The extraordinary specificity of xanthine phosphoribosyltransferase from Bacillus subtilis elucidated by reaction kinetics, ligand binding, and crystallography

Xanthine phosphoribosyltransferase (XPRTase) from Bacillus subtilis is a representative of the highly xanthine specific XPRTases found in Gram-positive bacteria. These XPRTases constitute a distinct subclass of 6-oxopurine PRTases, which deviate strongly from the major class of H(X)GPRTases with respect to sequence, PRPP binding motif, and oligomeric structure. They are more related with the PurR repressor of Gram-positive bacteria, the adenine PRTase, and orotate PRTase. The catalytic function and high specificity for xanthine of B. subtilis XPRTase were investigated by ligand binding studies and reaction kinetics as a function of pH with xanthine, hypoxanthine, and guanine as substrates. The crystal structure of the dimeric XPRTase-GMP complex was determined to 2.05 A resolution. In a sequential reaction mechanism XPRTase binds first PRPP, stabilizing its active dimeric form, and subsequently xanthine. The XPRTase is able also to react with guanine and hypoxanthine albeit at much lower (10(-)(4)-fold) catalytic efficiency. Different pK(a) values for the bases and variations in their electrostatic potential can account for these catalytic differences. The unique base specificity of XPRTase has been related to a few key residues in the active site. Asn27 can in different orientations form hydrogen bonds to an amino group or an oxo group at the 2-position of the purine base, and Lys156 is positioned to make a hydrogen bond with N7. This and the absence of a catalytic carboxylate group near the N7-position require the purine base to dissociate a proton spontaneously in order to undergo catalysis.     

Capture and Retention of Cryptosporidium parvum Oocysts by Pseudomonas aeruginosa Biofilms

The association of Cryptosporidium oocysts with biofilm communities can influence the propagation of this pathogen through both environmental systems and water treatment systems. We observed the capture and retention of C. parvum oocysts in Pseudomonas aeruginosa biofilms using laboratory flow cells. Biofilms were developed in two different growth media using two different strains of P. aeruginosa, a wild-type strain (PAO1) and a strain that overproduces the exopolysaccharide alginate (PDO300). Confocal laser-scanning microscopy was used in conjunction with image analysis to assess the structure of the biofilms prior to introducing oocysts into the flow cells. More oocysts were captured by the biofilm-coated surfaces than the abiotic glass surface in both media. There was no significant difference in capture across the two strains of P. aeruginosa biofilm, but the fraction of oocysts captured was positively related to biofilm roughness and surface-area-to-volume ratio. Once captured, oocysts were retained in the biofilm for more than 24 h and were not released after a 40-fold increase in the system flow rate. We believe the capture and retention of oocysts by biofilm communities can impact the environmental transmission of C. parvum, and this interaction should be taken into consideration when predicting the migration of pathogens in the environment.     

Phenotypic screening of hepatocyte nuclear factor (HNF) 4-gamma receptor knockout mice

Using the mouse as a model organism in pharmaceutical research presents unique advantages as its physiology in many ways resembles the human physiology, it also has a relatively short generation time, low breeding and maintenance costs, and is available in a wide variety of inbred strains. The ability to genetically modify mouse embryonic stem cells to generate mouse models that better mimic human disease is another advantage. In the present study, a comprehensive phenotypic screening protocol is applied to elucidate the phenotype of a novel mouse knockout model of hepatocyte nuclear factor (HNF) 4-gamma. HNF4-gamma is expressed in the kidneys, gut, pancreas, and testis. The first level of the screen is aimed at general health, morphologic appearance, normal cage behaviour, and gross neurological functions. The second level of the screen looks at metabolic characteristics and lung function. The third level of the screen investigates behaviour more in-depth and the fourth level consists of a thorough pathological characterisation, blood chemistry, haematology, and bone marrow analysis. When compared with littermate wild-type mice (HNF4-gamma(+/+)), the HNF4-gamma knockout (HNF4-gamma(-/-)) mice had lowered energy expenditure and locomotor activity during night time that resulted in a higher body weight despite having reduced intake of food and water. HNF4-gamma(-/-) mice were less inclined to build nest and were found to spend more time in a passive state during the forced swim test.     

The corticosteroid hormone induced factor: a new modulator of KCNQ1 channels?

The corticosteroid hormone induced factor (CHIF) is a member of the one-transmembrane segment protein family named FXYD, which also counts phospholemman and the Na,K-pump gamma-subunit. Originally it was suggested that CHIF could induce the expression of the I(Ks) current when expressed in Xenopus laevis oocytes, but recently CHIF has attracted attention as a modulatory subunit of the Na,K-pump. In renal and intestinal epithelia, the expression of CHIF is dramatically up-regulated in response to aldosterone stimulation, and regulation of epithelial ion channels by CHIF is an attractive hypothesis. To study a potential regulatory effect of the CHIF subunit on KCNQ1 channels, co-expression experiments were performed in Xenopus laevis oocytes and mammalian CHO-K1 cells. Electrophysiological characterization was obtained by two-electrode voltage-clamp and patch-clamp, respectively. In both expression systems, we find that CHIF drastically modulates the KCNQ1 current; in the presence of CHIF, the KCNQ1 channels open at all membrane potentials. Thereby, CHIF is the first accessory subunit shown to be capable of modulating both the Na,K-pump and an ion channel. To find a possible physiological function of the constitutively open KCNQ1/CHIF complex, the precise localization of KCNQ1 and CHIF in distal colon and kidney from control and salt-depleted rats was determined by confocal microscopy. However, in these tissues, we did not detect an obvious overlap in expression between KCNQ1 and CHIF. In conclusion, the hormone-regulated subunit CHIF modulates the voltage sensitivity of the KCNQ channels, but so far evidence for an actual co-localization of CHIF and KCNQ1 channels in native tissue is lacking.     

Observations on the behavior of vitreous ice at approximately 82 and approximately 12 K

In an attempt to determine why cooling with liquid helium actually proved disadvantageous in our electron cryotomography experiments, further tests were performed to explore the differences in vitreous ice at approximately 82 and approximately 12 K. Electron diffraction patterns showed clearly that the vitreous ice of interest in biological electron cryomicroscopy (i.e., plunge-frozen, buffered protein solutions) does indeed collapse into a higher density phase when irradiated with as few as 2-3 e-/A2 at approximately 12 K. The high density phase spontaneously expanded back to a state resembling the original, low density phase over a period of hours at approximately 82 K. Movements of gold fiducials and changes in the lengths of tunnels drilled through the ice confirmed these phase changes, and also revealed gross changes in the concavity of the ice layer spanning circular holes in the carbon support. Brief warmup-cooldown cycles from approximately 12 to approximately 82 K and back, as would be required by the flip-flop cryorotation stage, did not induce a global phase change, but did allow certain local strains to relax. Several observations including the rates of tunnel collapse and the production of beam footprints suggested that the high density phase flows more readily in response to irradiation. Finally, the patterns of bubbling were different at the two temperatures. It is concluded that the collapse of vitreous ice at approximately 12 K around macromolecules is too rapid to account alone for the problematic loss of contrast seen, which must instead be due to secondary effects such as changes in the mobility of radiolytic fragments and water.     

Artemisinin: The biosynthetic pathway and its regulation in Artemisia annua, a terpenoid-rich species

Summary Artemisinin is a sesquiterpene lactone isolated from the aerial parts of Artemisia annua L. plants. Besides being currently the best therapeutic against both drug-resistant and cerebral malaria-causing strains of Plasmodium falciparum, the drug has also been shown to be effective against other infections diseases including schistosomiasis and hepatitis. More recently, it has also been shown to be effective against numerous types of tumors. Although chemical synthesis of artemisinin is possible, it is not economically feasible. The relatively low yield (0.01–0.8%) of artemisinin in A. annua is a further serious limitation to the commercialization of the drug. Therffore, the enhanced production of artemisinin either in cell/tissue culture or in the whole plant of A. annua is highly desirable. A better understanding of the biochemical pathway leading to the synthesis of artemisinin and its regulation by both exogenous and endogenous factors is essential for facilitating increased yield. Two genes of the artemisinin biosynthetic pathway have now been identified. This critical review covers recent developments related to the biosynthesis of this important compound and related terpenoids, their regulation, and the production of these compounds both in vitro and in whole plants.     

A genomewide screen for petite-negative yeast strains yields a new subunit of the i-AAA protease complex

Unlike many other organisms, the yeast Saccharomyces cerevisiae can tolerate the loss of mitochondrial DNA (mtDNA). Although a few proteins have been identified that are required for yeast cell viability without mtDNA, the mechanism of mtDNA-independent growth is not completely understood. To probe the relationship between the mitochondrial genome and cell viability, we conducted a microarray-based, genomewide screen for mitochondrial DNA-dependent yeast mutants. Among the several genes that we discovered is MGR1, which encodes a novel subunit of the i-AAA protease complex located in the mitochondrial inner membrane. mgr1Delta mutants retain some i-AAA protease activity, yet mitochondria lacking Mgr1p contain a misassembled i-AAA protease and are defective for turnover of mitochondrial inner membrane proteins. Our results highlight the importance of the i-AAA complex and proteolysis at the inner membrane in cells lacking mitochondrial DNA.     

Anopheles and Plasmodium: from laboratory models to natural systems in the field

Parasites that cause malaria must complete a complex life cycle in Anopheles vector mosquitoes in order to be transmitted from human to human. Previous gene-silencing studies have shown the influence of mosquito immunity in controlling the development of Plasmodium. Thus, parasite survival to the oocyst stage increased when the parasite antagonist gene LRIM1 (leucine-rich repeat immune protein 1) of the mosquito was silenced, but decreased when the C-type lectin agonist gene CTL4 or CTLMA2 (CTL mannose binding 2) was silenced. However, such effects were shown for infections of the human mosquito vector Anopheles gambiae with the rodent parasite Plasmodium berghei. Here, we report the first results of A. gambiae gene silencing on infection by sympatric field isolates of the principal human pathogen P. falciparum. In contrast with the results obtained with the rodent parasite, silencing of the same three genes had no effect on human parasite development. These results highlight the importance of following up discoveries in laboratory model systems with studies on natural parasite-mosquito interactions.