Rapid identification of malaria vaccine candidates based on alpha-helical coiled coil protein motif

To identify malaria antigens for vaccine development, we selected alpha-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally "native" epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high alpha-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens.     

An autocrine loop involving ret and glial cell-derived neurotrophic factor mediates retinoic acid-induced neuroblastoma cell differentiation

In several neuroblastoma cell lines, retinoic acid (RA)-induced differentiation is coupled to increased expression of functional neurotrophic factor receptors, including Trk family receptors and the glial cell-derived neurotrophic factor receptor, Ret. In several cases, increased expression is dependent on signaling through TrkB. Unlike TrkA and TrkB, Ret has never been implicated as a prognostic marker for neuroblastomas. SK-N-BE(2) cells do not express any of Trk family receptors; therefore, they are a choice system to study the specific role of Ret in RA-induced differentiation. Using a 2'-fluoro-RNA aptamer and a truncated Ret protein as specific inhibitors of Ret, we show that RA-induced differentiation is mediated by a positive autocrine loop that sustains Ret downstream signaling and depends on glial cell-derived neurotrophic factor expression and release. This report shows that in SK-N-BE(2) cells, stimulation of Ret is a major upstream mechanism needed to mediate RA-induced differentiation. These results provide important insights on the molecular mechanism of RA action, which might be relevant for the development of biologically based therapeutic strategies.     

Longitudinal axons are guided by Slit/Robo signals from the floor plate

Longitudinal axons grow long distances along precise pathways to connect major CNS regions. However, during embryonic development, it remains largely undefined how the first longitudinal axons choose specific positions and grow along them. Here, we review recent evidence identifying a critical role for Slit/Robo signals to guide pioneer longitudinal axons in the embryonic brain stem. These studies indicate that Slit/Robo signals from the floor plate have dual functions: to repel longitudinal axons away from the ventral midline, and also to maintain straight longitudinal growth. These dual functions likely cooperate with other guidance cues to establish the major longitudinal tracts in the brain.Key words: Slit, Robo, longitudinal axon, hindbrain, axon guidance     

4E10-Resistant HIV-1 Isolated from Four Subjects with Rare Membrane-Proximal External Region Polymorphisms

Human antibody 4E10 targets the highly conserved membrane-proximal external region (MPER) of the HIV-1 transmembrane glycoprotein, gp41, and has extraordinarily broad neutralizing activity. It is considered by many to be a prototype for vaccine development. In this study, we describe four subjects infected with viruses carrying rare MPER polymorphisms associated with resistance to 4E10 neutralization. In one case resistant virus carrying a W680G substitution was transmitted from mother to infant. We used site-directed mutagenesis to demonstrate that the W680G substitution is necessary for conferring the 4E10-resistant phenotype, but that it is not sufficient to transfer the phenotype to a 4E10-sensitive Env. Our third subject carried Envs with a W680R substitution causing variable resistance to 4E10, indicating that residues outside the MPER are required to confer the phenotype. A fourth subject possessed a F673L substitution previously associated with 4E10 resistance. For all three subjects with W680 polymorphisms, we observed additional residues in the MPER that co-varied with position 680 and preserved charged distributions across this region. Our data provide important caveats for vaccine development targeting the MPER. Naturally occurring Env variants described in our study also represent unique tools for probing the structure-function of HIV-1 envelope.     

Using genetic markers to estimate the pollen dispersal curve

Pollen dispersal is a critical process that shapes genetic diversity in natural populations of plants. Estimating the pollen dispersal curve can provide insight into the evolutionary dynamics of populations and is essential background for making predictions about changes induced by perturbations. Specifically, we would like to know whether the dispersal curve is exponential, thin-tailed (decreasing faster than exponential), or fat-tailed (decreasing slower than the exponential). In the latter case, rare events of long-distance dispersal will be much more likely. Here we generalize the previously developed TWOGENER method, assuming that the pollen dispersal curve belongs to particular one- or two-parameter families of dispersal curves and estimating simultaneously the parameters of the dispersal curve and the effective density of reproducing individuals in the population. We tested this method on simulated data, using an exponential power distribution, under thin-tailed, exponential and fat-tailed conditions. We find that even if our estimates show some bias and large mean squared error (MSE), we are able to estimate correctly the general trend of the curve - thin-tailed or fat-tailed - and the effective density. Moreover, the mean distance of dispersal can be correctly estimated with low bias and MSE, even if another family of dispersal curve is used for the estimation. Finally, we consider three case studies based on forest tree species. We find that dispersal is fat-tailed in all cases, and that the effective density estimated by our model is below the measured density in two of the cases. This latter result may reflect the difficulty of estimating two parameters, or it may be a biological consequence of variance in reproductive success of males in the population. Both the simulated and empirical findings demonstrate the strong potential of TWOGENER for evaluating the shape of the dispersal curve and the effective density of the population (d(e)).     

Acyl Transfer Activity of an Amidase from Rhodococcus sp. Strain R312: Formation of a Wide Range of Hydroxamic Acids

The enantioselective amidase from Rhodococcus sp. strain R312 was produced in Escherichia coli and was purified in one chromatographic step. This enzyme was shown to catalyze the acyl transfer reaction to hydroxylamine from a wide range of amides. The optimum working pH values were 7 with neutral amides and 8 with α-aminoamides. The reaction occurred according to a Ping Pong Bi Bi mechanism. The kinetic constants demonstrated that the presence of a hydrophobic moiety in the carbon side chain considerably decreased the K_mamide values (e.g., K_mamide = 0.1 mM for butyramide, isobutyramide, valeramide, pivalamide, hexanoamide, and benzamide). Moreover, very high turnover numbers (k_cat) were obtained with linear aliphatic amides (e.g., k_cat = 333 s⁻¹ with hexanoamide), whereas branched-side-chain-, aromatic cycle- or heterocycle-containing amides were sterically hindered. Carboxylic acids, α-amino acids, and methyl esters were not acyl donors or were very bad acyl donors. Only amides and hydroxamic acids, both of which contained amide bonds, were determined to be efficient acyl donors. On the other hand, the highest affinities of the acyl-enzyme complexes for hydroxylamine were obtained with short, polar or unsaturated amides as acyl donors (e.g., K_mNH2OH = 20, 25, and 5 mM for acetyl-, alanyl-, and acryloyl-enzyme complexes, respectively). No acyl acceptors except water and hydroxylamine were found. Finally, the purified amidase was shown to be l-enantioselective towards α-hydroxy- and α-aminoamides.Many bacterial amidases (EC 3.5.1.4) have been described previously because of their amide hydrolysis activities. Wide-spectrum amidases from Rhodococcus sp. strain R312 (26) and Pseudomonas aeruginosa (1), which are very similar, hydrolyze only short-chain amides. These enzymes are made up of four and six identical subunits having molecular weights of about 45,000 and 35,000, respectively. Based on the results of experiments performed with inhibitors, they have been classified as belonging to a branch of sulfhydryl enzymes (1, 26). The other amidases, the enantioselective amidases from Pseudomonas chlororaphis B23 (5), Rhodococcus erythropolis MP50 (12, 27), Rhodococcus sp. strain R312 (20), Rhodococcus sp. strain N-774 (10), Rhodococcus sp. (21), and Rhodococcus rhodochrous J1 (14), belong to a group of amidases containing a GGSS signature in the amino acid sequence (4) and are made up of two (or eight) identical subunits. The corresponding genes are located in clusters containing genes encoding the two subunits of a nitrile hydratase (EC 4.2.1.84). These amidases were also previously classified as sulfhydryl enzymes (5, 15), but no active amino acid residue was identified in any of them. Recently, Kobayashi et al. (15) showed that the real active site residues of the amidase from R. rhodochrous J1 were Asp-191 and Ser-195 rather than the generally accepted Cys-203 residue. These authors showed that aspartic acid and serine residues of this enzyme were also present in the active site sequences of aspartic proteinases and suggested that there is an evolutionary relationship between amidases and aspartic proteinases.All of the different amidases also exhibit an acyl transfer activity in the presence of hydroxylamine: RCONH₂ + NH₂OH ↔ RCONHOH + NH₃. This kind of reaction was previously described for the wide-spectrum amidase from Rhodococcus sp. strain R312 (6), but there has been no detailed study examining the acyl transfer reaction of amidases belonging to the GGSS signature-containing group. The final reaction products (hydroxamic acids) are known to possess high chelating properties. Some of them (particularly α-aminohydroxamic acid derivatives) are potent inhibitors of matrix metalloproteases, a family of zinc endopeptidases involved in tissue remodelling (3). Some other hydroxamic acids (α-aminohydroxamic acids, synthetic siderophores, acetohydroxamic acid, etc.) have also been investigated as anti-human immunodeficiency virus agents or antimalarial agents or have been recommended for treatment of ureaplasma infections and anemia (2, 8, 13, 28). Moreover, some fatty hydroxamic acids have been studied as inhibitors of cylooxygenase and 5-lipooxygenase with potent antiinflammatory activity (9).Apart from these medical applications, some hydroxamic acids (particularly polymerizable unsaturated hydroxamic acids and mid-chain or long-chain hydroxamic acids) have also been extensively investigated in wastewater treatment and nuclear technology studies as a way to eliminate contaminating metal ions (11, 16, 18).In this paper we describe the formation of a wide range of hydroxamic acids with the enantioselective amidase (a 120,000-dalton homodimer) from Rhodococcus sp. strain R312, and we provide some additional information which enhanced our comprehension of the reaction mechanism of this amidase.     

UK-73,093: A non-peptide neurotensin receptor antagonist

UK-73,093 was identified in a screening program as a compound able to displace [³H]-neurotensin from its bovine brain receptor. We describe the discovery of this compound, species differences in receptor affinity and its characterization as a functional neurotensin antogonist in vitro and in vivo.     

Miniaturized analytical assays in biotechnology

Biotechnology today is a well-established paradigm in many areas of human endeavor, such as the pharmaceutical industry, agriculture, management of the environment and many others. Meanwhile, biology is undergoing a spectacular transition: whereas systematic biology was replaced gradually by molecular biology, the latter is rapidly being transformed into a new systematic era in which entire genomes are being charted by ever more sophisticated analytical techniques.In the wake of this onslaught of data, new fields are germinating, such as bioinformatics in an attempt to find answers to fundamental questions, answers that may be hidden in the massive amounts of data already available today.     

Interaction between Erbin and a Catenin-related protein in epithelial cells.

Integrity of epithelial tissues relies on the proper apical-basolateral polarity of epithelial cells. Members of the LAP (LRR and PDZ) protein family such as LET-413 and Scribble are involved in maintaining epithelial cell polarity in Caenorhabditis elegans and Drosophila melanogaster, respectively. We previously described Erbin as a mammalian LET-413 homologue interacting with ERBB2/HER2, an epidermal growth factor receptor family member. Erbin and ERBB2/HER2 are located in the basolateral membranes of epithelial cells. We show here that Erbin interacts with p0071 (also called plakophilin-4), an armadillo repeat protein linked to the cytoskeleton. Erbin binds to p0071 in vitro and in vivo in a PDZ domain-dependent manner, and both proteins colocalized in desmosomes of epithelial cells. Using a dominant negative approach, we found that integrity of epithelial cell monolayer is impaired when interaction between Erbin and p0071 is disrupted. We propose that Erbin is connected by p0071 to cytoskeletal networks in an interaction crucial for epithelial homeostasis.