Peptide libraries: at the crossroads of proteomics and bioinformatics

Peptide libraries offer a valuable means for providing functional information regarding protein-modifying enzymes and protein interaction domains. Library approaches have become increasingly useful as high-throughput strategies for the analysis of large numbers of new proteins identified as a result of genome-sequencing efforts. Recent developments in the field have produced faster methods with broadened applicability. Crucially, new computational and biochemical tools have emerged that facilitate identification of interaction partners and substrates for proteins on the basis of their peptide selectivity profiles. Such combinations of proteomics-scale experimental approaches with bioinformatics tools hold great promise for the elucidation of protein interaction networks and signal transduction pathways in living cells.     

Use of expression constructs to dissect the functional domains of the CHS/beige protein: identification of multiple phenotypes

The Chediak-Higashi Syndrome (CHS) and the orthologous murine disorder beige are characterized at the cellular level by the presence of giant lysosomes. The CHS1/Beige protein is a 3787 amino acid protein of unknown function. To determine functional domains of the CHS1/Beige protein, we generated truncated constructs of the gene/protein. These truncated proteins were transiently expressed in Cos-7 or HeLa cells and their effect on membrane trafficking was examined. Beige is apparently a cytosolic protein, as are most transiently expressed truncated Beige constructs. Expression of the Beige construct FM (amino acids 1-2037) in wild-type cells led to enlarged lysosomes. Similarly, expression of a 5.5-kb region (amino acids 2035-3787) of the carboxyl terminal of Beige (22B) also resulted in enlarged lysosomes. Expression of FM solely affected lysosome size, whereas expression of 22B led to alterations in lysosome size, changes in the Golgi and eventually cell death. The two constructs could be used to further dissect phenotypes resulting from loss of the Beige protein. CHS or beigej fibroblasts show an absence of nuclear staining using a monoclonal antibody directed against phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5) P2]. Transformation of beige j fibroblasts with a YAC containing the full-length Beige gene resulted in the normalization of lysosome size and nuclear PtdIns(4,5)P2 staining. Expression of the carboxyl dominant negative construct 22B led to loss of nuclear PtdIns(4,5)P2 staining. Expression of the FM dominant negative clone did not alter nuclear PtdIns(4,5) P2 localization. These results suggest that the Beige protein interacts with at least two different partners and that the Beige protein affects cellular events, such as nuclear PtdIns(4,5)P2 localization, in addition to lysosome size.     

Thermodynamic analysis of the dissociation of the aldolase tetramer substituted at one or both of the subunit interfaces

The fructose-1,6-bis(phosphate) aldolase isologous tetramer tightly associates through two different subunit interfaces defined by its 222 symmetry. Both single- and double-interfacial mutant aldolases have a destabilized quaternary structure, but there is little effect on the catalytic activity. These enzymes are however thermolabile. This study demonstrates the temperature-dependent dissociation of the mutant enzymes and determines the dissociation free energies of both mutant and native aldolase. Subunit dissociation is measured by sedimentation equilibrium in the analytical ultracentrifuge. At 25 degrees C the tetramer-dimer dissociation constants for each single-mutant enzyme are similar, about 10(-6) M. For the double-mutant enzyme, sedimentation velocity experiments on sucrose density gradients support a tetramer-monomer equilibrium. Furthermore, sedimentation equilibrium experiments determined a dissociation constant of 10(-15) M3 for the double-mutant enzyme. By the same methods the upper limit for the dissociation constant of wild-type aldolase A is approximately 10(-28) M3, which indicates an extremely stable tetramer. The thermodynamic values describing monomer-tetramer and dimer-tetramer equilibria are analyzed with regard to possible cooperative interaction between the two subunit interfaces.     

Nitrogen Dynamics in Ice Storm-Damaged Forest Ecosystems: Implications for Nitrogen Limitation Theory

Despite the widely recognized importance of disturbance in accelerating the loss of elements from land, there have been few empirical studies of the effects of natural disturbances on nitrogen (N) dynamics in forest ecosystems. We were provided the unusual opportunity for such study, partly because the intensively monitored watersheds at the Hubbard Brook Experimental Forest (HBEF), New Hampshire, experienced severe canopy damage following an ice storm. Here we report the effects of this disturbance on internal N cycling and loss for watershed 1 (W1) and watershed 6 (W6) at the HBEF and patterns of N loss from nine other severely damaged watersheds across the southern White Mountains. This approach allowed us to test one component of N limitation theory, which suggests that N losses accompanying natural disturbances can lead to the maintenance of N limitation in temperate zone forest ecosystems. Prior to the ice storm, fluxes of nitrate (NO₃ ^–) at the base of W1 and W6 were similar and were much lower than N inputs in atmospheric deposition. Following the ice storm, drainage water NO₃ ^– concentrations increased to levels that were seven to ten times greater than predisturbance values. We observed no significant differences in N mineralization, nitrification, or denitrification between damaged and undamaged areas in the HBEF watersheds, however. This result suggests that elevated NO₃ ^- concentrations were not necessarily due to accelerated rates of N cycling by soil microbes but likely resulted from decreased plant uptake of NO₃ ^-. At the regional scale, we observed high variability in the magnitude of NO₃ ^- losses: while six of the surveyed watersheds showed accelerated rates of NO₃ ^– loss, three did not. Moreover, in contrast to the strong linear relationship between NO₃ ^– loss and crown damage within HBEF watersheds [r ²: (W1 = 0.91, W6 = 0.85)], stream water NO₃ ^– concentrations were weakly related to crown damage (r ² = 0.17) across our regional sites. The efflux of NO₃ ^– associated with the ice storm was slightly higher than values reported for soil freezing and insect defoliation episodes, but was approximately two to ten times lower than NO₃ ^– fluxes associated with forest harvesting. Because over one half of the entire years worth of N deposition was lost following the ice storm, we conclude that catastrophic disturbances contribute synergistically to the maintenance of N limitation and widely observed delays of N saturation in northern, temperate zone forest ecosystems. Present address: Department of Ecology and Evolutionary Biology, Princeton University, Guyot Hall, Princeton, New Jersey 08544, USA.     

Exogenous reference RNA for normalization of real-time quantitative PCR

We have utilized an in vitro transcribed 3' mRNA fragment of the plant gene ribulose bisphosphate carboxylase (RuBisCO) as an exogenous standard for normalization of quantitative PCR data. Both K562 cells and primary erythroid CD34+ progenitor cells were treated with sodium butyrate and changes in gamma-globin mRNA levels were assayed using a previously published TaqMan probe and primer set, while RuBisCO levels were assayed by a SYBR Green detection assay. The data presented show that a correction to measured gamma-globin induction was necessary with both cell types. The correction for the CD34+ progenitor cells was a striking 95% increase, while that for the K562 cells was 44%. The use of an exogenous reference such as in vitro transcribed mRNA for the RuBisCO plant gene provides a robust and sample-independent method for the normalization of quantitative PCR data in bacterial and animal cells.     

Evolutionary dynamics of oncogenes and tumor suppressor genes: higher intensities of purifying selection than other genes

Oncogenes and tumor suppressor genes (hereafter referred to as "cancer genes") result in cancer when they experience substitutions that prevent or distort their normal function. We examined evolutionary pressures acting on cancer genes and other classes of disease-related genes and compared our results to analyses of genes without known association to disease. We compared synonymous and nonsynonymous substitution rates in 3,035 human genes-approximately 10% of the genome-measuring the intensity of purifying selection on 311 human disease genes, including 122 cancer-related genes. Although the genes examined are similar to nondisease genes in product, expression, function, and pathway affiliation, we found intriguing differences in the selective pressures experienced by cancer genes relative to other (noncancer) disease-related and non-disease-related genes. We found a statistically significant increase in the intensity of purifying selection exerted on cancer genes (the average ratio of nonsynonymous to synonymous substitutions, omega, was 0.079) relative to all other disease-related genes groups (omega = 0.101) and non-disease-related genes (omega = 0.100). This difference indicates a striking increase in selection against nonsynonymous substitutions in oncogenes and tumor suppressor genes. This finding provides insight into the etiology of cancer and the differences between genes involved in cancer and those implicated in other human diseases. Specifically, we found a significant overlap between human oncogenes and tumor suppressor genes and "essential genes," human homologs of mouse lethal genes identified by knockout experiments. This insight may improve our ability to identify cancer-related genes and enhances our understanding of the nature of these genes.     

Wound fluid bacterial levels exceed tissue bacterial counts in controlled porcine partial-thickness burn infections

In the present study, an established controlled burn wound model was used to test the hypothesis that controlled surface contamination with is capable of generating a noninvasive method for the creation of a reproducible deep tissue burn wound infection. Using a liquid tight-wound chamber in Yorkshire pigs, partial-thickness burns were inoculated with saline-immersed for 24 hours. Noninoculated burns and unwounded skin immersed in normal saline served as controls. Bacterial cultures of wound fluid were performed daily, and tissue biopsies for bacteriological and histological evaluations were performed on days 1, 3, and 5. was only recovered from -inoculated wounds (tissue and fluid), whereas all controls contained endogenous only. The number of colony-forming units per gram of wound tissue did not correlate with the bacterial counts found in the overlying wound fluid for any wounds. Fluid counts were consistently higher than tissue counts by two logs. -inoculated wounds showed three times deeper tissue destruction than control wounds. Obtaining consistently deep tissue colonization without cross-contamination among wounds, this study introduces a noninvasive model for controlled burn wound infection suitable for future investigations regarding the efficacy of topical antibiotic wound treatment in experimental burns.     

Massive light-dependent cycling of inorganic carbon between oxygenic photosynthetic microorganisms and their surroundings

Membrane inlet mass spectrometry indicated massive light-dependent cycling of inorganic carbon between the medium and the cells of various phytoplankton species representing the main groups of aquatic primary producers. These included diatoms, symbiotic and free living dinoflagellates, a coccolithophorid, a green alga and filamentous and single cell cyanobacteria. These organisms could maintain an ambient CO₂ concentration substantially above or below that expected at chemical equilibrium with HCO₃ ⁻. The coccolithophorid Emiliania huxleyishifted from net CO₂ uptake to net CO₂ efflux with rising light intensity. Differing responses of CO₂ uptake and CO₂ fixation to changing light intensity supported the notion that these two processes are not compulsorily linked. Simultaneous measurements of CO₂ and O₂ exchange and of the fluorescence parameters in Synechococcus sp. strain PCC 7942, showed that CO₂ uptake can serve as a sensitive probe of the energy status of the photosynthetic reaction centers. However, during transitions in light intensity, changes in CO₂ uptake did not accord with those expected from fluorescence change. Quantification of the net fluxes of CO₂, HCO₃ ⁻ and of photosynthesis at steady-state revealed that substantial HCO₃ ⁻ efflux accompanied CO₂ uptake and fixation in the case of `CO<sub>2</sub> users'. On the other hand, `HCO₃ ⁻ users' were characterized by a rate of net CO₂ uptake below that of CO₂ fixation. The results support the notion that entities associated with the CCM function not only in raising the CO₂ concentration at the site of Rubisco; they may also serve as a means of diminishing photodynamic damage by dissipating excess light energy. This revised version was published online in August 2006 with corrections to the Cover Date.     

Variation between observed and true Terminal Restriction Fragment length is dependent on true TRF length and purine content

Terminal Restriction Fragment (TRF) pattern analysis has become a widely used and informative tool for studying microbial communities. Variation between sequence-determined or true TRF length and observed TRF length (TRF drift) has been previously reported and can significantly affect identification of bacterial species using TRF lengths predicted from sequence databases. In this study TRF drift was determined for 21 bacterial species using an ABI 310 Genetic Analyzer. TRF drift was positively correlated with true TRF length and negatively correlated with TRF purine content. This implies that subtle differences in molecular weight, whether from purine content or dye label, can significantly affect the observed TRF length.