A life history model for the San Francisco Estuary population of the Chinese mitten crab, <Emphasis Type="Italic">Eriocheir sinensis</Emphasis> (Decapoda: Grapsoidea)

First discovered in San Francisco Bay in 1992, the Chinese mitten crab,Eriocheir sinensis, has become established over hundreds of km² of the San Francisco Estuary. Ecological and economic impacts of this invasive species motivated our search for a greater understanding of the crabs life history as an important step in better management and control. Data for this life history model comes from the authors research and scientific literature. Juvenile crabs migrate from the Estuary into fresh water where they develop into adults. Environmental signals may stimulate gonad development that is followed by a downstream migration beginning at the end of summer. Mating occurs after the crabs reach saline water. Embryos are carried until hatching, and the larvae undergo five zoeal stages before settlement. Our model projects rates of development at various temperatures and growth increments, supports a minimum of 2 years in low salinity or freshwater habitat, and predicts that most California mitten crabs are at least 3 years old before becoming sexually mature. Environmental factors strongly influence the timing and duration of the crabs life stages, and are discussed in the context of a gradient of development times for worldwide populations of this important invasive species.     

Repeated high doses of avermectins cause prolonged sterilisation, but do not kill, Onchocerca ochengi adult worms in African cattle

Background

Ivermectin (Mectizan?, Merck and CO. Inc.) is being widely used in the control of human onchocerciasis (Onchoverca volvulus) because of its potent effect on microfilariae. Human studies have suggested that, at the standard dose of 150 μg/kg an annual treatment schedule of ivermectin reversibly interferes with female worm fertility but is not macrofilaricidal. Because of the importance of determining whether ivermectin could be macrofilaricidal, the efficacy of high and prolonged doses of ivermectin and a related avermectin, doramectin, were investigated in cattle infected with O. ochengi.

Methods

Drugs with potential macrofilaricidal activity, were screened for the treatment of human onchocerciasis, using natural infections of O. ochengi in African cattle. Three groups of 3 cows were either treated at monthly intervals (7 treatments) with ivermectin (Ivomec^®, Merck and Co. Inc.) at 500 μg/kg or doramectin (Dectamax^®, Pfizer) at 500 μg/kg or not treated as controls. Intradermal nodules were removed at 6 monthly intervals and adult worms were examined for signs of drug activity.

Results

There was no significant decline in nodule diameter, the motility of male and female worms, nor in male and female viability as determined by the ability to reduce tetrazolium, compared with controls, at any time up to 24 months from the start of treatments (mpt). Embryogenesis, however, was abrogated by treatment, which was seen as an accumulation of dead and dying intra-uterine microfilariae (mf) persisting for up to 18 mpt. Skin mf densities in treated animals had fallen to zero by <3 mpt, but by 18 mpt small numbers of mf were found in the skin of some treated animals and a few female worms were starting to produce multi-cellular embryonic stages. Follow-up of the doramectin treated group at 36 mpt showed that mf densities had still only regained a small proportion of their pre-treatment levels.

Conclusion

These results have important implications for onchocerciasis control in the field. They suggest that ivermectin given at repeated high does may sterilise O. volvulus female worms for prolonged periods but is unlikely to kill them. This supports the view that control programmes may need to continue treatments with ivermectin for a period of decades and highlights the need to urgently identify new marcofiliaricidal compounds.     

Functional demonstration of reverse transsulfuration in the Mycobacterium tuberculosis complex reveals that methionine is the preferred sulfur source for pathogenic Mycobacteria

Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of (35)S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine gamma-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine gamma-synthase (CGS), but demonstration of a gamma-elimination reaction with cystathionine as well as the gamma-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.     

The apo and ternary complex structures of a chemotherapeutic target: human glycinamide ribonucleotide transformylase

Glycinamide ribonucleotide transformylase (GART; 10-formyltetrahydrofolate:5'-phosphoribosylglycinamide formyltransferase, EC 2.1.2.2), an essential enzyme in de novo purine biosynthesis, has been a chemotherapeutic target for several decades. The three-dimensional structure of the GART domain from the human trifunctional enzyme has been solved by X-ray crystallography. Models of the apoenzyme, and a ternary complex with the 10-formyl-5,8-dideazafolate cosubstrate and a glycinamide ribonucleotide analogue, hydroxyacetamide ribonucleotide [alpha,beta-N-(hydroxyacetyl)-d-ribofuranosylamine], are reported to 2.2 and 2.07 A, respectively. The model of the apoenzyme represents the first structure of GART, from any source, with a completely unoccupied substrate and cosubstrate site, while the ternary complex is the first structure of the human GART domain that is bound at both the substrate and cosubstrate sites. A comparison of the two models therefore reveals subtle structural differences that reflect substrate and cosubstrate binding effects and implies roles for the invariant residues Gly 133, Gly 146, and His 137. Preactivation of the DDF formyl group appears to be key for catalysis, and structural flexibility of the active end of the substrate may facilitate nucleophilic attack. A change in pH, rather than folate binding, correlates with movement of the folate binding loop, whereas the phosphate binding loop position does not vary with pH. The electrostatic surface potentials of the human GART domain and Escherichia coli enzyme explain differences in the binding affinity of polyglutamylated folates, and these differences have implications to future chemotherapeutic agent design.     

Calpain-dependent endoproteolytic cleavage of PrPSc modulates scrapie prion propagation

Previous studies using post-mortem human brain extracts demonstrated that PrP in Creutzfeldt-Jakob disease (CJD) brains is cleaved by a cellular protease to generate a C-terminal fragment, referred to as C2, which has the same molecular weight as PrP-(27-30), the protease-resistant core of PrP(Sc) (1). The role of this endoproteolytic cleavage of PrP in prion pathogenesis and the identity of the cellular protease responsible for production of the C2 cleavage product has not been explored. To address these issues we have taken a combination of pharmacological and genetic approaches using persistently infected scrapie mouse brain (SMB) cells. We confirm that production of C2 is the predominant cleavage event of PrP(Sc) in the brains of scrapie-infected mice and that SMB cells faithfully recapitulate the diverse intracellular proteolytic processing events of PrP(Sc) and PrP(C) observed in vivo. While increases in intracellular calcium (Ca(2+)) levels in prion-infected cell cultures stimulate the production of the PrP(Sc) cleavage product, pharmacological inhibitors of calpains and overexpression of the endogenous calpain inhibitor, calpastatin, prevent the production of C2. In contrast, inhibitors of lysosomal proteases, caspases, and the proteasome have no effect on C2 production in SMB cells. Calpain inhibition also prevents the accumulation of PrP(Sc) in SMB and persistently infected ScN2A cells, whereas bioassay of inhibitor-treated cell cultures demonstrates that calpain inhibition results in reduced prion titers compared with control-treated cultures assessed in parallel. Our observations suggest that calpain-mediated endoproteolytic cleavage of PrP(Sc) may be an important event in prion propagation.     

Invasive cleavage reactions on DNA-modified diamond surfaces

Recently developed DNA-modified diamond surfaces exhibit excellent chemical stability to high-temperature incubations in biological buffers. The stability of these surfaces is substantially greater than that of gold or silicon surfaces, using similar surface attachment chemistry. The DNA molecules attached to the diamond surfaces are accessible to enzymes and can be modified in surface enzymatic reactions. An important application of these surfaces is for surface invasive cleavage reactions, in which target DNA strands added to the solution may result in specific cleavage of surface-bound probe oligonucleotides, permitting analysis of single nucleotide polymorphisms (SNPs). Our previous work demonstrated the feasibility of performing such cleavage reactions on planar gold surfaces using PCR-amplified human genomic DNA as target. The sensitivity of detection in this earlier work was substantially limited by a lack of stability of the gold surface employed. In the present work, detection sensitivity is improved by a factor of approximately 100 (100 amole of DNA target compared with 10 fmole in the earlier work) by replacing the DNA-modified gold surface with a more stable DNA-modified diamond surface.     

Characterization and metabolic function of a peroxisomal sarcosine and pipecolate oxidase from Arabidopsis

Sarcosine oxidase (SOX) is known as a peroxisomal enzyme in mammals and as a sarcosine-inducible enzyme in soil bacteria. Its presence in plants was unsuspected until the Arabidopsis genome was found to encode a protein (AtSOX) with approximately 33% sequence identity to mammalian and bacterial SOXs. When overexpressed in Escherichia coli, AtSOX enhanced growth on sarcosine as sole nitrogen source, showing that it has SOX activity in vivo, and the recombinant protein catalyzed the oxidation of sarcosine to glycine, formaldehyde, and H(2) O(2) in vitro. AtSOX also attacked other N-methyl amino acids and, like mammalian SOXs, catalyzed the oxidation of l-pipecolate to Delta(1)-piperideine-6-carboxylate. Like bacterial monomeric SOXs, AtSOX was active as a monomer, contained FAD covalently bound to a cysteine residue near the C terminus, and was not stimulated by tetrahydrofolate. Although AtSOX lacks a typical peroxisome-targeting signal, in vitro assays established that it is imported into peroxisomes. Quantitation of mRNA showed that AtSOX is expressed at a low level throughout the plant and is not sarcosine-inducible. Consistent with a low level of AtSOX expression, Arabidopsis plantlets slowly metabolized supplied [(14)C]sarcosine to glycine and serine. Gas chromatography-mass spectrometry analysis revealed low levels of pipecolate but almost no sarcosine in wild type Arabidopsis and showed that pipecolate but not sarcosine accumulated 6-fold when AtSOX expression was suppressed by RNA interference. Moreover, the pipecolate catabolite alpha-aminoadipate decreased 30-fold in RNA interference plants. These data indicate that pipecolate is the endogenous substrate for SOX in plants and that plants can utilize exogenous sarcosine opportunistically, sarcosine being a common soil metabolite.     

Reorganization of the Mu transpososome active sites during a cooperative transition between DNA cleavage and joining

Transposition of mobile genetic elements proceeds through a series of DNA phosphoryl transfer reactions, with multiple reaction steps catalyzed by the same set of active site residues. Mu transposase repeatedly utilizes the same active site DDE residues to cleave and join a single DNA strand at each transposon end to a new, distant DNA location (the target DNA). To better understand how DNA is manipulated within the Mu transposase-DNA complex during recombination, the impact of the DNA immediately adjacent to the Mu DNA ends (the flanking DNA) on the progress of transposition was investigated. We show that, in the absence of the MuB activator, the 3 '-flanking strand can slow one or more steps between DNA cleavage and joining. The presence of this flanking DNA strand in just one active site slows the joining step in both active sites. Further evidence suggests that this slow step is not due to a change in the affinity of the transpososome for the target DNA. Finally, we demonstrate that MuB activates transposition by stimulating the reaction step between cleavage and joining that is otherwise slowed by this flanking DNA strand. Based on these results, we propose that the 3 '-flanking DNA strand must be removed from, or shifted within, both active sites after the cleavage step; this movement is coupled to a conformational change within the transpososome that properly positions the target DNA simultaneously within both active sites and thereby permits joining.     

Crystal structures of the Apo and penicillin-acylated forms of the BlaR1 beta-lactam sensor of Staphylococcus aureus

Staphylococcus aureus is among the most prevalent and antibiotic-resistant of pathogenic bacteria. The resistance of S. aureus to prototypal beta-lactam antibiotics is conferred by two mechanisms: (i) secretion of hydrolytic beta-lactamase enzymes and (ii) production of beta-lactam-insensitive penicillin-binding proteins (PBP2a). Despite their distinct modes of resistance, expression of these proteins is controlled by similar regulation systems, including a repressor (BlaI/MecI) and a multidomain transmembrane receptor (BlaR1/MecR1). Resistance is triggered in response to a covalent binding event between a beta-lactam antibiotic and the extracellular sensor domain of BlaR1/MecR1 by transduction of the binding signal to an intracellular protease domain capable of repressor inactivation. This study describes the first crystal structures of the sensor domain of BlaR1 (BlaRS) from S. aureus in both the apo and penicillin-acylated forms. The structures show that the sensor domain resembles the beta-lactam-hydrolyzing class D beta-lactamases, but is rendered a penicillin-binding protein due to the formation of a very stable acyl-enzyme. Surprisingly, conformational changes upon penicillin binding were not observed in our structures, supporting the hypothesis that transduction of the antibiotic-binding signal into the cytosol is mediated by additional intramolecular interactions of the sensor domain with an adjacent extracellular loop in BlaR1.     

Vav GEFs are required for beta2 integrin-dependent functions of neutrophils

Integrin regulation of neutrophils is essential for appropriate adhesion and transmigration into tissues. Vav proteins are Rho family guanine nucleotide exchange factors that become tyrosine phosphorylated in response to adhesion. Using Vav1/Vav3-deficient neutrophils (Vav1/3ko), we show that Vav proteins are required for multiple beta2 integrin-dependent functions, including sustained adhesion, spreading, and complement-mediated phagocytosis. These defects are not attributable to a lack of initial beta2 activation as Vav1/3ko neutrophils undergo chemoattractant-induced arrest on intercellular adhesion molecule-1 under flow. Accordingly, in vivo, Vav1/3ko leukocytes arrest on venular endothelium yet are unable to sustain adherence. Thus, Vav proteins are specifically required for stable adhesion. beta2-induced activation of Cdc42, Rac1, and RhoA is defective in Vav1/3ko neutrophils, and phosphorylation of Pyk2, paxillin, and Akt is also significantly reduced. In contrast, Vav proteins are largely dispensable for G protein-coupled receptor-induced signaling events and chemotaxis. Thus, Vav proteins play an essential role coupling beta2 to Rho GTPases and regulating multiple integrin-induced events important in leukocyte adhesion and phagocytosis.