Genetic modifiers of the Drosophila blue cheese gene link defects in lysosomal transport with decreased life span and altered ubiquitinated-protein profiles

Defects in lysosomal trafficking pathways lead to decreased cell viability and are associated with progressive disorders in humans. Previously we have found that loss-of-function (LOF) mutations in the Drosophila gene blue cheese (bchs) lead to reduced adult life span, increased neuronal death, and widespread CNS degeneration that is associated with the formation of ubiquitinated-protein aggregates. To identify potential genes that participate in the bchs functional pathway, we conducted a genetic modifier screen based on alterations of an eye phenotype that arises from high-level overexpression of Bchs. We found that mutations in select autophagic and endocytic trafficking genes, defects in cytoskeletal and motor proteins, as well as mutations in the SUMO and ubiquitin signaling pathways behave as modifiers of the Bchs gain-of-function (GOF) eye phenotype. Individual mutant alleles that produced viable adults were further examined for bchs-like phenotypes. Mutations in several lysosomal trafficking genes resulted in significantly decreased adult life spans and several mutants showed changes in ubiquitinated protein profiles as young adults. This work represents a novel approach to examine the role that lysosomal transport and function have on adult viability. The genes characterized in this study have direct human homologs, suggesting that similar defects in lysosomal transport may play a role in human health and age-related processes.     

The Ability of Flux Balance Analysis to Predict Evolution of Central Metabolism Scales with the Initial Distance to the Optimum

The most powerful genome-scale framework to model metabolism, flux balance analysis (FBA), is an evolutionary optimality model. It hypothesizes selection upon a proposed optimality criterion in order to predict the set of internal fluxes that would maximize fitness. Here we present a direct test of the optimality assumption underlying FBA by comparing the central metabolic fluxes predicted by multiple criteria to changes measurable by a ¹³C-labeling method for experimentally-evolved strains. We considered datasets for three Escherichia coli evolution experiments that varied in their length, consistency of environment, and initial optimality. For ten populations that were evolved for 50,000 generations in glucose minimal medium, we observed modest changes in relative fluxes that led to small, but significant decreases in optimality and increased the distance to the predicted optimal flux distribution. In contrast, seven populations evolved on the poor substrate lactate for 900 generations collectively became more optimal and had flux distributions that moved toward predictions. For three pairs of central metabolic knockouts evolved on glucose for 600–800 generations, there was a balance between cases where optimality and flux patterns moved toward or away from FBA predictions. Despite this variation in predictability of changes in central metabolism, two generalities emerged. First, improved growth largely derived from evolved increases in the rate of substrate use. Second, FBA predictions bore out well for the two experiments initiated with ancestors with relatively sub-optimal yield, whereas those begun already quite optimal tended to move somewhat away from predictions. These findings suggest that the tradeoff between rate and yield is surprisingly modest. The observed positive correlation between rate and yield when adaptation initiated further from the optimum resulted in the ability of FBA to use stoichiometric constraints to predict the evolution of metabolism despite selection for rate.     

Evolution and Allometry of Calcaneal Elongation in Living and Extinct Primates

Specialized acrobatic leaping has been recognized as a key adaptive trait tied to the origin and subsequent radiation of euprimates based on its observed frequency in extant primates and inferred frequency in extinct early euprimates. Hypothesized skeletal correlates include elongated tarsal elements, which would be expected to aid leaping by allowing for increased rates and durations of propulsive acceleration at takeoff. Alternatively, authors of a recent study argued that pronounced distal calcaneal elongation of euprimates (compared to other mammalian taxa) was related primarily to specialized pedal grasping. Testing for correlations between calcaneal elongation and leaping versus grasping is complicated by body size differences and associated allometric affects. We re-assess allometric constraints on, and the functional significance of, calcaneal elongation using phylogenetic comparative methods, and present an evolutionary hypothesis for the evolution of calcaneal elongation in primates using a Bayesian approach to ancestral state reconstruction (ASR). Results show that among all primates, logged ratios of distal calcaneal length to total calcaneal length are inversely correlated with logged body mass proxies derived from the area of the calcaneal facet for the cuboid. Results from phylogenetic ANOVA on residuals from this allometric line suggest that deviations are explained by degree of leaping specialization in prosimians, but not anthropoids. Results from ASR suggest that non-allometric increases in calcaneal elongation began in the primate stem lineage and continued independently in haplorhines and strepsirrhines. Anthropoid and lorisid lineages show stasis and decreasing elongation, respectively. Initial increases in calcaneal elongation in primate evolution may be related to either development of hallucal-grasping or a combination of grasping and more specialized leaping behaviors. As has been previously suggested, subsequent increases in calcaneal elongation are likely adaptations for more effective acrobatic leaping, highlighting the importance of this behavior in early euprimate evolution.     

Developmental specialization and geographic structure of host plant use in a polyphagous grasshopper, Schistocerca emarginata (=lineata) (Orthoptera: Acrididae)

Host plant use and availability were determined in early nymphal and adult-stage Schistocerca emarginata (=lineata) (Orthoptera: Acrididae) populations at six localities in Texas, USA. Early instar nymphal populations were feeding almost exclusively on either Ptelea trifoliata (Rutaceae) or Rubus trivialis (Rosaceae). This study represents the first demonstration of a geographic structure of host plant specificity in a polyphagous grasshopper. Recognizing this geographic structure required investigations of both developmental and geographical variation in host plant use. Nymphal diet breadths were significantly less than adult diet breadths at four of six localities and smaller overall when pooled nymphal and adult diet breadths were compared among sites. Neither restricted nymphal mobility nor host plant availability accounted for the observed differences in host plant use between developmental stages and among localities. Evidence suggests that the differences in host use among populations are due to host-plant-associated genetic differentiation. Received: 4 June 1998 / Accepted: 10 March 1999     

Hydraulic redistribution: limitations for plants in saline soils

Hydraulic redistribution (HR), the movement of water from wet to dry patches in the soil via roots, occurs in different ecosystems and plant species. By extension of the principle that HR is driven by gradients in soil water potential, HR has been proposed to occur for plants in saline soils. Despite the inherent spatial patchiness and salinity gradients in these soils, the lack of direct evidence of HR in response to osmotic gradients prompted us to ask the question: are there physical or physiological constraints to HR for plants in saline environments? We propose that build‐up of ions in the root xylem sap and in the leaf apoplast, with the latter resulting in a large predawn disequilibrium of water potential in shoots compared with roots and soil, would both impede HR. We present a conceptual model that illustrates how processes in root systems in heterogeneous salinity with water potential gradients, even if equal to those in non‐saline soils, will experience a dampened magnitude of water potential gradients in the soil–plant continuum, minimizing or preventing HR. Finally, we provide an outlook for understanding the relevance of HR for plants in saline environments by addressing key research questions on plant salinity tolerance.     

Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling

A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.     

Mate familiarity and social learning in a monogamous lizard

Oecologia - Social learning is thought to be advantageous as it allows an animal to gather information quickly without engaging in costly trial-and-error learning. However, animals should be...     

Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain

AIM: To establish a novel, sensitive and high-throughput gelatinolytic assay to define new inhibitors and compare domain deletion mutants of gelatinase B/matrix metalloproteinase (MMP)-9. METHODS: Fluorogenic Dye-quenched (DQ)TM-gelatin was used as a substrate and biochemical parameters (substrate and enzyme concentrations, DMSO solvent concentrations) were optimized to establish a highthroughput assay system. Various small-sized libraries (ChemDiv, InterBioScreen and ChemBridge) of hetero-cyclic, drug-like substances were tested and compared with prototypic inhibitors. RESULTS: First, we designed a test system with gelatin as a natural substrate. Second, the assay was validated by selecting a novel pyrimidine-2,4,6-trione (barbitu- rate) inhibitor. Third, and in line with present structural data on collagenolysis, it was found that deletion of the O-glycosylated region significantly decreased gelatinolytic activity (kcat/kM ± 40% less than full-length MMP-9). CONCLUSION: The DQTM-gelatin assay is useful in high-throughput drug screening and exosite targeting. We demonstrate that flexibility between the catalytic and hemopexin domain is functionally critical for gelatinolysis.