minimum dataset for soil quality assessment based on soil properties and land-use changes

To assess soil quality, a minimum dataset (MDS) of soil properties has to be proposed commonly through calculating the total load of each candidate soil parameter on all of the qualified principal components by use of principal component analysis (PCA) and Norm-value computation. Considering intensive land-use changes, the method introduced in this study on MDS establishment integrates the quantified contributions of land-use type and land-use duration on each soil parameter by using multivariate analysis and mean multiple comparison. In this way, a MDS maximally representing all candidates with minimal loss of the soil quality information contained by those non-MDS soil parameters is established. The MDS proposed can not only well integrate the quantified influence of land-use changes and land-use duration on soil parameters, but is also quite flexible and extendable with the potential to be extrapolated to assess soil quality in other regions. Based on two sets of soil database obtained separately in 1985 and 2004, two MDSs established are compared with each other. It is found that only quite a small change in MDS components occurs during a 20-year period. For a better assessment of soil quality, it seems necessary to examine on what kind of temporal scale and how much MDS will change for a site-specific area with intensive land-use changes.     

Urea sensitization caused by separation of Helicobacter pylori RNA polymerase beta and beta' subunits

BACKGROUND: The beta and beta' subunits of RNA polymerase are fused in all Helicobacters, but separate in most other taxa. Prior studies had shown that this fusion is not essential for viability in culture or in vivo, but had not tested it for potentially important quantitative effects on phenotype. METHODS: The effect of separating rpoB and rpoC sequences on Helicobacter pylori growth was tested in culture and during mouse infection. RESULTS: Derivatives of strains X47 and SS1 carrying this "rpoBCsplit" allele colonized mice less vigorously than their wild-type parents in competition tests. With X47 rpoBCsplit, this reduced vigor was evident in wild-type mice, whereas with SS1 rpoBCsplit it was seen only in cytokine IL-10- and IL-12beta-deficient mice. In culture, the rpoBCsplit allele sensitized each of four strains tested (X47, SS1, 88-3887, and AM1) to urea, a metabolite that is secreted into the gastric mucosa; urea sensitization was more severe in X47 than in SS1 genetic backgrounds. The rpoBCsplit allele also caused poorer growth on Ham's F12 agar, a nutritionally limiting medium, but had little effect on sensitivity to mild acidity. CONCLUSIONS: H. pylori's normal RNA polymerase beta-beta' subunit fusion contributes quantitatively to fitness. We propose that urea, although important to H. pylori in vivo, also be considered inhibitory; and that H. pylori's natural beta-beta' subunit fusion helps it cope with urea exposure.     

Production and characterization of human CTLA4Ig expressed in transgenic rice cell suspension cultures

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4I g) fusion protein, a novel immunosuppressive agent, was expressed in transgenic rice cell suspension culture and its characteristics and in vitro activities were investigated. The expression vector pMYN409 was constructed to express hCTLA4I g under the control of rice alpha-amylase 3D (RAmy3D) promoter. Transgenic calli were prepared by particle bombardment mediated transformation and were screened for hCTLA4I g expression using ELISA. Under the induction condition by sugar starvation, suspension-cultured rice cells secreted hCTLA4I g into the media up to 31.4 mg/L in flask culture. The rice-derived hCTLA4Ig (hCTLA4IgP) was purified from the culture media with affinity chromatography using protein A and compared with CHO-derived hCTLA4Ig (hCTLA4IgM). Recombinant hCTLA4IgP has molecular weight of approximately 50 kDa on SDS-PAGE under reducing condition, which is a little different from that of hCTLA4IgM probably due to the difference of carbohydrate chain structures. Purified hCTLA4IgP was biologically active and was confirmed to suppress T-cell proliferation.     

Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.     

Ubiquinone-10 production using Agrobacterium tumefaciens dps gene in Escherichia coli by coexpression system

Ubiquinone (Coenzyme Q; abbreviation, UQ) acts as a mobile component of the respiratory chain by playing an essential role in the electron transport system, and has been widely used in pharmaceuticals. The biosynthesis of UQ involves 10 sequential reactions brought about by various enzymes. In this study we have cloned, expressed the decaprenyl diphosphate synthase, designated dps gene, from Agrobacterium tumefaciens, and succeeded in detecting UQ-10 in addition to innate UQ-8 in Escherichia coli. Furthermore, the production of UQ-10 was higher than UQ-8. To establish an efficient expression system for UQ-10 production, we used genes, including ubiC, ubiA, and ubiG involved in UQ biosynthesis in E. coli, to construct a better co-expression system. The expression coupled by dps and ubiCA was effective for increasing UQ-10 production by five times than that by expressing single dps gene in the shake flask culture. To study for a large-scale production of UQ-10 in E. coli, fed-batch fermentations were implemented to achieve a high cell density culture. A cell concentration of 85.40 g/L and 94.58 g/L dry cell weight (DCW), and UQ-10 content of 50.29 mg/L and 45.86 mg/L was obtained after 32.5 h and 27.5 h of cultivation, subsequent to isopropyl-β-d-thiogalactopy ranoside and lactose induction, respectively. In addition, plasmid stability was maintained at high level throughout the fermentation.     

Production of 1,3-propanediol from Glycerol by Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Using Incompatible Plasmids System

1,3-Propanediol (1,3-PD) has numerous applications in polymers, cosmetics, foods, lubricants, and medicines as a bifunctional organic compound. The genes for the production of 1,3-PD in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, and gdrAB, which encodes glycerol dehydratase reactivating factor, are naturally under the control of different promoters and are transcribed in different directions. These genes were coexpressed in E. coli using two incompatible plasmids (pET28a and pET22b) in the presence of selective pressure. The recombinant E. coli coexpressed the glycerol dehydratase, 1,3-propanediol oxidoreductase and reactivating factor for the glycerol dehydratase at high levels. In a fed-batch fermentation of glycerol and glucose, the recombinant E. coli containing these two incompatible plasmids consumed 14.3 g/l glycerol and produced 8.6 g/l 1,3-propanediol. In the substitution case of yqhD (encoding alcohol dehydrogenase from E. coli) for dhaT, the final 1,3-propanediol concentration of the recombinant E. coli could reach 13.2 g/l.