Oligopeptide Signaling through TbGPR89 Drives Trypanosome Quorum Sensing

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Consumer attitudes toward food crops developed by CRISPR/Cas9 in Costa Rica

Plant Cell, Tissue and Organ Culture (PCTOC) - In the original publication, only affiliation number 2 was indicated as the last author’s affiliation. In fact, the author works for both...     

FtsZ filament structures in different nucleotide states reveal the mechanism of assembly dynamics

Treadmilling protein filaments perform essential cellular functions by growing from one end while shrinking from the other, driven by nucleotide hydrolysis. Bacterial cell division relies on the primitive tubulin homolog FtsZ, a target for antibiotic discovery that assembles into single treadmilling filaments that hydrolyse GTP at an active site formed upon subunit association. We determined high-resolution filament structures of FtsZ from the pathogen Staphylococcus aureus in complex with different nucleotide analogs and cations, including mimetics of the ground and transition states of catalysis. Together with mutational and biochemical analyses, our structures reveal interactions made by the GTP γ-phosphate and Mg²⁺ at the subunit interface, a K⁺ ion stabilizing loop T7 for co-catalysis, new roles of key residues at the active site and a nearby crosstalk area, and rearrangements of a dynamic water shell bridging adjacent subunits upon GTP hydrolysis. We propose a mechanistic model that integrates nucleotide hydrolysis signaling with assembly-associated conformational changes and filament treadmilling. Equivalent assembly mechanisms may apply to more complex tubulin and actin cytomotive filaments that share analogous features with FtsZ.

Bacterial cell division critically relies on the tubulin homolog FtsZ, which assembles into filaments that treadmill, fuelled by GTP hydrolysis. This structural and biochemical study of FtsZ from Staphylocuccus aureus reveals the mechanism of GTP hydrolysis and its connection with filament dynamics.     

Effect of beta-N-oxalylamino-l-alanine on cerebellar cGMP level in vivo

Beta-N-oxalylamino-l-alanine (BOAA), a non-protein amino acid present in the seeds of Lathyrus Sativus (LS), is one of several neuroactive glutamate analogs reported to stimulate excitatory receptors and, in high concentrations, cause neuronal degeneration. In the present study, the in vivo acute effects of synthetic BOAA and LS seed extract were investigated on rat cerebellar cyclic GMP following intraperitoneal (10–100 mg/kg) or oral (100 mg/kg) administration of subconvulsive doses of toxin. Furthermore, the BOAA content in LS seeds and in the cerebellum of injected rats was determined by high performance liquid chromatograph analysis. A dose- and time-dependent increase of cerebellar cyclic guanosine monophosphate (cGMP) level was observed after intraperitoneal administration of synthetic BOAA or LS extract. The neurotoxin evoked a maximum stimulation 90 min after injection within the dose range of 50–75 mg/kg, elevating cGMP from basal levels of 5.3±0.5 pmol/mg protein to 15±1.3 pmol/mg protein. Similarly, the oral intake of LS-extracted neurotoxin resulted in the elevation of cGMP content. Kynurenic acid (300 mg/kg i.p.), a non specific excitatory amino acid antagonist, was effective in blocking LS BOAA-elicited cGMP enhancement. The data suggest that in the cerebellum acute administration of low concentrations of BOAA exert in vivo activation of glutamate receptors involved in the regulation of cGMP level.     

MAPK-dependent degradation of G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors (GPCR). Altered expression of GRK2 has been described to occur during pathological conditions characterized by impaired GPCR signaling. We have reported recently that GRK2 is rapidly degraded by the proteasome pathway and that beta-arrestin function and Src-mediated phosphorylation are involved in targeting GRK2 for proteolysis. In this report, we show that phosphorylation of GRK2 by MAPK also triggers GRK2 turnover by the proteasome pathway. Modulation of MAPK activation alters the degradation of transfected or endogenous GRK2, and a GRK2 mutant that mimics phosphorylation by MAPK shows an enhanced degradation rate, thus indicating a direct effect of MAPK on GRK2 turnover. Interestingly, MAPK-mediated modulation of wild-type GRK2 stability requires beta-arrestin function and is facilitated by previous phosphorylation of GRK2 on tyrosine residues by c-Src. Consistent with an important physiological role, interfering with this GRK2 degradation process results in altered GPCR responsiveness. Our data suggest that both c-Src and MAPK-mediated phosphorylation would contribute to modulate GRK2 degradation, and put forward the existence of new feedback mechanisms connecting MAPK cascades and GPCR signaling.     

Xanthomonas campestris pv. campestris gum Mutants: Effects on Xanthan Biosynthesis and Plant Virulence

Xanthan is an industrially important exopolysaccharide produced by the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. It is composed of polymerized pentasaccharide repeating units which are assembled by the sequential addition of glucose-1-phosphate, glucose, mannose, glucuronic acid, and mannose on a polyprenol phosphate carrier (L. Ielpi, R. O. Couso, and M. A. Dankert, J. Bacteriol. 175:2490–2500, 1993). A cluster of 12 genes in a region designated xpsI or gum has been suggested to encode proteins involved in the synthesis and polymerization of the lipid intermediate. However, no experimental evidence supporting this suggestion has been published. In this work, from the biochemical analysis of a defined set of X. campestris gum mutants, we report experimental data for assigning functions to the products of the gum genes. We also show that the first step in the assembly of the lipid-linked intermediate is severely affected by the combination of certain gum and non-gum mutations. In addition, we provide evidence that the C-terminal domain of the gumD gene product is sufficient for its glucosyl-1-phosphate transferase activity. Finally, we found that alterations in the later stages of xanthan biosynthesis reduce the aggressiveness of X. campestris against the plant.     

Small hydroxyethylene-based peptidomimetics inhibiting both HIV-1 and C. albicans aspartic proteases

We have extended a highly flexible method for rapidly assembling aspartic protease inhibitors to produce symmetric and asymmetric monohydroxyethylene peptidomimetics. This method is based on the prior synthesis of the central non-cleavable peptide-bond isostere [NH(2)-P(1)psiP1'-NH(2); psi=hydroxyethylene isostere, HNCH(Bz)CHOHCH(2)CH(Bz)NH], with the possibility of accurately controlling its stereochemistry (S,S,S or S,R,S), and subsequently adding appropriate flanking units, chosen from commercially available amino acids, aromatic carboxylic acids, or phenoxyacetic acid (Poa) derivatives. The method was used to make asymmetric inhibitors of general formula Kyn-Xaa-PhepsiPhe-dmPoa, (Kyn=kynurenic acid, Xaa=Val, Thr or D-thienylglycine, M(r)=716-754) and symmetric inhibitors of formula xPoa-PhepsiPhe-xPoa (xPoa=Poa or dimethyl-, hydroxy-, formyl- or acetyl-Poa, M(r)=553-609), with logP(o/w) values ranging from 4.1 to 7.6. Inhibition of HIV-PR did not depend on the stereochemistry of the hydroxyl group, while it depended markedly on the substituents present on the Poa residues, with dmPoa being preferred over Poa or its more hydrophilic derivatives. Conversely, inhibition of Candida albicans Sap2 was higher for the S,S,S epimers, and Poa or its hydrophilic derivatives were preferred over dmPoa.     

Comparative Analysis of Three Different Total Nucleic Acid Extraction Protocols for the Diagnosis of Geminiviruses in Squash (Cucurbita moschata)

Squash (Cucurbita moschata) is one of the most important crops in tropical countries. Geminiviruses are an important group of plant pathogens. In 2002 a new begomovirus was reported to naturally infect squash and some other crops in Costa Rica. Our objective was to compare, using molecular techniques, the extraction and further purification of DNA from squash by different extraction protocols and storage methods. A single infected sample was collected, half of the material was stored frozen at ?70°C, and the remainder was stored dehydrated in silica gel (SG). Total nucleic acids (TNAs) were extracted by three different protocols and were quantified by fluorometry, and the quality was analysed by electrophoresis in agarose gels, polymerase chain reaction (PCR) of the virus genome, dot blot and Southern blot hybridization. Even though the tissue stored in SG yielded a higher amount of TNAs, the genetic material exhibited lower integrity and this made it useful exclusively for the detection of geminiviral DNA by PCR amplification of short viral sequences and by hybridization with short viral probes. The Dellaporta method proved to be the most effective for the detection of geminiviral DNA in infected squash tissue. Although the cetyltrimethylammonium bromide method showed similar results, the procedure is more time‐consuming. Surprisingly, the citrate method showed either similar or worse results than the other methods.     

Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase.

The gene fprA of Mycobacterium tuberculosis, encoding a putative protein with 40% identity to mammalian adrenodoxin reductase, was expressed in Escherichia coli and the protein purified to homogeneity. The 50-kDa protein monomer contained one tightly bound FAD, whose fluorescence was fully quenched. FprA showed a low ferric reductase activity, whereas it was very active as a NAD(P)H diaphorase with dyes. Kinetic parameters were determined and the specificity constant (kcat/Km) for NADPH was two orders of magnitude larger than that of NADH. Enzyme full reduction, under anaerobiosis, could be achieved with a stoichiometric amount of either dithionite or NADH, but not with even large excess of NADPH. In enzyme titration with substoichiometric amounts of NADPH, only charge transfer species (FAD-NADPH and FADH2-NADP+) were formed. At NADPH/FAD ratios higher than one, the neutral FAD semiquinone accumulated, implying that the semiquinone was stabilized by NADPH binding. Stabilization of the one-electron reduced form of the enzyme may be instrumental for the physiological role of this mycobacterial flavoprotein. By several approaches, FprA was shown to be able to interact productively with [2Fe-2S] iron-sulfur proteins, either adrenodoxin or plant ferredoxin. More interestingly, kinetic parameters of the cytochrome c reductase reaction catalyzed by FprA in the presence of a 7Fe ferredoxin purified from M. smegmatis were determined. A Km value of 30 nm and a specificity constant of 110 microM(-1) x s(-1) (10 times greater than that for the 2Fe ferredoxin) were determined for this ferredoxin. The systematic name for FprA is therefore NADPH-ferredoxin oxidoreductase.