Compressibility and uncoupling of cytochrome P450cam: high pressure FTIR and activity studies

The effect of the hydrostatic pressure on the CO ligand stretch vibration in cytochrome P450cam-CO bound with various substrates is studied by FTIR. The vibration frequency is linearily shifted to lower values with increasing pressure. The slope of the shift gives the isothermal compressibility of the heme pocket and is found to be related to the high-spin state content in an opposite direction to that previously observed from the pressure-induced shift of the Soret band. This opposite behaviour is explained by the dual effect of heme pocket water molecules both on the CO ligand and on electrostatic potentials produced by the protein at the distal side. The latter effect disturbs ligand-distal side contacts which are needed for a specific proton transfer in oxygen activation when dioxygen is the ligand. Their loss results in uncoupled H(2)O(2) formation.     

Charge displacements during ATP-hydrolysis and synthesis of the Na+-transporting FoF1-ATPase of Ilyobacter tartaricus

Transient electrical currents generated by the Na(+)-transporting F(o)F(1)-ATPase of Ilyobacter tartaricus were observed in the hydrolytic and synthetic mode of the enzyme. Two techniques were applied: a photochemical ATP concentration jump on a planar lipid membrane and a rapid solution exchange on a solid supported membrane. We have identified an electrogenic reaction in the reaction cycle of the F(o)F(1)-ATPase that is related to the translocation of the cation through the membrane bound F(o) subcomplex of the ATPase. In addition, we have determined rate constants for the process: For ATP hydrolysis this reaction has a rate constant of 15-30 s(-1) if H(+) is transported and 30-60 s(-1) if Na(+) is transported. For ATP synthesis the rate constant is 50-70 s(-1).     

Laminin alpha5 chain is required for intestinal smooth muscle development

Laminins (comprised of alpha, beta, and gamma chains) are heterotrimeric glycoproteins integral to all basement membranes. The function of the laminin alpha5 chain in the developing intestine was defined by analysing laminin alpha5(-/-) mutants and by grafting experiments. We show that laminin alpha5 plays a major role in smooth muscle organisation and differentiation, as excessive folding of intestinal loops and delay in the expression of specific markers are observed in laminin alpha5(-/-) mice. In the subepithelial basement membrane, loss of alpha5 expression was paralleled by ectopic or accelerated deposition of laminin alpha2 and alpha4 chains; this may explain why no obvious defects were observed in the villous form and enterocytic differentiation. This compensation process is attributable to mesenchyme-derived molecules as assessed by chick/mouse alpha5(-/-) grafted associations. Lack of the laminin alpha5 chain was accompanied by a decrease in epithelial alpha3beta1 integrin receptor expression adjacent to the epithelial basement membrane and of Lutheran blood group glycoprotein in the smooth muscle cells, indicating that these receptors are likely mediating interactions with laminin alpha5-containing molecules. Taken together, the data indicate that the laminin alpha5 chain is essential for normal development of the intestinal smooth muscle and point to possible mesenchyme-derived compensation to promote normal intestinal morphogenesis when laminin alpha5 is absent.     

Worldwide distribution of transposable element copy number in natural populations of Drosophila simulans

Abstract.— Transposable elements (TEs), which promote various kinds of mutations, constitute a large fraction of the genome. How they invade natural populations and species is therefore of fundamental importance for understanding the dynamics of genetic diversity and genome composition. On the basis of 85 samples of natural populations of Drosophila simulans , we report the distributions of the genome insertion site numbers of nine TEs that were chosen because they have a low average number of sites. Most populations were found to have 0–3 insertion sites, but some of them had a significantly higher number of sites for a given TE. The populations located in regions outside Africa had the highest number of sites for all elements except HMS Beagle and Coral , suggesting a recent increase in the activity of some TEs associated with the colonization patterns of Drosophila simulans . The element Tirant had a very distinctive pattern of distribution: it was identified mainly in populations from East Africa and some islands in the Indian Ocean, and its insertion site number was low in all these populations. The data suggest that the genome of the entire species of Drosophila simulans may be being invaded by TEs from populations in which they are present in high copy number.     

Early apoptosis-related changes triggered by HSV-1 in individual neuronlike cells

Early events of apoptosis following HSV-1 infection were investigated at the single-cell level using intensified fluorescence digital-imaging microscopy. The results provide evidence that infection of differentiated ND7 neuronlike cells by HSV-1 triggers detectable alterations indicative of physiological changes associated with the early stages of apoptosis. Less than 1 h after infection with HSV-1 (KOS strain) or K26GFP (GFP being fused to HSV-1 capsid protein VP26) we observed (i) moderate decrease in mitochondrial membrane potential (about 20%), (ii) exposure of phosphatidyl serine, (iii) morphological change in the mitochondria that became spherical instead of filamentous, and (iv) activation of caspase-8. Within 3 h changes reverted to normal, which indicated that apoptosis was counteracted very early following HSV-1 infection. Similar results were obtained with KOS-TK27GFP, lacking TK and UL24 proteins, suggesting that TK and UL24 play no role in apoptosis. In Vero cells mitochondrial changes characteristic of the apoptotic process were not observed following HSV-1 infection. The UV-inactivated K26GFP had the capacity to induce apoptosis in neuronlike cells. This real-time multiparametric analysis, in combination with relevant viral mutants, could be a useful approach for dissecting the roles of various viral genes in modulating apoptotic pathways during infection.     

Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi)

For reversal of MDR1 gene-dependent multidrug resistance (MDR), two small interfering RNA (siRNA) constructs were designed to inhibit MDR1 expression by RNA interference. SiRNA duplexes were used to treat human pancreatic carcinoma (EPP85-181RDB) and gastric carcinoma (EPG85-257RDB) cells. In both cellular systems, siRNAs could specifically inhibit MDR1 expression up to 91% at the mRNA and protein levels. Resistance against daunorubicin was decreased to 89% (EPP85-181RDB) or 58% (EPG85-257RDB). The data indicate that this approach may be applicable to cancer patients as a specific means to reverse tumors with a P-glycoprotein-dependent MDR phenotype back to a drug-sensitive one.     

Misfolded plant virus proteins: elicitors and targets of ubiquitylation

Mutant tobacco mosaic virus (TMV) coat proteins (CPs) with known amino acid replacements provide well defined examples of destabilized tertiary structures. Here we show that misfolded TMV CPs, but not functional wild-type CPs, induce massive ubiquitylation in tobacco cells and that denatured, insoluble CP subunits are the main substrates of ubiquitin conjugation. As TMV CPs can be easily manipulated they are unique tools to study the molecular basis of the plant cell's response to aberrant protein structures and the associated intracellular stress reactions.     

Transposition of Tn5367 in Mycobacterium marinum,using a conditionally recombinant mycobacteriophage

Mycobacterium marinum is a close relative of the obligate human pathogen Mycobacterium tuberculosis. As with M. tuberculosis, M. marinum causes intracellular infection of poikilothermic vertebrates and skin infection in humans. It is considered a valid model organism for the study of intracellular pathogenesis of mycobacteria. Low transformation efficiencies for this species have precluded approaches using mutant libraries in pathogenesis studies. We have adapted the conditionally replicating mycobacteriophage phAE94, originally developed as a transposon mutagenesis tool for M. tuberculosis, to meet the specific requirements of M. marinum. Conditions permissive for phage replication in M. tuberculosis facilitated highly efficient transposon delivery in M. marinum. Using this technique we succeeded in generating a representative mutant library of this species, and we conclude that TM4-derived mycobacteriophages are temperature-independent suicide vectors for M. marinum.     

How Spike Synchronization Among Olfactory Neurons Can Contribute to Sensory Discrimination

Recent studies in honeybees have demonstrated that, when odor-evoked action potentials in antennal lobe neurons are pharmacologically desynchronized, the bees are impaired in their ability to discriminate chemically similar odor stimuli. Using a reduced computational model of the honeybee antennal lobe, we show how changes in spike-synchronization properties alone, independent of changes in overall spike-discharge rate or differences in activity levels among responsive neurons, can produce changes in associative learning similar to those observed experimentally.     

B2 receptor activation reduces Erk1 and Erk2 phosphorylation induced by insulin-like growth factor-1, platelet-derived growth factor-BB,and high glucose in rat isolated glomeruli

Several experimental data document an activation of the mitogen-activated protein kinases Erk1 and Erk2 by bradykinin (BK), an agonist of the kinin B2 receptor (B2R). In contrast, other reports showed an inhibitory modulation of mitogenesis by BK. Therefore, we explored in the isolated glomeruli the effect of B2R activation on the signaling of insulin-like growth factor-1 (IGF-1), platelet-derived growth factor-BB (PDGF-BB), and high glucose (HG), three factors that are believed to be involved in the development of glomerulosclerosis via the phosphorylation of Erk1 and Erk2. We observed that the activation of B2R negatively modulates the phosphorylation of Erk1 and Erk2 induced by IGF-1, PDGF-BB, and HG in the glomerulus. These effects are consistent with the hypothesis of a protective role for BK in the kidney during development of glomerulosclerosis and renal pathologies associated with a hyperproliferative state.