A putative twin-arginine translocation pathway in Legionella pneumophila

Legionella pneumophila is a facultative intracellular human pathogen causing Legionnaires' disease, a severe form of pneumonia. Because of the importance of secretion pathways in virulence, we were interested in the possible presence of the twin-arginine translocation (Tat) pathway in L. pneumophila. This secretion pathway is used to transport folded proteins, characterized by two arginines in their signal peptide, across the cytoplasmic membrane. We describe here the presence of a putative Tat pathway in L. pneumophila. Three genes encoding Escherichia coli TatA, TatB, and TatC homologues were identified. The tatA and tatB genes were shown to constitute an operon while tatC is monocistronic. RT-PCR analysis revealed expression of the tat genes during both exponential and stationary growth as well as during intracellular growth in Acanthamoeba castellanii. A search for the conserved twin-arginine motif in predicted signal peptides resulted in a list of putative Tat substrates.     

Increased cerebral uptake and oxidation of exogenous betaHB improves ATP following traumatic brain injury in adult rats

There is growing evidence of the brain's ability to increase its reliance on alternative metabolic substrates under conditions of energy stress such as starvation, hypoxia and ischemia. We hypothesized that following traumatic brain injury (TBI), which results in immediate changes in energy metabolism, the adult brain increases uptake and oxidation of the alternative substrate beta-hydroxybutyrate (betaHB). Arterio-venous differences were used to determine global cerebral uptake of betaHB and production of 14CO2 from [14C]3-betaHB 3 h after controlled cortical impact (CCI) injury. Quantitative bioluminescence was used to assess regional changes in ATP concentration. As expected, adult sham and CCI animals with only endogenously available betaHB showed no significant increase in cerebral uptake of betaHB or 14CO2 production. Increasing arterial betaHB concentrations 2.9-fold with 3 h of betaHB infusion failed to increase cerebral uptake of betaHB or 14CO2 production in adult sham animals. Only CCI animals that received a 3-h betaHB infusion showed an 8.5-fold increase in cerebral uptake of betaHB and greater than 10.7-fold increase in 14CO2 production relative to sham betaHB-infused animals. The TBI-induced 20% decrease in ipsilateral cortical ATP concentration was alleviated by 3 h of betaHB infusion beginning immediately after CCI injury.     

Proficiencies of two artificial substrates in collecting stream macroinvertebrates

Proficiencies in collecting stream macroinvertebrates by two types of artificial substrates, #200 3M conservation webbing and cone-shaped concrete blocks, were compared with bottom net collections. Conservation webbing collected more individuals than cone substrates, but fewer taxa than either cone substrates or bottom nets. Diversity indices calculated for collections made with the three techniques were lowest for webbing collections, highest for net collections and intermediate for cone collections. It was concluded that collections made with webbing substrates gave an extremely distorted view of macroinvertebrate community balance due to a preponderance of net spinning and case building forms, and that cone substrates were better suited for use in the particular situation studied.Department of Biology, Virginia Polytechnic Institute and State University.Center for Environmental Studies, Virginia Polytechnic Institute and State University.     

Purification, characterization and inhibition of sterol C24-methyltransferase from Candida albicans

Solubilized sterol C24-methyltransferase (24-SMT) was purified to homogeneity from a cell extract of the yeast Candida albicans (Ca) by anion exchange chromatography, gel permeation chromatography and fast performance liquid chromatography using a Mono Q column. The purified enzyme has an apparent molecular mass of 178 kDa on gel permeation chromatography and 43 kDa on SDS/PAGE, indicating that it is composed of four identical subunits. The substrate requirement of the native enzyme has an optimal specificity for zymosterol with associated kinetic constants of K_m 50 μM and k_cat of 0.01 s⁻¹. The product of the enzyme incubated with zymosterol was fecosterol. Inhibition of the catalyst was observed with substrate analogs designed as transition state analogs (25-azalanosterol, K_i = 54 nM and 24 (R,S),25-epiminolanosterol, K_i = 11 nM) or as mechanism-based inactivators (26,27-dehydrozymosterol, K_i 9 μM) and k_inact = 0.03 min⁻¹) of the C24-methylation reaction. Product analogs ergosterol and fecosterol, but neither cholesterol nor sitosterol, inhibited activity affording K_i values of 20 and 72 μM, respectively. Ammonium and thia analogs of the intermediates of the sterol C24-methyl reaction sequence were effective growth inhibitors exhibiting IC₅₀ values that ranged from 3 to 20 μM.     

Spotting optimization for oligo microarrays on aldehyde-glass

Low-density microarrays that utilize short oligos (<100 nt) for capture are highly attractive for use in diagnostic applications, yet these experiments require strict quality control and meticulous reproducibility. However, a survey of current literature indicates vast inconsistencies in the spotting and processing procedures. In this study, spotting and processing protocols were optimized for aldehyde-functionalized glass substrates. Figures of merit were developed for quantitative comparison of spot quality and reproducibility. Experimental variables examined included oligo concentration in the spotting buffer, composition of the spotting buffer, postspotting "curing" conditions, and postspotting wash conditions. Optimized conditions included the use of 3-4 microM oligo in a 3x standard saline citrate/0.05% sodium dodecyl sulfate/0.001% (3-[(3-cholamidopropyl) dimethylammonia]-1-propane sulfonate) spotting buffer, 24-h postspotting reaction at 100% relative humidity, and a four-step wash procedure. Evaluation of six types of aldehyde-functionalized glass substrates indicated that those manufactured by CEL Associates, Inc. yield the highest oligo coverage.     

Chemoenzymatic preparation of dermatan sulfate oligosaccharides as arylsulfatase B and α-L-iduronidase substrates

Dermatan sulfate was partially depolymerized with chondroitin ABC lyase to obtain an oligosaccharide mixture from which an unsaturated disulfated tetrasaccharide was purified and characterized using nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. Chemical removal of the unsaturated uronate residue with mercuric acetate, followed by de-4-O-sulfation with arylsulfatase B (N-acetylgalactosamine 4-sulfatase) and N- acetylhexo-saminidase catalyzed removal of the 2-acetamido-2-deoxy-D-galactospyranosyl residue at the non-reducing end afforded a monosulfated disaccharide of the structure -L-idopyranosyluronic acid (13)-,-D-2-acetamido-2-deoxy-4-O-sulfo galactopyranose. This monosulfated disaccharide serves as a substrate for mammalian -L-iduronidase as demonstrated using fluorophore assisted carbohydrate electrophoresis.     

Substrate dependence of the postischemic cardiomyocyte recovery: Dissociation between functional, metabolic and injury markers

Defining the substrate that influences the most favourably the myocardial post-ischemic recovery is subject of debates, due to dissociation between functional and biochemical benefits. Hence, we studied the effects of either glucose or different fatty acids on the functional and metabolic recovery of post-ischemic cardiomyocytes in a substrate-free hypoxia model of simulated ischemia-reperfusion. Rat cardiomyocytes were submitted to a 2.5 h simulated ischemia followed by a 2 h reoxygenation without substrate (control), or with either glucose, octanoic acid, oleic acid, or elaidic acid. During simulated ischemia, electromechanical function gradually disappeared while the cellular viability and mitochondrial function declined. During control simulated reperfusion, cardiomyocytes recovered near normal function but a significant reduction in the action potential amplitude and rate persisted. The addition of glucose or oleic acid during simulated reperfusion promoted a faster, better and sustain functional recovery. Amongst the fatty acids, the functional recovery was slower with elaidic and octanoic acids as compared with oleic acid. The mitochondrial function was better improved during simulated reperfusion with glucose than with the tested fatty acids, among which elaidic acid was the less unfavourable. Paradoxically, the addition of whichever substrate during simulated reperfusion tended to worsen the cellular viability. Thus, cardiomyocytes recovery strongly relies on the characteristics of the substrate supplied at the onset of simulated reperfusion: glucidic or lipidic nature, chain-length, insaturation degree. Moreover, these data suggest that defining the appropriateness of a given substrate for the post-ischemic cardiomyocyte recovery is closely related to the functional and the biological endpoints in consideration.     

MET meet adaptors: Functional and structural implications in downstream signalling mediated by the Met receptor

The tyrosin kinase Met receptor regulates multiple cellular events, ranging from cell motility and angiogenesis to morphological differentiation and tissue regeneration. To conduce these activities, the cytoplasmic C-terminal region of this receptor acts as a docking site for multiple protein substrates, including Grb2, Gab1, STAT3, Shc, SHIP-1 and Src. These substrates are characterised by the presence of multiple domains, including the PH, PTB, SH2 and SH3 domains, which directly interact with the multisubstrate C-terminal region of Met. How this receptor recognises and binds a specific substrate in a space-temporal mode is a central question in cell signalling. The recently solved crystal structure of the tyrosine kinase domain of the Met receptor and that of domains of diverse Met substrates provides the molecular framework to understand Met substrate specificity. This structural information also gives new insights on the plasticity of Met signalling and the implications of Met deregulation in tumorigenic processes. In the light of these advances, the present work discusses the molecular basis of Met-substrate recognition and its functional implications in signalling events mediated by this pleiotropic receptor. (Mol Cell Biochem 276: 149–157, 2005)     

Modulation by substrates of the protective effect of cyclosporin A on mitochondrial damage

The influence of substrates on the role of cyclosporin A, to promote the closure of the permeability transition pore, was studied. It was found that in succinate-oxidizing mitochondria, cyclosporin inhibited pore opening as induced by carboxyatractyloside. The opposite occurred when mitochondrial respiration was supported by malate-glutamate, i.e., cyclosporin A was unable to block pore opening promoted by carboxyatractyloside. We propose that the failure of cyclosporin A to induce pore closure could be due to a low NADH matrix content.     

Synthesis of peptidyl methylcoumarin esters as substrates and active-site titrants for the prohormone processing proteases Kex2 and PC2

Peptidyl methylcoumarin amides are well established as model substrates for understanding protease specificity, but the corresponding methylcoumarin esters have attracted scant attention despite their potential utility in active-site titration and mechanistic characterization. We have devised techniques for the synthesis and deprotection of extended peptidyl methylcoumarin esters in good to moderate yields, and we have demonstrated their suitability for steady-state characterization and active-site titration of the Saccharomyces cerevisiae processing protease Kex2. Additionally, we have used one of these compounds to active-site titrate the homologous enzyme PC2, which had not previously been feasible using other types of substrates. These compounds should thus prove widely suitable for use as substrates and active-site titrants not only for proteases of the prohormone processing family but also for a wide range of other serine proteases.