VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia

Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. It controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells.     

Biodiversity of 52 chicken populations assessed by microsatellite typing of DNA pools

In a project on the biodiversity of chickens funded by the European Commission (EC), eight laboratories collaborated to assess the genetic variation within and between 52 populations from a wide range of chicken types. Twenty-two di-nucleotide microsatellite markers were used to genotype DNA pools of 50 birds from each population. The polymorphism measures for the average, the least polymorphic population (inbred C line) and the most polymorphic population (Gallus gallus spadiceus) were, respectively, as follows: number of alleles per locus, per population: 3.5, 1.3 and 5.2; average gene diversity across markers: 0.47, 0.05 and 0.64; and proportion of polymorphic markers: 0.91, 0.25 and 1.0. These were in good agreement with the breeding history of the populations. For instance, unselected populations were found to be more polymorphic than selected breeds such as layers. Thus DNA pools are effective in the preliminary assessment of genetic variation of populations and markers. Mean genetic distance indicates the extent to which a given population shares its genetic diversity with that of the whole tested gene pool and is a useful criterion for conservation of diversity. The distribution of population-specific (private) alleles and the amount of genetic variation shared among populations supports the hypothesis that the red jungle fowl is the main progenitor of the domesticated chicken.     

In vitro effects of leptin on human adipocyte metabolism

OBJECTIVE: Leptin receptors are expressed in adipocytes, suggesting potential autocrine/paracrine effects. Studies on the direct effects of leptin on adipose tissue metabolism in different species have yielded controversial data. To assess the in vitro effects of leptin on human adipocyte metabolism: lipolysis, the insulin-induced inhibition of lipolysis and lipogenesis were studied in adipocytes obtained from infants and adults. METHODS: Lipolysis was studied by incubating adipocytes with increasing concentrations of leptin or isoprenaline. Glycerol in the incubation medium was measured as an indicator of lipolysis. For the lipogenesis and insulin-induced inhibition of lipolysis experiments, the cells were preincubated with 0, 25, or 250 ng/ml of leptin for 2 h. RESULTS: Leptin did not stimulate lipolysis in human adipocytes, either in children or adults. Preincubation with leptin did not affect the insulin-induced inhibition of lipolysis, but decreased the insulin-induced lipogenesis (p < 0.05). CONCLUSIONS: This study shows that leptin has no direct lipolytic effect in human adipocytes. The lack of effect on the insulin-induced inhibition of lipolysis and the negative effect on lipogenesis indicates that the effect of leptin is not at the proximal insulin-signalling pathway but further downstream.     

Quantitative protein profiling using two-dimensional gel electrophoresis,isotope-coded affinity tag labeling,and mass spectrometry

Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, and comprehensively identify and quantify the proteins contained in biological samples. We describe a new strategy for quantitative protein profiling that is based on the separation of proteins labeled with isotope-coded affinity tag reagents by two-dimensional gel electrophoresis and their identification and quantification by mass spectrometry. The method is based on the observation that proteins labeled with isotopically different isotope-coded affinity tag reagents precisely co-migrate during two-dimensional gel electrophoresis and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel. By analyzing changes in the proteome of yeast (Saccharomyces cerevisiae) induced by a metabolic shift we show that this simple method accurately quantifies changes in protein abundance even in cases in which multiple proteins migrate to the same gel coordinates. The method is particularly useful for the quantitative analysis and structural characterization of differentially processed or post-translationally modified forms of a protein and is therefore expected to find wide application in proteomics research.     

Selective mortality of a coral reef damselfish: role of predator–competitor synergisms

Phenotypic variability within cohorts of juvenile organisms can serve as the basis for selective mortality. Previous studies have demonstrated the important role that predators play in this process but not the impact of competitors on selective predation. We use a combination of lab and field studies to evaluate the effect of the presence of adult competitor damselfish (Dascyllus aruanus and Pomacentrus moluccensis; family Pomacentridae) on the selective mortality of newly-arrived (settled) lemon damselfish (P. moluccensis) by resident predator fishes (Pseudochromis fuscus Pseudochromidae and Cheilodipterus quinquelineatus Apogonidae). Lab trials consisted of mesocosm experiments in which the behaviour, mortality, and physiological condition (measured as lipid content) of surviving P. moluccensis settlers from each of three treatments: (1) predators absent, (2) predators present, and (3) predators and competitors present, were compared. The field study involved stocking newly settled P. moluccensis on natural bommies (patch reefs) which had either been subject to a partial removal of resident fish (predators and competitors) or left alone. Results indicated there was very strong condition-based selective mortality in both the lab and field trials. In both cases there was a strong positive relationship between mortality and the lipid content of surviving fish; implying low-condition fish were selectively removed. The mesocosm trials indicated that the strength of mortality as well as condition selectivity was higher when competitors were present than when they were absent. Behavioural observations in the mesocosm study suggest that attention by juvenile P. moluccensis to the movements and occasional chases of the competitors (especially D. aruanus) reduced their vigilance to the predators. These results suggest the important and interactive roles which condition of newly settled reef fish and interspecific competition can play in the outcomes of post-settlement predation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Breast Cancer‐Derived Exosomes Reflect the Cell‐of‐Origin Phenotype

A manner in which cells can communicate with each other is via secreted nanoparticles termed exosomes. These vesicles contain lipids, nucleic acids, and proteins, and are said to reflect the cell‐of‐origin. However, for the exosomal protein content, there is limited evidence in the literature to verify this statement. Here, proteomic assessment combined with pathway‐enrichment analysis is used to demonstrate that the protein cargo of exosomes reflects the epithelial/mesenchymal phenotype of secreting breast cancer cells. Given that epithelial‐mesenchymal plasticity is known to implicate various stages of cancer progression, the results suggest that breast cancer subtypes with distinct epithelial and mesenchymal phenotypes may be distinguished by directly assessing the protein content of exosomes. Additionally, the work is a substantial step toward verifying the statement that cell‐derived exosomes reflect the phenotype of the cells‐of‐origin.     

Gaining knowledge from previously unexplained spectra-application of the PTM-Explorer software to detect PTM in HUPO BPP MS/MS data

A novel software tool named PTM-Explorer has been applied to LC-MS/MS datasets acquired within the Human Proteome Organisation (HUPO) Brain Proteome Project (BPP). PTM-Explorer enables automatic identification of peptide MS/MS spectra that were not explained in typical sequence database searches. The main focus was detection of PTMs, but PTM-Explorer detects also unspecific peptide cleavage, mass measurement errors, experimental modifications, amino acid substitutions, transpeptidation products and unknown mass shifts. To avoid a combinatorial problem the search is restricted to a set of selected protein sequences, which stem from previous protein identifications using a common sequence database search. Prior to application to the HUPO BPP data, PTM-Explorer was evaluated on excellently manually characterized and evaluated LC-MS/MS data sets from Alpha-A-Crystallin gel spots obtained from mouse eye lens. Besides various PTMs including phosphorylation, a wealth of experimental modifications and unspecific cleavage products were successfully detected, completing the primary structure information of the measured proteins. Our results indicate that a large amount of MS/MS spectra that currently remain unidentified in standard database searches contain valuable information that can only be elucidated using suitable software tools.     

West Nile Virus and California Breeding Bird Declines

Since it was first detected in 1999, West Nile virus (WNV) quickly spread, becoming the dominant vector-borne disease in North America. Sometimes fatal to humans, WNV is even more widespread among birds, with hundreds of species known to be susceptible to WNV infection in North America alone. However, despite considerable mortality and local declines observed in American crows (Corvus brachyrhynchos), there has been little evidence of a large regional association between WNV susceptibility and population declines of any species. Here we demonstrate a correlation between susceptibility to WNV measured by large-scale testing of dead birds and two indices of overall population change among bird species following the spread of WNV throughout California. This result was due primarily to declines in four species of Corvidae, including all species in this family except common ravens (Corvus corax). Our results support the hypothesis that susceptibility to WNV may have negative population consequences to most corvids on regional levels. They also provide confirmation that dead animal surveillance programs can provide important data indicating populations most likely to suffer detrimental impacts due to WNV.     

High CO(2) Requiring Mutant of Anacystis nidulans R(2)

Some physiological characteristics of a mutant (E₁) of Anacystis nidulans R₂, incapable of growing at air level of CO₂, are described. E₁ is capable of accumulating inorganic carbon (C_i) internally as efficiently as the wild type (R₂). The apparent photosynthetic affinity for C_i in E₁, however, is some 1000 times lower than that of R₂. The kinetic parameters of ribulose 1,5-bisphosphate carboxylase/oxygenase from E₁ are similar to those observed in R₂. The mutant appears to be defective in its ability to utilize the intracellular C_i pool for photosynthesis and depends on extracellular supply of Ci in the form of CO₂. The very high apparent photosynthetic K_m (CO₂) of the mutant indicate a large diffusion resistance for CO₂. Data obtained here are used to calculate the permeability coefficient for CO₂ between the bulk medium and the carboxylation site of cyanobacteria.