Identifying vulnerable pathways in Mycobacterium tuberculosis by using a knockdown approach

We constructed recombinant strains of Mycobacterium tuberculosis in which expression of specific genes was downregulated to identify vulnerable drug targets. Growth phenotypes in macrophages and culture were used to rank targets: the dprE1, clpP1, and fadD32 operons were the best targets and glnA1, glnE, pknL, regX3, and senX3 were poor targets.     

Indicators to support environmental sustainability of bioenergy systems

Indicators are needed to assess environmental sustainability of bioenergy systems. Effective indicators will help in the quantification of benefits and costs of bioenergy options and resource uses. We identify 19 measurable indicators for soil quality, water quality and quantity, greenhouse gases, biodiversity, air quality, and productivity, building on existing knowledge and on national and international programs that are seeking ways to assess sustainable bioenergy. Together, this suite of indicators is hypothesized to reflect major environmental effects of diverse feedstocks, management practices, and post-production processes. The importance of each indicator is identified. Future research relating to this indicator suite is discussed, including field testing, target establishment, and application to particular bioenergy systems. Coupled with such efforts, we envision that this indicator suite can serve as a basis for the practical evaluation of environmental sustainability in a variety of bioenergy systems.     

Identification and characterisation of a novel adhesin Ifp in <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>

Background  

In order to identify new virulence determinants in Y. pseudotuberculosis a comparison between its genome and that of Yersinia pestis was undertaken. This reveals dozens of pseudogenes in Y. pestis, which are still putatively functional in Y. pseudotuberculosis and may be important in the enteric lifestyle. One such gene, YPTB1572 in the Y. pseudotuberculosis IP32953 genome sequence, encodes a protein with similarity to invasin, a classic adhesion/invasion protein, and to intimin, the attaching and effacing protein from enteropathogenic (EPEC) and enterohaemorraghic (EHEC) Escherichia coli.     

Ookinete-interacting proteins on the microvillar surface are partitioned into detergent resistant membranes of Anopheles gambiae midguts

Lipid raft microdomains, a component of detergent resistant membranes (DRMs), are routinely exploited by pathogens during host-cell entry. Multiple membrane-surface proteins mediate Plasmodium ookinete invasion of the Anopheles midgut, a critical step in the parasite life cycle that is successfully targeted by transmission-blocking vaccines (TBV). Given that lipid rafts are a common feature of host-pathogen interactions, we hypothesized that they promote the partitioning of midgut surface proteins and thus facilitate ookinete invasion. In support of this hypothesis, we found that five of the characterized Anopheles TBV candidates, including the leading Anopheles TBV candidate, AgAPN1, are present in Anopheles gambiae DRMs. Therefore, to extend the repertoire of putative midgut ligands that can be targeted by TBVs, we analyzed midgut DRMs by tandem mass spectrometry. We identified 1452 proteins including several markers of DRMs. Since glycosylphosphotidyl inositol (GPI)-anchored proteins partition to DRMs, we characterized the GPI subproteome of An. gambiae midgut brush-border microvilli and found that 96.9% of the proteins identified in the GPI-anchored fractions were also present in DRMs. Our study vastly expands the number of candidate malarial TBV targets for subsequent analysis by the broader community and provides an inferred role for midgut plasmalemma microdomains in ookinete cell invasion.     

Wnt5a regulates midbrain dopaminergic axon growth and guidance

During development, precise temporal and spatial gradients are responsible for guiding axons to their appropriate targets. Within the developing ventral midbrain (VM) the cues that guide dopaminergic (DA) axons to their forebrain targets remain to be fully elucidated. Wnts are morphogens that have been identified as axon guidance molecules. Several Wnts are expressed in the VM where they regulate the birth of DA neurons. Here, we describe that a precise temporo-spatial expression of Wnt5a accompanies the development of nigrostriatal projections by VM DA neurons. In mice at E11.5, Wnt5a is expressed in the VM where it was found to promote DA neurite and axonal growth in VM primary cultures. By E14.5, when DA axons are approaching their striatal target, Wnt5a causes DA neurite retraction in primary cultures. Co-culture of VM explants with Wnt5a-overexpressing cell aggregates revealed that Wnt5a is capable of repelling DA neurites. Antagonism experiments revealed that the effects of Wnt5a are mediated by the Frizzled receptors and by the small GTPase, Rac1 (a component of the non-canonical Wnt planar cell polarity pathway). Moreover, the effects were specific as they could be blocked by Wnt5a antibody, sFRPs and RYK-Fc. The importance of Wnt5a in DA axon morphogenesis was further verified in Wnt5a-/- mice, where fasciculation of the medial forebrain bundle (MFB) as well as the density of DA neurites in the MFB and striatal terminals were disrupted. Thus, our results identify a novel role of Wnt5a in DA axon growth and guidance.     

Polo-like kinase 4 controls centriole duplication but does not directly regulate cytokinesis

Centrioles organize the centrosome, and accurate control of their number is critical for the maintenance of genomic integrity. Centrioles duplicate once per cell cycle, and duplication is coordinated by Polo-like kinase 4 (Plk4). We previously demonstrated that Plk4 accumulation is autoregulated by its own kinase activity. However, loss of heterozygosity of Plk4 in mouse embryonic fibroblasts has been proposed to cause cytokinesis failure as a primary event, leading to centrosome amplification and gross chromosomal abnormalities. Using targeted gene disruption, we show that human epithelial cells with one inactivated Plk4 allele undergo neither cytokinesis failure nor increase in centrosome amplification. Plk4 is shown to localize exclusively at the centrosome, with none in the spindle midbody. Substantial depletion of Plk4 by small interfering RNA leads to loss of centrioles and subsequent spindle defects that lead to a modest increase in the rate of cytokinesis failure. Therefore, Plk4 is a centriole-localized kinase that does not directly regulate cytokinesis.     

A novel MYB-related gene from Arabidopsis thaliana expressed in developing anthers.

A novel MYB-like gene (AtMYB103) was isolated from a genomic library of Arabidopsis. Plants transgenic for chimeric AtMYB103 promoter/GUS genes expressed the enzyme in early anthers. In situ hybridization of flower sections showed a high level of AtMYB103 mRNA in the tapetum and middle layer of developing anthers.     

Regulation of sterol biosynthesis and of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in cultured cells by progesterone

Treatment of rat intestinal epithelial cells in culture (IEC-6) with progesterone (10 micrograms/ml) caused a strong inhibition of cholesterol biosynthesis as indicated by a decreased incorporation of radiolabel from [3H]acetate. This inhibition was accompanied by an accumulation of radioactivity in an intermediate which coeluted with authentic desmosterol upon high performance liquid chromatography (HPLC). In addition, treatment of cells with progesterone caused lesser accumulation of radiolabel in products with retention times (RT) of 7.9 and 13.5 min on reverse-phase HPLC. The RT-13.5 compound was tentatively identified as cholesta-5,7,24-trien-3 beta-ol based on its relative retention and on its conversion to cholesterol upon incubation with untreated cells. The RT-7.9 compound was identified as 24 (S),25-epoxycholesterol (S-EC) based on its coelution with authentic S-EC and by its conversion to 25-hydroxycholesterol upon reduction with LiAlH4. Incubation of IEC-6 cells with chemically prepared S-EC resulted in dose-dependent suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity within 6 h (I50 = 0.3 microM). Pretreatment of cells with progesterone prevented this suppressive effect. No suppression of reductase activity was observed in progesterone-treated cells in spite of obvious accumulation of S-EC in amounts sufficient to effect regulation; instead, a 2-3-fold increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity occurred within a 24-h period. Following the removal of progesterone from the culture medium, reductase activity declined rapidly over the next 6 h. However, IEC-6 cells could not metabolize S-EC, derived either endogenously or exogenously, during a similar time frame; nor did progesterone affect the uptake of exogenous S-EC by IEC-6 cells. These results show that although progesterone treatment of cultured cells promotes the synthesis of a natural oxysterol suppressor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, the continued presence of progesterone prevents the regulatory action of S-EC. The unique nature of this interference is high-lighted by the observation that progesterone could not prevent the suppression of reductase activity by either 25-hydroxycholesterol or mevalonolactone.     

Sex steroid content and metabolism in Trichostrongylus colubriformis (Nematoda)

Testosterone, progesterone and cholesterol were found in mixed sexes of the nematode Trichostrongylus colubriformis from goats, according to thin-layer, gas-liquid and high-performance liquid chromatography. The structure of these steroids was confirmed by proton nuclear magnetic resonance and mass spectroscopy. Melting points of the worms' steroids were similar to authentic standards of the steroids. Estradiol was not detected in worms from either goat sex. Cholesterol was about 0.08% of the worms' dry weight in helminths from either sex of host. Testosterone was 0.02% of the dry weight when worms were taken from male goats, but only 0.005% from female goats. Progesterone was not detected in worms from male goats, but was 0.005% of the dry weight of helminths from female hosts. Incubation of a worm preparation with tritiated steroids showed that progesterone was converted to 17-alpha-hydroxyprogesterone, based on retention during radioactive thin-layer and gas-liquid chromatography, and co-crystallization. Testosterone, cholesterol and 17-beta-estradiol were not metabolized.