The molecular basis of phosphite and hypophosphite recognition by ABC-transporters

Inorganic phosphate is the major bioavailable form of the essential nutrient phosphorus. However, the concentration of phosphate in most natural habitats is low enough to limit microbial growth. Under phosphate-depleted conditions some bacteria utilise phosphite and hypophosphite as alternative sources of phosphorus, but the molecular basis of reduced phosphorus acquisition from the environment is not fully understood. Here, we present crystal structures and ligand binding affinities of periplasmic binding proteins from bacterial phosphite and hypophosphite ATP-binding cassette transporters. We reveal that phosphite and hypophosphite specificity results from a combination of steric selection and the presence of a P-H…π interaction between the ligand and a conserved aromatic residue in the ligand-binding pocket. The characterisation of high affinity and specific transporters has implications for the marine phosphorus redox cycle, and might aid the use of phosphite as an alternative phosphorus source in biotechnological, industrial and agricultural applications

Absolute quantification of cellular levels of photosynthesis-related proteins in Synechocystis sp. PCC 6803

Quantifying cellular components is a basic and important step for understanding how a cell works, how it responds to environmental changes, and for re-engineering cells to produce valuable metabolites and increased biomass. We quantified proteins in the model cyanobacterium Synechocystis sp. PCC 6803 given the general importance of cyanobacteria for global photosynthesis, for synthetic biology and biotechnology research, and their ancestral relationship to the chloroplasts of plants. Four mass spectrometry methods were used to quantify cellular components involved in the biosynthesis of chlorophyll, carotenoid and bilin pigments, membrane assembly, the light reactions of photosynthesis, fixation of carbon dioxide and nitrogen, and hydrogen and sulfur metabolism. Components of biosynthetic pathways, such as those for chlorophyll or for photosystem II assembly, range between 1000 and 10,000 copies per cell, but can be tenfold higher for CO2 fixation enzymes. The most abundant subunits are those for photosystem I, with around 100,000 copies per cell, approximately 2 to fivefold higher than for photosystem II and ATP synthase, and 5–20 fold more than for the cytochrome b6f complex. Disparities between numbers of pathway enzymes, between components of electron transfer chains, and between subunits within complexes indicate possible control points for biosynthetic processes, bioenergetic reactions and for the assembly of multisubunit complexes

The ChlD subunit links the motor and porphyrin binding subunits of magnesium chelatase

Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP2- dependent insertion of a Mg2+ ion into protoporphyin IX. The catalytic core of this large enzyme complex consists of three subunits: Bch/ChlI, Bch/ChlD and Bch/ChlH (in bacteriochlorophyll and chlorophyll producing species respectively). The D and I subunits are members of the AAA+ (ATPases associated with various cellular activities) superfamily of enzymes, and they form a complex that binds to H, the site of metal ion insertion. In order to investigate the physical coupling between ChlID and ChlH in vivo and in vitro , ChlD was FLAG-tagged in the cyanobacterium Synechocystis sp. PCC 6803 and co-immunoprecipitation experiments showed interactions with both ChlI and ChlH. Co-production of recombinant ChlD and ChlH in Escherichia coli yielded a ChlDH. Quantitative analysis using microscale thermophoresis (MST) showed magnesium-dependent binding ( K d 331 ± 58 nM) between ChlD and H. The physical basis for a ChlD-H interaction was investigated using chemical crosslinking coupled with mass spectrometry (XL-MS), together with modifications that either truncate ChlD or modify single residues. We found that the C-terminal integrin I domain of ChlD governs association with ChlH, the Mg2+ dependence of which also mediates the cooperative response of the Synechocystis chelatase to magnesium. Our work, showing the interaction site between the AAA+ motor and the chelatase domain of magnesium chelatase, will be essential for understanding how free energy from the hydrolysis of ATP on the AAA+ ChlI subunit is transmitted via the bridging subunit ChlD to the active site on ChlH

Schistosome and liver fluke derived catechol-estrogens and helminth associated cancers

Infection with helminth parasites remains a persistent public health problem in developing countries. Three of these pathogens, the liver flukes Clonorchis sinensis, Opisthorchis viverrini and the blood fluke Schistosoma haematobium, are of particular concern due to their classification as Group 1 carcinogens: infection with these worms is carcinogenic. Using liquid chromatography-mass spectrometry (LC-MS/MS) approaches, we identified steroid hormone like (e.g., oxysterol-like, catechol estrogen quinone-like, etc.) metabolites and related DNA-adducts, apparently of parasite origin, in developmental stages including eggs of S. haematobium, in urine of people with urogenital schistosomiasis, and in the adult stage of O. viverrini. Since these kinds of sterol derivatives are metabolized to active quinones that can modify DNA, which in other contexts can lead to breast and other cancers, helminth parasite associated sterols might induce tumor-like phenotypes in the target cells susceptible to helminth parasite associated cancers, i.e., urothelial cells of the bladder in the case of urogenital schistosomiasis and the bile duct epithelia or cholangiocytes, in the case of O. viverrini and C. sinensis. Indeed we postulate that helminth induced cancers originate from parasite estrogen-host epithelial/urothelial cell chromosomal DNA adducts, and here we review recent findings that support this conjecture.José M. Correia da Costa, Maria J. Gouveia, Mónica C. Botelho, Lúcio L. Santos, and Júlio H. Santos thank FCT for Pest- OE/AGR/UI0211/2011 and Strategic Project UI211-2011- 2013, Clínica Sagrada Esperança and Hospital Américo Boavida, Luanda, Angola. Nuno Vale thanks to Fundação para a Ciência e Tecnologia (FCT, Portugal) and FEDER (European Union) for funding through project grants CONCREEQ/275/QUI and PEstC/QUI/UI0081/2011. Nuno Vale also thanks FCT for Post-Doc grant SFRH/BPD/48345/2008. The research findings reviewed here were supported by award R01CA155297 (Paul J. Brindley, Gabriel Rinaldi, Banchob Sripa) from the National Cancer Institute, NIH and P50 P50AI098639 (Banchob Sripa, Paul J. Brindley) from the National Institute of Allergy and Infectious Diseases, NIH

Urinary Estrogen Metabolites and Self-Reported Infertility in Women Infected with Schistosoma haematobium

Schistosomiasis is a neglected tropical disease, endemic in 76 countries, that afflicts more than 240 million people. The impact of schistosomiasis on infertility may be underestimated according to recent literature. Extracts of Schistosoma haematobium include estrogen-like metabolites termed catechol-estrogens that down regulate estrogen receptors alpha and beta in estrogen responsive cells. In addition, schistosome derived catechol-estrogens induce genotoxicity that result in estrogen-DNA adducts. These catechol estrogens and the catechol-estrogen-DNA adducts can be isolated from sera of people infected with S. haematobium. The aim of this study was to study infertility in females infected with S. haematobium and its association with the presence of schistosome-derived catechol-estrogens

Blended versus face-to-face: comparing student performance in a therapeutics class

Therapeutics is a very complex subject for every pharmacy student, since it requires the application of knowledge from several other disciplines. The study of therapeutics is often done in case-based learning in order to promote reflective thinking and give a scenario as real as possible. The objective of this study was to compare student performance between faceto-face (n = 54) and blended learning (n = 56) approaches to the teaching of therapeutics. They can confirm that there are statistically significant differences (p < 0.05) between the final exam scores from both groups, being that the b learning group achieved higher scores. Blended learning seems to be an effective way to teach therapeutics, following pre established teaching methods, and above all, does not negatively affect student performance. It also provides new learning environments and strategies, and promotes the development of new skills such as learning and collaborating online, which may be relevant in a networked knowledge society.info:eu-repo/semantics/publishedVersio

Genetic Control of Liability to Infection with Nematospiroides Dubius in Mice: Direct and Correlated Responses to Selection of Mice for Faecal Parasite Egg Count

Mice selected as liable (L) and refractory (R) over 10 generations voided significantly more and less Nematospiroides dubius eggs compared with randomly mated (Rd) mice after primary infection with 100 larvae. Strong positive correlation was found between the numbers of N. dubius eggs in mouse faeces and the numbers of adult N. dubius recovered from mice culled from the R, Rd and L colonies. Selection limit based on a faecal e.p.g. was reached in the L mice after 9 generations whereas the faecal N. dubius e.p.g. voided by the R mice continued to decline throughout selection. In contrast, no change in worm numbers was found in L or R mice after 6 generations. The refractory state of the trait, liability to infection with N. dubius, was inherited as a dominant character with a realized heritability value of ˜ 0.2. Differential correlated responses from N. dubius infectivity compared with N. dubius fecundity and growth indicated murine genetic control of this trait by 2 major genetic units. In general, there were negative phenotypic and genetic correlations between the faecal N. dubius e.p.g. of mice and murine morphological conformation, but positive correlations between the faecal N. dubius e.p.g. of selected mice and establishment, growth and fecundity of N. dubius populations in these mice. There was little correlation between the faecal N. dubius e.p.g. after primary infection and anti-N. dubius antibody titres and parasite female/male sex-ratio

Specificity of passive serum protection against Nippostrongylus brasiliensis and Nematospiroides dubius in mice

Fewer and smaller worms were recovered from, and significantly fewer parasite eggs were voided by, mice injected with mouse antiserum raised against Nippostrongylus brasiliensis or Nematospiroides dubius after infection with the homologous nematode species compared with the control mice. The passive transfer of protective immunity was specific for the species which induced the antibody response in the donor mice. Passively immunized mice infected with the heterologous nematode species harboured the same number of worms, with the same epg output, as parasites in the control mice, but were stunted. Serological cross-reactions were observed between the two donor antiserum pools and N. dubius and N. brasiliensis antigens