Expression, purification, and characterization of authentic monoferric and apo-human serum transferrins

Transferrin is a bilobal protein with the ability to bind iron in two binding sites situated at the bottom of a cleft in each lobe. We have previously described the production of recombinant non-glycosylated human serum transferrins (hTF-NG), containing a factor Xa cleavage site and a hexa-His tag at the amino-terminus. Constructs in this background that contain strategic mutations to completely prevent iron binding in each lobe or in both lobes have now been produced. These monoferric hTFs will allow dissection of the contribution of each lobe to transferrin function. In addition, the construct completely lacking in the ability to bind iron in either lobe provides an opportunity to assess whether hTF has any other functions in addition to iron transport. Following insertion of the His-tagged hTF molecules into the pNUT vector, transfection into baby hamster kidney cells and selection with methotrexate, the secreted recombinant proteins were isolated from the tissue culture medium and characterized with regard to their iron binding properties. Significant improvements over our previous protocol include: (1) addition of butyric acid at a level of 1mM which leads to a substantial increase in protein production (as much as a 65% increase compared to control cells); and (2) elimination of an anion exchange column prior to isolation on a Qiagen Ni-NTA column which makes purification of the His-tagged constructs faster and therefore more efficient. These improvements should be applicable to expression of other recombinant proteins in mammalian cells.     

Candidatus "Scalindua brodae", sp. nov., Candidatus "Scalindua wagneri", sp. nov., two new species of anaerobic ammonium oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is both a promising process in wastewater treatment and a long overlooked microbial physiology that can contribute significantly to biological nitrogen cycling in the world's oceans. Anammox is mediated by a monophyletic group of bacteria that branches deeply in the Planctomycetales. Here we describe a new genus and species of anaerobic ammonium oxidizing planctomycetes, discovered in a wastewater treatment plant (wwtp) treating landfill leachate in Pitsea, UK. The biomass from this wwtp showed high anammox activity (5.0 +/- 0.5 nmol/mg protein/min) and produced hydrazine from hydroxylamine, one of the unique features of anammox bacteria. Eight new planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass. Four of these were affiliated to known anammox 16S rRNA gene sequences, but branched much closer to the root of the planctomycete line of descent. Fluorescence in situ hybridization (FISH) with oligonucleotide probes specific for these new sequences showed that two species (belonging to the same genus) together made up > 99% of the planctomycete population which constituted 20% of the total microbial community. The identification of these organisms as typical anammox bacteria was confirmed with electron microscopy and lipid analysis. The new species, provisionally named Candidatus "Scalindua brodae" and "Scalindua wagneri" considerably extend the biodiversity of the anammox lineage on the 16S rRNA gene level, but otherwise resemble known anammox bacteria. Simultaneously, another new species of the same genus, Candidatus "Scalindua sorokinii", was detected in the water column of the Black Sea, making this genus the most widespread of all anammox bacteria described so far.     

Some observations on the effect of Daflon (micronized purified flavonoid fraction of Rutaceae aurantiae) in bancroftian filarial lymphoedema

BACKGROUND: Morbidity management is a core component of the global programme for the elimination of lymphatic filariasis. In a double-blind clinical trial, the tolerability and efficacy of Daflon (500 mg) + DEC (25 mg) or DEC (25 mg) alone, twice daily for 90 days, was studied in 26 patients with bancroftian filarial lymphoedema. RESULTS: None of the patients in either drug group reported any adverse reaction throughout the treatment period (90 days). Haematological and biochemical parameters were within normal limits and there was no significant difference between the pre-treatment (day 0) and post-treatment (day 90) values. The group receiving Daflon showed significant reduction in oedema volume from day 90 (140.6 PlusMinus; 18.8 ml) to day 360 (71.8 PlusMinus; 20.7 ml) compared to the pre-treatment (day 0, 198.4 PlusMinus; 16.5 ml) value. This accounted for a 63.8% reduction in oedema volume by day 360 (considering the pre-treatment (day 0) as 100%). In the DEC group, the changes in oedema volume (between day 1 and day 360) were not significant when compared to the pre-treatment (day 0) value. The percentage reduction at day 360 was only 9%, which was not significant (P > 0.05). CONCLUSION: This study has shown that Daflon (500 mg, twice a day for 90 days) is both safe and efficacious in reducing oedema volume in bancroftian filarial lymphoedema. Further clinical trials are essential for strengthening the evidence base on the role of this drug in the morbidity management of lymphatic filariasis.     

Sediment microbial enzyme activity as an indicator of nutrient limitation in the great rivers of the Upper Mississippi River basin

We compared extracellular enzyme activity (EEA) of microbial assemblages in river sediments at 447 sites along the Upper Mississippi, Missouri, and Ohio Rivers with sediment and water chemistry, atmospheric deposition of nitrogen and sulfate, and catchment land uses. The sites represented five unique river reaches—impounded and unimpounded reaches of the Upper Mississippi River, the upper and lower reaches of the Missouri River, and the entire Ohio River. Land use and river chemistry varied significantly between rivers and reaches. There was more agriculture in the two Upper Mississippi River reaches, and this was reflected in higher nutrient concentrations at sites in these reaches. EEA was highest in the two Upper Mississippi River reaches, followed by the lower Missouri River reach. EEA was generally lowest in the upper Missouri River reach. Canonical correlation analysis revealed a strong correlation between EEA and the suite of water and sediment chemistry variables, and the percent of the catchment in anthropogenically dominated land uses, including agriculture and urban development. Nutrient ratios of the waters and sediments suggested carbon (C), nitrogen (N), or phosphorus (P) limitation at a large number of sites in each reach. C-limitation was most pronounced in the unimpounded Mississippi River and lower Missouri River reaches; N-limitation was prevalent in the two Missouri River reaches; and P-limitation dominated the Ohio River. Linking microbial enzyme activities to regional-scale anthropogenic stressors in these large river ecosystems suggests that microbial enzyme regulation of carbon and nutrient dynamics may be sensitive indicators of anthropogenic nutrient and carbon loading.     

Brothers in arms? Common and contrasting themes in pathogen perception by plant NB-LRR and animal NACHT-LRR proteins

Both plant and animal genomes encode proteins with nucleotide binding domains fused to leucine-rich repeat domains that are involved in responses to pathogens. While these domain structures are probably an example of convergent evolution, there are a number of similarities in the core mechanisms by which these proteins are regulated.     

The role of the maternal immune system in the regulation of human birthweight

Human birthweight is subject to stabilizing selection. Large babies are at risk of obstetric complications such as obstructed labour, which endangers both mother and child. Small babies are also at risk with reduced survival. Fetal growth requires remodelling of maternal spiral arteries to provide an adequate maternal blood supply to the placenta. This arterial transformation is achieved by placental trophoblast cells, which invade into the uterine wall. Under-invasion is associated with fetal growth restriction; but if invasion is excessive large babies can result. A growing body of evidence suggests that this process is controlled by interactions between killer-cell immunoglobulin-like receptors (KIRs) expressed on maternal uterine natural killer cells (uNK) and their corresponding human leukocyte antigen-C (HLA-C) ligands on invading trophoblast. Mothers with the KIR AA genotype and a fetus with a paternal HLA-C2 allele tend to have small babies, because this combination inhibits cytokine secretion by uNK. Mothers with the activating KIR2DS1 gene and an HLA-C2 fetus are more likely to have large babies. When KIR2DS1 binds to HLA-C2 this increases secretion of cytokines that enhance trophoblast invasion. We conclude that specific combinations of the highly polymorphic gene systems, KIR and HLA-C, contribute to successful reproduction by maintaining birthweight between two extremes.