Determining Protein Complex Connectivity Using a Probabilistic Deletion Network Derived from Quantitative Proteomics

Protein complexes are key molecular machines executing a variety of essential cellular processes. Despite the availability of genome-wide protein-protein interaction studies, determining the connectivity between proteins within a complex remains a major challenge. Here we demonstrate a method that is able to predict the relationship of proteins within a stable protein complex. We employed a combination of computational approaches and a systematic collection of quantitative proteomics data from wild-type and deletion strain purifications to build a quantitative deletion-interaction network map and subsequently convert the resulting data into an interdependency-interaction model of a complex. We applied this approach to a data set generated from components of the Saccharomyces cerevisiae Rpd3 histone deacetylase complexes, which consists of two distinct small and large complexes that are held together by a module consisting of Rpd3, Sin3 and Ume1. The resulting representation reveals new protein-protein interactions and new submodule relationships, providing novel information for mapping the functional organization of a complex.     

Arthropods of a semi-natural grassland in an urban environment: the John F. Kennedy International Airport,New York

Semi-natural grassland habitat fragments, such as those found on airports, might be important for arthropod conservation and biodiversity in urban ecosystems. The objectives of this study were to: (1) describe the arthropod communities present within the grasslands on the John F. Kennedy International Airport and (2) assess spatial and temporal variation in those arthropod communities. We collected arthropods using a vacuum sampler during 2003 and using sweep-net collection methods during 2003 and 2004. During 2003, a total of 1,467 arthropods, representing 17 orders and 68 families were found in vacuum samples. A total of 3,784 arthropods, representing 12 orders and 94 families were collected in sweep-net samples during 2003. In 2004, a total of 3,281 arthropods, representing 12 orders and 85 families were collected in sweep-net samples. Hemiptera, Orthoptera, and Diptera were the most abundant taxa, accounting for 47, 18, and 14% of all arthropods captured, respectively. We found evidence of spatial and temporal variation in arthropod abundance, in particular as noted by fluctuations in Orthoptera: Acrididae and Hemiptera: Auchenorrhyncha. Hemipteran family diversity was also influenced by habitat type. Grassland habitats on airfields, although influenced by anthropogenic factors (e.g., mowing), have the potential to provide abundant and diverse arthropod communities and might serve as a refugium for such species within urban ecosystems.     

In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum‐infected red blood cells

The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle‐like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake byP. falciparum‐infected erythrocytes shows that at R and S stages, a time‐increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time‐increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.     

Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae

We have devised an approach for analyzing shotgun proteomics datasets based on the normalized spectral abundance factor that can be used for quantitative proteomics analysis. Three biological replicates of samples enriched for plasma membranes were isolated from S. cerevisiae grown in 14N-rich media and 15N-minimal media and analyzed via quantitative multidimensional protein identification technology. The natural log transformation of NSAF values from S. cerevisiae cells grown in 14N YPD media and 15N-minimal media had a normal distribution. The t-test analysis demonstrated 221 of 1316 proteins were significantly overexpressed in one or the other growth conditions with a p value <0.05. Notably, amino acid transporters were among the 14 membrane proteins that were significantly upregulated in cells grown in minimal media, and we functionally validated these increases in protein expression with radioisotope uptake assays for selected proteins.     

Analyzing chromatin remodeling complexes using shotgun proteomics and normalized spectral abundance factors

Mass spectrometry-based approaches are commonly used to identify proteins from multiprotein complexes, typically with the goal of identifying new complex members or identifying post-translational modifications. However, with the recent demonstration that spectral counting is a powerful quantitative proteomic approach, the analysis of multiprotein complexes by mass spectrometry can be reconsidered in certain cases. Using the chromatography-based approach named multidimensional protein identification technology, multiprotein complexes may be analyzed quantitatively using the normalized spectral abundance factor that allows comparison of multiple independent analyses of samples. This study describes an approach to visualize multiprotein complex datasets that provides structure function information that is superior to tabular lists of data. In this method review, we describe a reanalysis of the Rpd3/Sin3 small and large histone deacetylase complexes previously described in a tabular form to demonstrate the normalized spectral abundance factor approach.     

Functional symmetries in the schmelzmuster and morphology of rootless rodent molars

Morphology and schmelzmuster of rootless cheek teeth of 25 extant rodent genera were studied in relation to jaw movement. A differentiation between leading and trailing edges is observed regularly in enamel thickness and schmelzmuster. Similarities between antagonists are interpreted as 'functional symmetries'. Differences in the enamel thickness, the schmelzmuster and orientation of cutting edges are controlled by functional and phylogenetic constraints. The heterogenous sample allows discrimination between these two constraints. The most obvious functional constraint leads to the almost regular occurrence of radial enamel on the push sides of cutting edges. The degree of functional symmetry seems to be determined by phylogenetic limitations.     

Block of outward current in cardiac purkinje fibers by injection of quaternary ammonium ions

We have studied the effects of iontophoretic injection of the quaternary ammonium compounds tetraethylammonium (TEA) and tetrabutylammonium (TBA) in cardiac purkinje fibers. We find that TBA(+) is a more effective blocker than TEA(+), but injection of either compound reduces the time-dependent outward plateau currents, transient outward current (I(to)), and the delayed rectifier (I(x)). Our findings provide evidence that these outward cardiac currents are carried by channels that in some respects are pharmacologically similar to squid axon potassium channels. We demonstrate that this procedure is a new tool that can be useful in the analysis of membrane currents in the heart.     

Genetic variation of physiological response to aflatoxin in Gallus domesticus

Summary A pedigreed, commercial broiler population of 31 sire families was administered dietary aflatoxin at levels of either 0.0 or 5.0 g of aflatoxin per g of diet from 7 to 21 days of age and their response assessed by various physiological parameters.Body weight, gain, packed red blood cell volume (PCV). plasma albumin, plasma protein and cholesterol responses were significantly reduced from control values by the 5.0 g/g aflatoxin diet. Males had greater body weights and gains in both dietary regimes than females. Females had significantly higher PCV, protein, albumin and cholesterol values in the 5.0 g/g aflatoxin group than their male counterparts. These differences resulted in significant sex × aflatoxin level interactions for these parameters. Coefficients of variation were increased for all parameters measured in the 5.0 g/g aflatoxin treatment compared to values for the control group. This increase was greatest for plasma protein, albumin, and cholesterol responses. Heritabilities were calculated for all responses within both treatment groups and were found to be increased in all cases by the 5.0 g/g aflatoxin diet. Highly significant phenotypic correlations were determined between body weight and gain and between plasma albumin and total plasma protein in both treatment groups. High phenotypic correlations among PCV, plasma cholesterol, plasma protein, and plasma albumin were noted in the 5.0 g/g aflatoxin group. Significant genetic correlations were determined between body weight and gain and between plasma albumin and plasma protein in the control group. Body weight and gain and plasma protein, albumin, cholesterol and PCV were genetically correlated in the 5.0 g/g aflatoxin group. Genetic correlations calculated across environments for the same traits were high for PCV, body weight and gain and much lower for plasma albumin, plasma protein, and plasma cholesterol.The results of this study demonstrate that genetic variability for resistance to aflatoxin exists in commercial broiler populations. Strong genetic and phenotypic relationships, and high heritabilities associated with plasma albumin and protein suggest their applicability as selection criteria for aflatoxin resistance. Genetic correlation for these traits across dietary environments indicate that responses for aflatoxin resistance should be measured during aflatoxin challenge and suggest that selection for growth and selection for aflatoxin resistance are not antagonistic.     

Ligament cells stretch-adapted on a microgrooved substrate increase intercellular communication in response to a mechanical stimulus

An in vitro model was used to investigate the effect of mechanical stimuli on adaptation to load and calcium signaling in aligned medial collateral ligament cells (MCL). This model used a patterned silicone membrane to align the cells parallel with the direction of the microgrooves. Alignment created an architecture that simulated a degree of cell orientation in native ligament tissue. It was hypothesized that aligned ligament cells would be more efficient at calcium wave propagation than cells that were randomly oriented. It was further hypothesized that calcium wave propagation would be greater among cells that were both aligned and subjected to mechanical stretch compared to cells that were aligned but not stretched. Rat MCL cells were loaded with Fura-2AM, a calcium-binding dye, and mechanically indented using a micropipette tip. A ratio-imaging fluorescence technique was used to quantitate the calcium (Ca2+) response. It was concluded that stretching ligament cells prior to stimulation increased their sensitivity to load and their ability to propagate a calcium wave. However, the ability of aligned cells to propagate this wave was not significantly different when compared to nonaligned cells. Treatment of cultures with inhibitors such as apyrase and suramin significantly reduced the number of cells recruited in the calcium response. Hence, it was concluded that ATP released from mechanically stimulated cells was a principal mediator responsible for the rise in intracellular calcium in ligament cells. Further, purinoceptor activation may amplify the signal to alert and recruit more cells in a response to mechanical stimulation.