Plasmodium falciparum: linkage disequilibrium between loci in chromosomes 7 and 5 and chloroquine selective pressure in Northern Nigeria.

In view of the recent discovery (Molecular Cell 6, 861-871) of a (Lys76Thr) codon change in gene pfcrt on chromosome 7 which determines in vitro chloroquine resistance in Plasmodium falciparum, we have re-examined samples taken before treatment in our study in Zaria, Northern Nigeria (Parasitology, 119, 343-348). Drug resistance was present in 5/5 cases where the pfcrt 76Thr codon change was seen (100% positive predictive value). Drug sensitivity was found in 26/28 cases where the change was absent (93% negative predictive value). Allele pfcrt 76Thr showed strong linkage disequilibrium with pfmdr1 Tyr86 on chromosome 5, more complete than that between pfcrt and cg2 alleles situated between recombination cross-over points on chromosome 7. Physical linkage of cg2 with pfcrt may account for linkage disequilibrium between their alleles but in the case of genes pfmdr1 and pfcrt, on different chromosomes, it is likely that this is maintained epistatically through the selective pressure of chloroquine

Comparison between methods for creating DEMs of physical models

Within physical modelling, it is often necessary to create DEMs (digital elevation models) when testing the stability of rock structures or the filter layers and scour protection around foundations and other marine structures. These DEMs are used to detect changes in the position of the structure or surrounding protective material. Several methods are available to create these models, yet no one technique has been selected as an industry standard. A comparison between three widely used methods – terrestrial laser scanner (TLS), combined laser scanner (CLS) and structure from motion (SfM) – are presented within this paper. The CLS in underwater mode gave low measurement errors and can be deployed without having to drain the facility but requires a traverser system. An area of approximately 7 m by 4 m can be measured in half an hour. The TLS can survey a much larger area in the same time, but requires the facility to be drained. SfM is cheapest method, but struggles to create a full shape and more care must be taken. The CLS in underwater mode has been chosen for use in scour studies in the Fast Flow Facility, with high volumes of water but a relatively limited area

Molecular modeling and inhibitory activity of cowpea cystatin against bean bruchid pests.

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Identification of a novel zinc metalloprotease through a global analysis of clostridium difficile extracellular proteins

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis

Identification of Nucleases and Phosphatases by Direct Biochemical Screen of the Saccharomyces cerevisiae Proteome

The availability of yeast strain collections expressing individually tagged proteins to facilitate one-step purification provides a powerful approach to identify proteins with particular biochemical activities. To identify novel exo- and endo-nucleases that might function in DNA repair, we undertook a proteomic screen making use of the movable ORF (MORF) library of yeast expression plasmids. This library consists of 5,854 yeast strains each expressing a unique yeast ORF fused to a tripartite tag consisting of His6, an HA epitope, a protease 3C cleavage site, and the IgG-binding domain (ZZ) from protein A, under the control of the GAL1 promoter for inducible expression. Pools of proteins were partially purified on IgG sepharose and tested for nuclease activity using three different radiolabeled DNA substrates. Several known nucleases and phosphatases were identified, as well as two new members of the histidine phosphatase superfamily, which includes phosphoglycerate mutases and phosphatases. Subsequent characterization revealed YDR051c/Det1 to be an acid phosphatase with broad substrate specificity, whereas YOR283w has a broad pH range and hydrolyzes hydrophilic phosphorylated substrates. Although no new nuclease activities were identified from this screen, we did find phosphatase activity associated with a protein of unknown function, YOR283w, and with the recently characterized protein Det1. This knowledge should guide further genetic and biochemical characterization of these proteins

The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation

The ability to ensure continuous availability of energy despite highly variable supplies in the environment is a major determinant of the survival of all species. In higher organisms, including mammals, the capacity to efficiently store excess energy as triglycerides in adipocytes, from which stored energy could be rapidly released for use at other sites, was developed. To orchestrate the processes of energy storage and release, highly integrated systems operating on several physiological levels have evolved. The adipocyte is no longer considered a passive bystander, because fat cells actively secrete many members of the cytokine family, such as leptin, tumor necrosis factor-alpha, and interleukin-6, among other cytokine signals, which influence peripheral fuel storage, mobilization, and combustion, as well as energy homeostasis. The existence of a network of adipose tissue signaling pathways, arranged in a hierarchical fashion, constitutes a metabolic repertoire that enables the organism to adapt to a wide range of different metabolic challenges, such as starvation, stress, infection, and short periods of gross energy excess

ATG5 is essential for ATG8-dependent autophagy and mitochondrial homeostasis in Leishmania major

Macroautophagy has been shown to be important for the cellular remodelling required for Leishmania differentiation. We now demonstrate that L. major contains a functional ATG12-ATG5 conjugation system, which is required for ATG8-dependent autophagosome formation. Nascent autophagosomes were found commonly associated with the mitochondrion. L. major mutants lacking ATG5 (Δatg5) were viable as promastigotes but were unable to form autophagosomes, had morphological abnormalities including a much reduced flagellum, were less able to differentiate and had greatly reduced virulence to macrophages and mice. Analyses of the lipid metabolome of Δatg5 revealed marked elevation of phosphatidylethanolamines (PE) in comparison to wild type parasites. The Δatg5 mutants also had increased mitochondrial mass but reduced mitochondrial membrane potential and higher levels of reactive oxygen species. These findings indicate that the lack of ATG5 and autophagy leads to perturbation of the phospholipid balance in the mitochondrion, possibly through ablation of membrane use and conjugation of mitochondrial PE to ATG8 for autophagosome biogenesis, resulting in a dysfunctional mitochondrion with impaired oxidative ability and energy generation. The overall result of this is reduced virulence

Exoerythrocytic Plasmodium Parasites Secrete a Cysteine Protease Inhibitor Involved in Sporozoite Invasion and Capable of Blocking Cell Death of Host Hepatocytes

Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death