In vivo evidence for the iron-binding activity of an iron-sulfur cluster assembly protein IscA in Escherichia coli

IscA is a key member of the iron-sulfur cluster assembly machinery in prokaryotic and eukaryotic organisms; however, the physiological function of IscA still remains elusive. In the present paper we report the in vivo evidence demonstrating the iron-binding activity of IscA in Escherichia coli cells. Supplement of exogenous iron (1 μM) in M9 minimal medium is sufficient to maximize the iron binding in IscA expressed in E. coli cells under aerobic growth conditions. In contrast, IscU, an iron-sulfur cluster assembly scaffold protein, or CyaY, a bacterial frataxin homologue, fails to bind any iron in E. coli cells under the same experimental conditions. Interestingly, the strong iron-binding activity of IscA is greatly diminished in E. coli cells under anaerobic growth conditions. Additional studies reveal that oxygen in medium promotes the iron binding in IscA, and that the iron binding in IscA in turn prevents formation of biologically inaccessible ferric hydroxide under aerobic conditions. Consistent with the differential iron-binding activity of IscA under aerobic and anaerobic conditions, we find that IscA and its paralogue SufA are essential for the iron-sulfur cluster assembly in E. coli cells under aerobic growth conditions, but not under anaerobic growth conditions. The results provide in vivo evidence that IscA may act as an iron chaperone for the biogenesis of iron-sulfur clusters in E. coli cells under aerobic conditions.     

Identification of a new QTL for <Emphasis Type="Italic">Fusarium</Emphasis> head blight resistance in the wheat genotype “Wang shui-bai”

Fusarium head blight (FHB) is one of the most devastating wheat diseases, causing both yield loss and quality reduction. To detect quantitative trait loci (QTL) responsible for FHB resistance, plants of the F _2:3 population derived from a ‘Wangshui-bai’ × ‘Sy95-7’ cross were artificially inoculated. Of 396 simple sequence repeats (SSRs), 125 amplified fragment length polymorphisms were used for FHB resistance QTL analysis. Five QTLs for FHB resistance were detected on chromosomes 3B, 6B, 7A, 1B and 2D. The effect of the QTL located on chromosome 3B on phenotypic variation was 31.69%, while that of the QTL found on 2D was the smallest and only accounted for 4.98% of the variation. The resistance alleles originated from ‘Wangshibai’ and association of the QTLs using these SSR markers may facilitate marker-assisted selection to improve FHB resistance in the wheat breeding programs of southwest China.     

Low penetrance of Leber's hereditary optic neuropathy in ten Han Chinese families carrying the ND6 T11484C mutation

Background

Leber’s hereditary optic neuropathy (LHON) is a maternally inherited disorder. The purpose of this investigation is to understand the role of mitochondrial haplotypes in the development of LHON associated with ND6 T14484C mutation in Chinese families.

Methods

One hundred fourteen subjects from ten Han Chinese families with LHON were studied by the clinical and genetic evaluation as well as molecular and biochemical analyses of mitochondrial DNA (mtDNA).

Results

Clinical evaluation revealed that ten families exhibited extremely low penetrance of visual impairment, with an average of 10%. In particular, ten (8 males/2 females) of 114 matrilineal relatives in these families exhibited the variable severity and age-at-onset in visual dysfunction. The average age-of-onset of vision loss was 19 years old. Molecular analysis of mitochondrial DNA (mtDNA) identified the homoplasmic T14484C mutation and distinct sets of variants, belonging to the Asian haplogroups B5b, D4, D4g1b, G3a2, R11, R11a and Z3, respectively. However, there was the absence of secondary LHON-associated mtDNA mutations in these ten Chinese families.

Conclusion

The low penetrance of vision loss in these Chinese pedigrees strongly indicated that the T14484C mutation was itself insufficient to produce a clinical phenotype. The absence of secondary LHON mtDNA mutations suggests that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the T14484C mutation in those Chinese families with low penentrace of vision loss. However, nuclear modifier genes and environmental factors appear to be modifier factors for the phenotypic manifestation of the T14484C mutation in these Chinese families.     

Parasiticidal activity of human α-defensin-5 against <Emphasis Type="Italic">Toxoplasma gondii</Emphasis>

Human defensins play a fundamental role in the initiation of innate immune responses to some microbial pathogens. In this paper, we show that human α-defensin-5 displays a parasiticidal role against Toxoplasma gondii, the causative agent of toxoplasmosis. Exposure of the tachyzoite form of T. gondii to defensin induced aggregation and significantly reduced parasite viability in a concentration-dependent peptide. Pre-incubation of tachyzoites with human α-defensin-5 followed by exposure to a mouse embryonal cell line (NIH/3T3) significantly reduced T. gondii infection in these cells. Thus, human α-defensin-5 is an innate immune molecule that causes severe toxocity to T. gondii and plays an important role in reducing cellular infection. This is the first report showing that human α-defensin-5 causes aggregation, leading to Toxoplasma destruction.     

Effect of chitosan‐alginate nanoparticles and ultrasound on the efficiency of gene transfection of human cancer cells

Background

Gene therapy has been used to treat a variety of health problems, but transfection inefficiency and the lack of safe vectors have limited clinical progress. Fabrication of a vector that is safe and has high transfection efficiency is crucial for the development of successful gene therapy. The present study aimed to synthesize chitosan‐alginate nanoparticles that can be used as carriers of the pAcGFP1‐C1 plasmid and to use these nanoparticles with an ultrasound protocol to achieve high efficiency gene transfection.

Methods

Chitosan was complexed with alginate and the pAcGFP1‐C1 plasmid at different charge ratios to create chitosan‐alginate‐DNA nanoparticles (CADNs). The average particle size and loading efficiency were measured. Plasmid DNA retardation and integrity were analysed on 1% agarose gels. The effect of CADNs and ultrasound on the efficiency of transfection of cells and subcutaneous tumors was evaluated.

Results

In the CADNs, the average size of incorporated plasmid DNA was 600–650 nm and the loading efficiency was greater than 90%. On the basis of the results of the plasmid DNA protection test, CADNs could protect the transgene from DNase I degradation. The transgene product expression could be enhanced efficiently if cells or tumor tissues were first given CADNs and then treated with ultrasound.

Conclusions

The use of CADNs combined with an ultrasound regimen is a promising method for safe and effective gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.

     

Purification and biochemical characterization of a 17 kDa fibrinolytic enzyme from <Emphasis Type="Italic">Schizophyllum commune</Emphasis>

A fibrinolytic enzyme of the mushroom, Schizophyllum commune was purified with chromatographic methods, including a DEAE-Sephadex A-50 ion-exchange column and gel filtrations with Sephadex G-75 and Sephadex G-50 columns. The analysis of fibrin-zymography and SDS-PAGE showed that the enzyme was a monomeric subunit that was estimated to be approximately 17 kDa in size. The fibrinolytic activity of the enzyme in plasminogen-rich and plasminogen-free fibrin plates was 1.25 and 0.44 U/ml, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as HYNIXNSWSSFID, which was highly distinguished from known fibrinolytic enzymes. The relative activity of the purified enzyme with an addition of 5 mM EDTA, Phosphoramidon, and Bestatin was about 76, 64, and 52%, respectively, indicating that it is a metalloprotease. The optimum temperature for the purified enzyme was approximately 45°C, and over 87% of the enzymatic activity was maintained as a stable state in a pH range from 4.0 to 6.0. Therefore, our results suggest that the potential thrombolytic agent from S. commune is a unique type of fibrinolytic enzyme.     

Management scenario evaluation for a large treatment wetland using a spatio-temporal phosphorus transport and cycling model

This paper describes the development of a two-dimensional, spatially distributed model to simulate coupled hydrologic and phosphorus (P) biogeochemical processes in a 147-ha cell of a 1544-ha stormwater treatment wetland designed to help protect the greater Everglades, FL, USA. The model was used to assess the effects of a suite of feasible management alternatives on the long-term ability of the wetland to sustain total P (TP) removal. The spatial and temporal dynamics of TP retention were simulated under historical (1995–2000) conditions, and under assumptions of removal of short-circuiting channels and ditches, changes in external hydraulic and TP loading, and long-term (>20 years) impacts on soil and water column TP dynamics under current and reduced load conditions. Internal hydrology and transport processes were calibrated against measured tracer concentrations, and subsequently validated against outflow discharge and spatial chloride concentration data. Cycling of P was simulated as first-order uptake and release, with different uptake coefficients for open water/sparse submerged aquatic vegetation (SAV) areas (0.2 day^?1) and dense SAV areas (0.4 day^?1), and a much lower, uniform release coefficient (1.97 × 10^?4 day^?1). The calibration and validation of the P model showed good agreement with field measurements of water column TP concentrations measured at the wetland outlet (calibration RMSE = 10.5 μg L^?1; validation RMSE = 15.6 μg L^?1). Under simulated conditions of preferential channels eliminated, average annual TP treatment effectiveness increased by 25%. When inflow TP loads were assumed to be eliminated after 6 years of loading, the release of accumulated soil P sustained predicted annual average outlet concentrations above 6.7 μg L^?1 for 18 years, decreasing at a rate of 0.16 μg L^?1 yr^?1. Sensitivity analyses indicate that the most critical model input factors include flow resistance parameters, initial soil TP content, and P cycling parameters compared to initial water level, initial TP concentration in water column, ET and transport parameters.     

Gender-specific fatty acid profiles in platelet phosphatidyl-choline and -ethanolamine

Previous studies suggested that women synthesise docosahexaenoic acid (DHA) more efficiently from their precursors than men. This study investigated the relationship between diet, platelet phospholipids fatty acids and gender. Dietary intake and platelet phosphatidyl-choline (PC) and phosphatidylethanolamine (PE) fatty acids were determined in Caucasian 40 men and 34 women. Absolute and %energy intakes of arachidonic acid (AA), eicosapentaenoic acid (EPA), and DHA, and the ratios of total n-6/n-3 PUFA and linoleic/alpha-linolenic acids did not differ between the sexes. However, women had higher DHA in PC (1.19 vs 1.05 wt%, p<0.05) and PE (3.62 vs 3.21 wt%, p<0.05) than men. Also EPA (1.10 vs 0.93 wt%, p<0.05) was higher in women's PE. Conversely, men had elevated AA and total n-6 fatty acids in PC. The higher platelet DHA levels and lower platelet AA/EPA and AA/DHA ratios in women of child-bearing age compared with men, may lead to less platelet aggregation and vaso-occlusion.     

Carbon sources-dependent carotenoid production in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

The effects of three phosphoenolpyruvate (PEP)-dependent PTS carbon sources (glucose, mannose and maltose) and three non-PTS carbon sources (glycerol, galactose, and lactose) on the formation of four carotenoids with diverse structures and on the cell growth of the recombinant Escherichia coli were investigated. The biosynthetic pathways of four carotenoids, C₃₀ diapolycopene, C₃₀ diapotorulene, C₄₀ lycopene, and C₄₀ beta-carotene, were engineered in E. coli. The resulting E. coli cells were grown in a mineral medium supplemented with each of the six carbon sources. Among the six carbon sources, non-PTS glycerol showed the highest performance in production of all four carotenoid structures, whereas PTS glucose showed the lowest performance. Based on the conversion yield, carotenoid-producing capability, and the cell density, we found that there was no close correlation between PTS and non-PTS transport mechanism and carotenoid formations in E. coli.