Thermo-osmoregulation of Heat-Labile Enterotoxin Expression by <Emphasis Type="Italic">Escherichia coli</Emphasis>

Abstract:Enterotoxigenic Escherichia coli causes diarrhea by producing several virulence factors including heat-labile enterotoxin (LT). LT is maximally expressed at 37°C. The histone-like nucleoid structuring protein (H-NS) appears to inhibit LT expression by binding to a downstream regulatory element (DRE) at low temperatures. An hns⁺ E. coli strain, X7026, carrying an LT–beta-galactosidase translational fusion plasmid (pLT-lac) was shown to be responsive to varying amounts of sodium chloride (NaCl) as well as sucrose or lithium chloride. Maximal responsiveness to the various osmolytes was obtained with cells grown at 37°C under microaerophilic conditions. Temperature-osmotic upshift experiments demonstrate LT expression is thermo-osmoregulated. pLT-lac was tested in an hns strain or its congenic hns⁺ strain for its response to NaCl. LT expression is elevated in the hns strain regardless of NaCl concentration and retains its osmoresponsiveness. The response of the DRE deletion plasmid (pLT-lacNC) to NaCl is similar to that of the undeleted plasmid.     

Effects of starvation on haemolymphatic glucose levels, glycogen contents and nucleotidase activities in different tissues of Helix aspersa (Müller, 1774) (Mollusca, Gastropoda)

In the present study, the glucose concentration in the haemolymph and glycogen levels were determined in the various body parts of the Helix aspersa snail after feeding lettuce ad libitum and after various periods of starvation. To characterize the effect of starvation on nucleotidase activity, enzyme assays were performed on membranes of the nervous ganglia and digestive gland. Results demonstrated the maintenance of the haemolymph glucose concentration for up to 30 days of starvation, probably due to the consumption of glycogen from the mantle. In the nervous ganglia, depletion of glycogen occurs progressively during the different periods of starvation. No significant changes were observed on ATP and ADP hydrolysis in the membranes of nervous ganglia and no alterations in Ca2+ -ATPase and Mg2+ -ATPase occurred in the membranes of the digestive gland of H. aspersa during the different periods of starvation. Although there were no changes in the enzyme activities during starvation, they could be modulated by effectors in situ with concomitant changes in products/reactants during starvation.     

Mitochondrial ribosomes in a trypanosome

The nature, and even the existence, of trypanosome mitochondrial ribosomes has been the subject of some debate. We investigated this further in the insect trypanosome, Crithidia fasciculata. In sucrose gradients of parasite lysates, mitochondrial ribosomal RNA co-sediments at approximately 35S with nascent peptides synthesized in the presence of the cytosolic translational inhibitor, cycloheximide. Co-sedimenting peptides in this peak are much reduced when the parasites are treated with the bacterial translational inhibitor, chloramphenicol. In CsCl gradients this peak resolves at a buoyant density of 1.42 g/cm(3), a value typical for mito-ribosomes. Electron microscopy of peak material shows particles smaller than cytosolic ribosomes, but with characteristic ribosomal shapes. We propose that these particles represent the parasite's mitochondrial ribosomes.     

Zinc-induced decrease of the thermal stability and regeneration of rhodopsin

Zinc is present at high concentrations in the photoreceptor cells of the retina where it has been proposed to play a role in the visual phototransduction process. In order to obtain more information about this role, the study of the effect of zinc on several properties of the visual photoreceptor rhodopsin has been investigated. A specific effect of Zn(2+) on the thermal stability of rhodopsin, obtained from bovine retinas and solubilized in dodecyl maltoside detergent, in the dark is reported. The thermal stability of rhodopsin in its ground state (dark state) is clearly reduced with increasing Zn(2+) concentrations (0-50 microm Zn(2+)). The thermal bleaching process is accelerated in the presence of Zn(2+) with k rate constants, at 55 degrees C, of 0.028 +/- 0.002 min(-1) (0 microm Zn(2+)) and 0.056 +/- 0.003 min(-1) (50 microm Zn(2+)), corresponding to t(12) values of 24.4 +/- 1.6 min and 11.8 +/- 0.1 min, respectively. Thermodynamic parameters derived from Arrhenius plots show a significant E(a) increase at 50 microm Zn(2+) for the process, with deltaG++ decrease and increase in deltaH++ and deltaS++ possibly reflecting conformational rearrangements and reordering of water molecules. The stability of the metarhodopsin II intermediate is also decreased and changes in the metarhodopsin II decay pathway are also detected. The extent of rhodopsin regeneration in vitro is also reduced by zinc. These effects, specific for zinc, are also seen for rhodopsin in native disc membranes, and may be relevant to the suggested role of Zn(2+) in normal and pathological retinal function.     

Single strand conformational polymorphism evaluation for the identification of TH3R alleles of the circumsporozoite protein gene of Plasmodium falciparum

Highly polymorphic regions of the circumsporozoite protein (CSP) of Plasmodium falciparum are associated with cellular immune responses. One of these regions, the TH3R polymorfic region of the csp gene codes for known T-cell epitopes. The present study tested the use of SSCP to determine sequence variations of the TH3R regions of four clones of P. falciparum (3D7, HB3, Dd2 and K1) which are known to have different TH3R regions. Single-strand conformation polymorphism (SSCP) technique was performed on amplified products labelled with fluorescent primer (both strands) and electrophoresed in an automated sequencer. Various gel compositions and electrophoresis conditions were tested. Even if some electrophoretogram differences were observed between clones, they could not distinguish between the alleles.     

Genome plasticity in Lactococcus lactis

Comparative genome analyses contribute significantly to our understanding of bacterial evolution and indicate that bacterial genomes are constantly evolving structures. The gene content and organisation of chromosomes of lactic acid bacteria probably result from a strong evolutionary pressure toward optimal growth of these microorganisms in milk. The genome plasticity of Lactococcus lactis was evaluated at inter- and intrasubspecies levels by different experimental approaches. Comparative genomics showed that the lactococcal genomes are not highly plastic although large rearrangements (a.o. deletions, inversions) can occur. Experimental genome shuffling using a new genetic strategy based on the Cre-loxP recombination system revealed that two domains are under strong constraints acting to maintain the original chromosome organisation: a large region around the replication origin, and a smaller one around the putative terminus of replication. Future knowledge of the rules leading to an optimal genome organisation could facilitate the definition of new strategies for industrial strain improvement.     

A direct pyrophosphatase-coupled assay provides new insights into the activation of the secreted adenylate cyclase from Bordetella pertussis by calmodulin

Continuous recording of the activity of recombinant adenylate cyclase (CyaA) of Bordetella pertussis (EC ) by conductimetric determination of enzyme-coupled pyrophosphate cleavage has enabled us to define a number of novel features of the activation of this enzyme by calmodulin and establish conditions under which valid activation data can be obtained. Activation either in the presence or absence of calcium is characterized by a concentration-dependent lag phase. The rate of formation and breakdown of the activated complex can be determined from an analysis of the lag phase kinetics and is in good agreement with thermodynamic data obtained by measuring the dependence of activation on calmodulin concentration, which show that calcium increases k(on) by about 30-fold. The rate of breakdown of the activated complex, formed either in the presence or absence of calcium, has been determined by dilution experiments and has been shown to be independent of the presence of calcium. The coupled assay is established as a rapid, convenient and safe method which should be readily applicable to the continuous assays of most other enzymes that catalyze reactions in which inorganic pyrophosphate is liberated.     

Characterization of pregnancy-associated glycoproteins extracted from zebu (Bos indicus) placentas removed at different gestational periods

In the present work, two biochemical approaches were used to characterize PAGs isolated from Bos indicus fetal cotyledons removed at different gestational ages. The first procedure included acidic and ammonium sulfate precipitations, anion and cation exchange chromatographies and the second included pepstatin-agarose affinity chromatography. A bovine PAG radioimmunoassay was used to monitor the immunoreactivity throughout the isolation procedures. The most immunoreactive fractions issued from cation exchange and affinity chromatographies were analyzed by SDS-PAGE and Western blotting, before transfer to a polyvinylidene difluoride (PVDF) membrane for NH2-microsequence determination. Use SDS-PAGE and Western blotting, different isoforms of PAG with apparent molecular masses of 51 to 69 kDa and isoelectric points varying from 4.4 to 6.7 were identified in the placentas from different gestational ages. N-terminal microsequencing (10 to 25 aa long) indicates the expression of one single terminal amino acid sequence in the Bos indicus placenta, which is 100% identical to the bovine PAG-1.