Maximum-entropy decomposition of fluorescence correlation spectroscopy data: application to liposome–human serum albumin association

Fluorescence correlation spectroscopy was used to measure the diffusion behavior of a mixture of DMPC or DMPC/DMPG liposomes with human serum albumin (HSA) and mesoporphyrin (MP), which was used as the fluorescent label for liposomes and HSA as well. For decomposing the fluorescence intensity autocorrelation function (ACF) into components corresponding to a liposome population, HSA and MP, we used a maximum entropy procedure that computes a distribution of diffusion times consistent with the ACF data. We found that a simple parametric non-linear fit with a discrete set of decay components did not converge to a stable parameter set. The distribution calculated with the maximum entropy method was stable and the average size of the particles calculated from the effective diffusion time was in good agreement with the data determined using the discrete-component fit.     

Seasonal Variation of Antifouling Activities of Marine Algae from the Brittany Coast (France)

The antifouling activity of extracts (aqueous, ethanol, and dichloromethane) of 9 marine macroalgae against bacteria, fungi, diatoms, macroalgal spores, mussel phenoloxidase activity, and barnacle cypris larvae has been investigated in relation to season in bimonthly samples from the Bay of Concarneau (France). Of the extracts tested, 48.2% were active against at least one of the fouling organisms, and of these extracts, 31.2% were seasonally active with a peak of activity in summer corresponding to maximal values for water temperature, light intensity, and fouling pressure, and 17% were active throughout the year. This seasonal activity may be adaptive as it coincides with maximal fouling pressure in the Bay of Concarneau. Dichloromethane extracts of Rhodophyceae were the most active in the antifouling assays.     

The ternary complex of Pseudomonas aeruginosa alcohol dehydrogenase with NADH and ethylene glycol

Pseudomonas aeruginosa alcohol dehydrogenase (PaADH; ADH, EC 1.1.1.1) catalyzes the reversible oxidation of primary and secondary alcohols to the corresponding aldehydes and ketones, using NAD as coenzyme. We crystallized the ternary complex of PaADH with its coenzyme and a substrate molecule and determined its structure at a resolution of 2.3 A, using the molecular replacement method. The PaADH tetramer comprises four identical chains of 342 amino acid residues each and obeys ~222-point symmetry. The PaADH monomer is structurally similar to alcohol dehydrogenase monomers from vertebrates, archaea, and bacteria. The stabilization of the ternary complex of PaADH, the coenzyme, and the poor substrate ethylene glycol (k(cat) = 4.5 sec(-1); Km > 200 mM) was due to the blocked exit of the coenzyme in the crystalline state, combined with a high (2.5 M) concentration of the substrate. The structure of the ternary complex presents the precise geometry of the Zn coordination complex, the proton-shuttling system, and the hydride transfer path. The ternary complex structure also suggests that the low efficiency of ethylene glycol as a substrate results from the presence of a second hydroxyl group in this molecule.     

TRACTS: A program to map oligopurine.oligopyrimidine and other binary DNA tracts

A program to map the locations and frequencies of DNA tracts composed of only two bases ('Binary DNA') is described. The program, TRACTS (URL http://bioportal.weizmann.ac.il/tracts/tracts.html and/or http://bip.weizmann.ac.il/miwbin/servers/tracts) is of interest because long tracts composed of only two bases are highly over-represented in most genomes. In eukaryotes, oligopurine.oligopyrimidine tracts ('R.Y tracts') are found in the highest excess. In prokaryotes, W tracts predominate (A,T 'rich'). A pre-program, ANEX, parses database annotation files of GenBank and EMBL, to produce a convenient one-line list of every gene (exon, intron) in a genome. The main unit lists and analyzes tracts of the three possible binary pairs (R.Y, K.M and S;W). As an example, the results of R.Y tract mapping of mammalian gene p53 is described.     

Direct injection of venom by a predatory wasp into cockroach brain

In this article, we provide direct evidence for injection of venom by a wasp into the central nervous system of its cockroach prey. Venomous predators use neurotoxins that generally act at the neuromuscular junction, resulting in different types of prey paralysis. The sting of the parasitoid wasp Ampulex compressa is unusual, as it induces grooming behavior, followed by a long-term lethargic state of its insect prey, thus ultimately providing a living meal for the newborn wasp larvae. These behavioral modifications are induced only when a sting is inflicted into the head. These unique effects of the wasp venom on prey behavior suggest that the venom targets the insect's central nervous system. The mechanism by which behavior modifying compounds in the venom transverse the blood-brain barrier to induce these central and long-lasting effects has been the subject of debate. In this article, we demonstrate that the wasp stings directly into the target ganglia in the head of its prey. To prove this assertion, we produced "hot" wasps by injecting them with (14)C radiolabeled amino acids and used a combination of liquid scintillation and light microscopy autoradiography to trace radiolabeled venom in the prey. To our knowledge, this is the first direct evidence documenting targeted delivery of venom by a predator into the brain of its prey.     

Elucidation of primary structure elements controlling early amyloid beta-protein oligomerization

Assembly of monomeric amyloid beta-protein (A beta) into oligomeric structures is an important pathogenetic feature of Alzheimer's disease. The oligomer size distributions of aggregate-free, low molecular weight A beta 40 and A beta 42 can be assessed quantitatively using the technique of photo-induced cross-linking of unmodified proteins. This approach revealed that low molecular weight A beta 40 is a mixture of monomer, dimer, trimer, and tetramer, in rapid equilibrium, whereas low molecular weight A beta 42 preferentially exists as pentamer/hexamer units (paranuclei), which self-associate to form larger oligomers. Here, photo-induced cross-linking of unmodified proteins was used to evaluate systematically the oligomerization of 34 physiologically relevant A beta alloforms, including those containing familial Alzheimer's disease-linked amino acid substitutions, naturally occurring N-terminal truncations, and modifications altering the charge, the hydrophobicity, or the conformation of the peptide. The most important structural feature controlling early oligomerization was the length of the C terminus. Specifically, the side-chain of residue 41 in A beta 42 was important both for effective formation of paranuclei and for self-association of paranuclei into larger oligomers. The side-chain of residue 42, and the C-terminal carboxyl group, affected paranucleus self-association. A beta 40 oligomerization was particularly sensitive to substitutions of Glu22 or Asp23 and to truncation of the N terminus, but not to substitutions of Phe19 or Ala21. A beta 42 oligomerization, in contrast, was largely unaffected by substitutions at positions 22 or 23 or by N-terminal truncations, but was affected significantly by substitutions of Phe19 or Ala21. These results reveal how specific regions and residues control A beta oligomerization and show that these controlling elements differ between A beta 40 and A beta 42.     

Evidence that phosphatidylinositol 3-kinase- and mitogen-activated protein kinase kinase-4/c-Jun NH2-terminal kinase-dependent Pathways cooperate to maintain lung cancer cell survival

Cancer cells in which the PTEN lipid phosphatase gene is deleted have constitutively activated phosphatidylinositol 3-kinase (PI3K)-dependent signaling and require activation of this pathway for survival. In non-small cell lung cancer (NSCLC) cells, PI3K-dependent signaling is typically activated through mechanisms other than PTEN gene loss. The role of PI3K in the survival of cancer cells that express wild-type PTEN has not been defined. Here we provide evidence that H1299 NSCLC cells, which express wild-type PTEN, underwent proliferative arrest following treatment with an inhibitor of all isoforms of class I PI3K catalytic activity (LY294002) or overexpression of the PTEN lipid phosphatase. In contrast, overexpression of a dominant-negative mutant of the p85alpha regulatory subunit of PI3K (Deltap85) induced apoptosis. Whereas PTEN and Delta85 both inhibited activation of AKT/protein kinase B, only Deltap85 inhibited c-Jun NH2-terminal kinase (JNK) activity. Cotransfection of the constitutively active mutant Rac-1 (Val12), an upstream activator of JNK, abrogated Deltap85-induced lung cancer cell death, whereas constitutively active mutant mitogen-activated protein kinase kinase (MKK)-1 (R4F) did not. Furthermore, LY294002 induced apoptosis of MKK4-null but not wild-type mouse embryo fibroblasts. Therefore, we propose that, in the setting of wild-type PTEN, PI3K- and MKK4/JNK-dependent pathways cooperate to maintain cell survival.     

Proteomic analysis of astrocytic secretion in the mouse. Comparison with the cerebrospinal fluid proteome

Astrocytes, the most abundant cell type in the central nervous system, are intimately associated with synapses. They play a pivotal role in neuronal survival and the brain inflammatory response. Some astrocytic functions are mediated by the secretion of polypeptides. Using a proteomic approach, we have identified more than 30 proteins released by cultured astrocytes. These include proteases and protease inhibitors, carrier proteins, and antioxidant proteins. Exposing astrocytes to brefeldin A, which selectively blocks secretory vesicle assembly, suppressed the release of some of these proteins. This indicates that astrocytes secrete these proteins by a classic vesicular mechanism and others by an alternative pathway. Astrocytes isolated from different brain regions secreted a similar pattern of proteins. However, the secretion of some of them, including metalloproteinase inhibitors and apolipoprotein E, was region-specific. In addition, pro-inflammatory treatments modified the profile of astrocytic protein secretion. Finally, more than two thirds of the proteins identified in the astrocyte-conditioned medium were detectable in the mouse cerebrospinal fluid, suggesting that astrocytes contribute to the cerebrospinal fluid protein content. In conclusion, this study provides the first unbiased characterization of the major proteins released by astrocytes, which may play a crucial role in the modulation of neuronal survival and function.     

Peroxidation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effects of surface charge on the oxidizability and on the potency of antioxidants

Peroxidation of membrane phospholipids is an important determinant of membrane function. Previously we studied the kinetics of peroxidation of the polyunsaturated fatty acid (PUFA) residues in model membranes (liposomes) made by sonication of palmitoyllinoleoylphosphatidylcholine (PLPC). Since most biomembranes are negatively-charged, we have now studied the effect of negative surface charge on the kinetics of peroxidation of liposomes made of PLPC and 9% of one of the negatively-charged phospholipids phosphatidylserine (PS) or phosphatidic acid (PA). Peroxidation was initiated by either CuCl2 or AAPH and continuously monitored spectrophotometrically. The following results were obtained: (i) The negative charge had only a slight effect on AAPH-induced peroxidation, but accelerated markedly copper-induced peroxidation of the liposomes, probably by increasing the binding of copper to the membrane surface. (ii) Ascorbic acid (AA) inhibited AAPH-induced but promoted copper-induced peroxidation in all the studied liposomes, probably by enhancing the production of free radicals upon reduction of Cu(II) to Cu(I). (iii) alpha-tocopherol (Toc) inhibited AAPH-induced peroxidation in all the studied liposomes, whereas the effect of tocopherol on copper-induced peroxidation varied from being pro-oxidative in PA-containing liposomes, to being extremely anti-oxidative in PS-containing liposomes, even at very low tocopherol concentrations. The significance of the latter unusual protective effect, which we attribute to recycling of tocopherol by a PS-Cu complex, requires further investigation.     

Synthesis and localization of the Salmonella SPI-1 type III secretion needle complex proteins PrgI and PrgJ

An essential component of type III secretion systems (TTSS) is a supramolecular structure termed the needle complex. In Salmonella enterica, at least four proteins make up this structure: InvG, PrgH, PrgK, and PrgI. Another protein, PrgJ, is thought to play a role in the assembly of this structure, but its function is poorly understood. We have analyzed the expression and localization of PrgJ and the needle protein PrgI in different S. enterica serovar Typhimurium mutant strains. We found that the levels of PrgI and PrgJ were significantly reduced in a TTSS-deficient invA mutant strain and that the decreased levels were due to protein instability. In addition, we found that PrgJ, although associated with the needle complex in wild-type S. enterica serovar Typhimurium, was absent from needle complexes obtained from an invJ mutant strain, which exhibits very long needle substructures. We suggest that PrgJ is involved in capping the needle substructure of the needle complex.