The actin cytoskeleton in presynaptic assembly

Dramatic morphogenetic processes underpin nearly every step of nervous system development, from initial neuronal migration and axon guidance to synaptogenesis. Underlying this morphogenesis are dynamic rearrangements of cytoskeletal architecture. Here we discuss the roles of the actin cytoskeleton in the development of presynaptic terminals, from the elaboration of terminal arbors to the recruitment of presynaptic vesicles and active zone components. The studies discussed here underscore the importance of actin regulation at every step in neuronal circuit assembly.     

Perturbation Biology: Inferring Signaling Networks in Cellular Systems

We present a powerful experimental-computational technology for inferring network models that predict the response of cells to perturbations, and that may be useful in the design of combinatorial therapy against cancer. The experiments are systematic series of perturbations of cancer cell lines by targeted drugs, singly or in combination. The response to perturbation is quantified in terms of relative changes in the measured levels of proteins, phospho-proteins and cellular phenotypes such as viability. Computational network models are derived de novo, i.e., without prior knowledge of signaling pathways, and are based on simple non-linear differential equations. The prohibitively large solution space of all possible network models is explored efficiently using a probabilistic algorithm, Belief Propagation (BP), which is three orders of magnitude faster than standard Monte Carlo methods. Explicit executable models are derived for a set of perturbation experiments in SKMEL-133 melanoma cell lines, which are resistant to the therapeutically important inhibitor of RAF kinase. The resulting network models reproduce and extend known pathway biology. They empower potential discoveries of new molecular interactions and predict efficacious novel drug perturbations, such as the inhibition of PLK1, which is verified experimentally. This technology is suitable for application to larger systems in diverse areas of molecular biology.     

Catch-up growth in Japanese quail (Coturnix Japonica): relationships with food intake, metabolic rate and sex

The effects of early environmental conditions can profoundly affect individual development and adult phenotype. In birds, limiting resources can affect growth as nestlings, but also fitness and survival as adults. Following periods of food restriction, individuals may accelerate development, undergoing a period of rapid “catch-up” growth, in an attempt to reach the appropriate size at adulthood. Previous studies of altricial birds have shown that catch-up growth can have negative consequences in adulthood, although this has not been explored in species with different developmental strategies. Here, we investigated the effects of resource limitation and the subsequent period of catch-up growth, on the morphological and metabolic phenotype of adult Japanese quail (Coturnix japonica), a species with a precocial developmental strategy. Because males and females differ in adult body size, we also test whether food restriction had sex-specific effects. Birds that underwent food restriction early in development had muscles of similar size and functional maturity, but lower adult body mass than controls. There was no evidence of sex-specific sensitivity of food restriction on adult body mass; however, there was evidence for body size. Females fed ad lib were larger than males fed ad lib, while females subjected to food restriction were of similar size to males. Adults that had previously experienced food restriction did not have an elevated metabolic rate, suggesting that in contrast to altricial nestlings, there was no metabolic carry-over effect of catch-up growth into adulthood. While Japanese quail can undergo accelerated growth after re-feeding, timing of food restriction may be important to adult size, particularly in females. However, greater developmental flexibility compared to altricial birds may contribute to the lack of metabolic carryover effects at adulthood.     

The impact of either 4-R-hydroxyproline or 4-R-fluoroproline on the conformation and SH3m-cort binding of HPK1 proline-rich peptide

SH3 domains are probably the most abundant molecular-recognition modules of the proteome. A common feature of these domains is their interaction with ligand proteins containing Pro-rich sequences. Crystal and NMR structures of SH3 domains complexes with Pro-rich peptides show that the peptide ligands are bound over a range of up to seven residues in a PPII helix conformation. Short proline-rich peptides usually adopt little or no ordered secondary structure before binding interactions, and consequently their association with the SH3 domain is characterized by unfavorable binding entropy due to a loss of rotational freedom on forming the PPII helix. With the aim to stabilize the PPII helix conformation into the proline-rich decapeptide PPPLPPKPKF (P2), we replaced some proline residues either with the 4(R)-4-fluoro-l-proline (FPro) or the 4(R)-4-hydroxy-l-proline (Hyp). The interactions of P2 analogues with the SH3 domain of cortactin (SH3_m-cort) were analyzed by circular dichroism spectroscopy, while CD thermal transition experiments have been used to determine their propensity to adopt a PPII helix conformation. Results show that the introduction of three residues of Hyp efficiently stabilizes the PPII helix conformation, while it does not improve the affinity towards the SH3 domain, suggesting that additional forces, e.g., electrostatic interactions, are involved in the SH3_m-cort substrate recognition.     

GPCR screening via ERK 1/2: a novel platform for screening G protein-coupled receptors

Discovery of novel agonists and antagonists for G protein-coupled receptors (GPCRs) relies heavily on cell-based assays because determination of functional consequences of receptor engagement is often desirable. Currently, there are several key parameters measured to achieve this, including mobilization of intracellular Ca2+ and formation of cyclic adenosine monophosphate or inositol triphosphate. However, no single assay platform is suitable for all situations, and all of the assays have limitations. The authors have developed a new high-throughput homogeneous assay platform for GPCR discovery as an alternative to current assays, which employs detection of phosphorylation of the key signaling molecule p42/44 MAP kinase (ERK 1/2). The authors show that ERK 1/2 is consistently activated in cells stimulated by Gq-coupled GPCRs and provides a new high-throughput platform for screening GPCR drug candidates. The activation of ERK 1/2 in Gq-coupled GPCR systems generates comparable pharmacological data for receptor agonist and antagonist data obtained by other GPCR activation measurement techniques.     

Fine structure of trophozoites of the gregarine Leidyana ephestiae (Apicomplexa: Eugregarinida) parasitic in Ephestia kuehniella larvae (Lepidoptera)

The ultrastructure of the eugregarine Leidyana ephestiae, parasitic in the larval gut of the flour moth, Ephestia kuehniella, is described. Guts of experimentally infected larvae of E. kuehniella were fixed and sectioned for electron microscope studies of young and mature trophozoites. Young unsegmented trophozoites were small, oval to ovoid, and possessed a simple, globular epimerite. The plasma membrane covering the epimerite region was continuous with the plasma membrane of the protodeutomerite and was in close contact with that of the host cell. Three cortical membranes covered the protodeutomerite region. Folding of the protodeutomeritic epicyte occurred after about 2 days of development of the gregarine. After 3–4 days the body of the trophozoite became differentiated into three segments. A septum was visible between protomerite and deutomerite, but there was nothing similar to this structure between epimerite and protomerite. Fully developed trophozoites showed a large ovoid epimerite containing many mitochondria and vesicles. The epimerite was situated on a short neck filled with fibrils. The cytoplasm of protomerite and deutomerite was rich in amylopectin granules and electron-dense bodies.     

Recombinant expression, purification, and functional analysis of two novel cystatins from sugarcane (Saccharum officinarum)

Phytocystatins are cysteine proteinase inhibitors from plants implicated in the endogenous regulation of protein turnover, programmed cell death, and in defense mechanisms against pathogens. To date, only few cystatin genes have been characterized in most plant species. We have previously characterized the protein Canecystatin, the first cystatin described in sugarcane. In an attempt to study novel Canecystatins, we identified two ORFs encoding cystatins (referred as CaneCPI-2 and CaneCPI-3) using the data from the Sugarcane EST genome project. These ORFs were then subcloned and expressed in Escherichia coli using pET28 expression vector. High amounts (approximately 20 mg/L) of pure recombinant proteins were obtained by affinity chromatography in a single step of purification. Polyclonal antibodies against the recombinant Canecystatins were raised, allowing the immunodetection of the endogenous proteins in the plant tissues. Moreover, the proteins were able to inhibit papain in a fluorometric assay with K(i) values of 0.2 and 0.25 microM for CaneCPI-2 and CaneCPI-3, respectively. These findings contribute to a better understanding of the activity of sugarcane cystatins and encourage future activity and structural studies of these proteins.     

Dental caries and antemortem tooth loss in the Northern Peten area,Mexico: a biocultural perspective on social status differences among the Classic Maya

Dental caries and antemortem tooth loss (AMTL) are investigated in a Classic Maya sample obtained from the sites of Calakmul, Dzibanché, and Kohunlich (Mexico). This study aims at assessing the effect that sex and social status had on the prevalence of oral pathologies. The lack of a direct relationship between caries, AMTL, and age-at-death led us to interpret the results in terms of the biological, socioeconomic, and behavioral conditions prevailing in these ancient Maya settlements. Benefits related to sex and social status are evident in the frequency of carious lesions, which appear less frequently in elite males than in low-status individuals of both sexes and in elite females. Individuals from problematic mortuary contexts and isolated bone assemblages, who could not be ascribed to any status group, showed the highest rates of caries. Sex discrimination in dietary preferences appears in the elite sample, while the homogeneity encountered between sexes in the low-status segment suggests a more uniform access to resources. Tooth loss clearly distinguishes elite individuals from commoners, regardless of sex, with the former bearing a much higher rate of loss. In individuals from the undefined mortuary assemblages and sacrificial contexts, it was even more pronounced than in the other groups, although its interpretation is problematic due to a lack of associated funerary data. The overall evidence from oral pathologies is interpreted to be the result of deficient oral hygiene coupled with a softer and more refined diet in the high-status population, particularly males. Whereas elite males' subsistence was apparently based more on animal proteins and relatively soft and refined foods, a diet relying on carbohydrates may account for the observed rate of oral pathologies in elite females and commoners.