The identification of protein-protein interactions of the nuclear pore complex of Saccharomyces cerevisiae using high throughput matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry

Mass spectrometry has become the technology of choice for detailed identification of proteins in complex mixtures. Although electrophoretic separation, proteolytic digestion, mass spectrometric analysis of unseparated digests, and database searching have become standard methods in widespread use, peptide sequence information obtained by collision-induced dissociation and tandem mass spectrometry is required to establish the most comprehensive and reliable results. Most tandem mass spectrometers in current use employ electrospray ionization. In this work a novel tandem mass spectrometer employing matrix-assisted laser desorption ionization-time-of-flight/time-of-flight operating at 200 Hz has been used to identify proteins interacting with known nucleoporins in the nuclear pore complex of Saccharomyces cerevisiae. Proteins interacting with recombinant proteins as bait were purified from yeast extracts and then separated by one-dimensional SDS-PAGE. Although peptide mass fingerprinting is sometimes sufficient to identify proteins, this study shows the importance of employing tandem mass spectrometry for identifying proteins in mixtures or as covalently modified forms. The rules for incorporating these features into MS-Tag are presented. In addition to providing an evaluation of the sensitivity and overall quality of collision-induced dissociation spectra obtained, standard conditions for ionization and fragmentation have been selected that would allow automatic data collection and analysis, without the need to adjust parameters in a sample-specific fashion. Other considerations essential for successful high throughput protein analysis are discussed.     

Genetic issues in aquatic species management: the shortnose sturgeon (Acipenser brevirostrum) in the southeastern United States

Anadromous species occupy multiple freshwater,estuarine, and marine habitats, which posesspecial challenges in wildlife management. Inparticular, the level of immigration betweendrainages can be a critical factor in thedefinition of management units and the designof population-specific stocking programs. Thesemi-anadromous shortnose sturgeon (Acipenser brevirostrum) is listed as anendangered species under the Endangered SpeciesAct in the United States. To assess populationstructure in this species as a guide toeffective management, tissue samples werecollected from adult specimens (N = 198) fromfive river systems in the southeastern U.S.,and augmented with a sample from New Brunswick,Canada (N = 13), the extreme northern end ofthe species' range. Comparisons of mtDNAcontrol region sequences reveal a shallow genegenealogy and modest, but significant,population structuring (_st = 4.3%or _st = 17.7% when Canadian samplesare included). Populations inhabiting riversystems in the southeastern U.S. are closelyrelated, a pattern consistent with more recentdivergences along evolutionary timeframes. Haplotype diversity is moderate to high in mostdrainages (h = 0.383–1.000), exceptfor the Savannah and Edisto rivers, indicatingthat historical levels of mtDNA diversity mightbe largely intact outside of these drainages. Low mtDNA diversity in the Savannah andneighboring Edisto drainages might stem from anexperimental stocking effort during 1984–1992 that depressed overall genetic diversityin the Savannah and established or augmentedthe Edisto River population with a relativelylimited number of matrilines.     

Edwardsiella ictaluri invasion of IEC-6, Henle 407, fathead minnow and channel catfish enteric epithelial cells

Invasion of Edwardsiella ictaluri into cultured mammalian, fish and enzymatically harvested catfish enteric epithelial cells is described. Gentamicin survival assays were used to demonstrate the ability of this catfish pathogen to invade IEC-6 (origin: rat small intestinal epithelium), Henle 407 (origin: human embryonic intestinal epithelium), fathead minnow (FHM, minnow epithelial cells) and trypsin/pepsin-harvested channel catfish enteric epithelial cells. Invasion of all cell types occurred within 2 h of contact at 26 degrees C, in contrast to Escherichia coli DH5 alpha, which did not invade cells tested. Eight Edwardsiella ictaluri isolates from diseased catfish and the ATCC (American Type Culture Collection) strain were evaluated for invasion efficiency using FHM cells. All isolates were invasive, but at differing efficiencies. Invasion blocking assays using chemical blocking agents were performed on a single isolate (LA 89-9) using IEC-6 epithelial cells. Preincubation of IEC-6 cells with cytochalasin D (microfilament depolymerizer) and monodansylcadaverine (blocks receptor-mediated endocytosis) significantly reduced invasion by E. ictaluri, whereas exposure to colchicine (microtubule depolymerizer) had no effect on bacterial internalization. Results indicate that actin polymerization and receptor-mediated endocytosis are involved in uptake of E. ictaluri by IEC-6 epithelial cells. Invasion trials using freshly harvested cells from the intestine of the natural host, Ictalurus punctatus, show that invasion occurs, but at a low efficiency. This is possibly due to loss of outer membrane receptors during enzymatic cell harvest. This study provides the first documentation of the invasion of cultured mammalian and fish cells by E. ictaluri, and identifies possible mechanisms used for intracellular access. Additionally, the study describes several functional in vitro invasion models using commercially available cell lines as well as cells from the natural host (channel catfish, I. punctatus).     

Oxygen transport capacity in the air-breathing fish,Megalops cyprinoides: compensations for strenuous exercise

Tarpon have high resting or routine hematocrits (Hct) (37.6+/-3.4%) and hemoglobin concentrations (120.6+/-7.3 gl(-1)) that increased significantly following bouts of angling-induced exercise (51.9+/-3.7% and 142.8+/-13.5 gl(-1), respectively). Strenuous exercise was accompanied by an approximately tenfold increase in blood lactate and a muscle metabolite profile indicative of a high energy demand teleost. Routine blood values were quickly restored only when this facultative air-breathing fish was given access to atmospheric air. In vitro studies of oxygen transport capacity, a function of carrying capacity and viscosity, revealed that the optimal Hct range corresponded to that observed in fish under routine behaviour. During strenuous exercise however, further increase in viscosity was largely offset by a pronounced reduction in the shear-dependence of blood which conformed closely to an ideal Newtonian fluid. The mechanism for this behaviour of the erythrocytes appears to involve the activation of surface adrenergic receptors because pre-treatment with propranolol abolished the response. High levels of activity in tarpon living in hypoxic habitats are therefore supported by an elevated Hct with adrenergically mediated viscosity reduction, and air-breathing behaviour that enables rapid metabolic recovery.     

Allometric analysis of the induced flavonols on the leaf surface of wild tobacco (Nicotiana attenuata)

Trichomes excrete secondary metabolites that may alter the chemical composition of the leaf surface, reducing damage caused by herbivores, pathogens and abiotic stresses. We examined the surface exudates produced by Nicotiana attenuata Torr. Ex Wats., a plant known to contain and secrete a number of secondary metabolites that are toxic or a deterrent to herbivorous insects. Extractions specific to the leaf surface, the trichomes, and the laminar components demonstrated the localization of particular compounds. Diterpene glycosides occurred exclusively in leaf mesophyll, whereas nicotine was found in both the trichomes and mesophyll. Neither rutin nor nicotine was found on the leaf surface. Quercetin and 7 methylated derivatives were found in the glandular trichomes and appeared to be excreted onto the leaf surface. We examined the elicitation of these flavonols on the leaf surface with a surface-area allometric analysis, which measures changes in metabolites independent of the effects of leaf expansion. The flavonols responded differently to wounding, methyl jasmonate (MeJA), herbivore attack and UV-C radiation, and the response patterns corresponded to their compound-specific allometries. Finding greater amounts of quercetin on younger leaves and reduced amounts after herbivore feeding and MeJA treatment, we hypothesized that quercetin may function as an attractant, helping the insects locate a preferred feeding site. Consistent with this hypothesis, mirids (Tupiocoris notatus) were found more often on mature leaves sprayed with quercetin at a concentration typical of young leaves than on unsupplemented mature leaves. The composition of metabolites on the leaf surface of N. attenuata changes throughout leaf development and in response to herbivore attack or environmental stress, and these changes are mediated in part by responses of the glandular trichomes.     

Nuclear ribosomal DNA sequence polymorphism and hybridization in checker mallows (Sidalcea, Malvaceae)

Checker mallows (Sidalcea, Malvaceae) constitute a western North American genus of annuals and perennials that have been regarded as taxonomically difficult because of complex patterns of morphological variation putatively stemming from hybridization and polyploidy. In recent molecular phylogenetic investigations extensive polymorphism was observed in the internal and external transcribed spacers (ITS and ETS) of 18S-26S nuclear ribosomal DNA for some Sidalcea samples. To resolve the evolutionary basis for this polymorphism and to readdress the evolutionary impact of hybridization in Sidalcea we cloned and sequenced the polymorphic DNAs and included the clones in phylogenetic analyses together with direct sequences of non-polymorphic samples. The positions of cloned spacer sequences in the phylogenetic trees suggest that S. reptans and two subspecies of S. malviflora may have been influenced by past hybridization with lineages of the "glaucescens" clade. Polymorphic sequence patterns in other taxa may be a result of extensive interbreeding within young clades, in keeping with the minimal sequence divergence, largely overlapping geographic distributions and morphology, and ploidy variation in these groups. Other possible explanations for polymorphic sequences in members of Sidalcea include slow concerted evolution relative to mutation rates, incomplete lineage sorting, and recent pseudogene formation.     

The p65 (RelA) subunit of NF-kappaB interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression

Regulation of NF-kappaB transactivation function is controlled at several levels, including interactions with coactivator proteins. Here we show that the transactivation function of NF-kappaB is also regulated through interaction of the p65 (RelA) subunit with histone deacetylase (HDAC) corepressor proteins. Our results show that inhibition of HDAC activity with trichostatin A (TSA) results in an increase in both basal and induced expression of an integrated NF-kappaB-dependent reporter gene. Chromatin immunoprecipitation (ChIP) assays show that TSA treatment causes hyperacetylation of the wild-type integrated NF-kappaB-dependent reporter but not of a mutant version in which the NF-kappaB binding sites were mutated. Expression of HDAC1 and HDAC2 repressed tumor necrosis factor (TNF)-induced NF-kappaB-dependent gene expression. Consistent with this, we show that HDAC1 and HDAC2 target NF-kappaB through a direct association of HDAC1 with the Rel homology domain of p65. HDAC2 does not interact with NF-kappaB directly but can regulate NF-kappaB activity through its association with HDAC1. Finally, we show that inhibition of HDAC activity with TSA causes an increase in both basal and TNF-induced expression of the NF-kappaB-regulated interleukin-8 (IL-8) gene. Similar to the wild-type integrated NF-kappaB-dependent reporter, ChIP assays showed that TSA treatment resulted in hyperacetylation of the IL-8 promoter. These data indicate that the transactivation function of NF-kappaB is regulated in part through its association with HDAC corepressor proteins. Moreover, it suggests that the association of NF-kappaB with the HDAC1 and HDAC2 corepressor proteins functions to repress expression of NF-kappaB-regulated genes as well as to control the induced level of expression of these genes.     

Mice unresponsive to GM-CSF are unexpectedly resistant to cutaneous Leishmania major infection

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to play a protective role in leishmanial infection. Mice with a null mutation in the gene for the beta common (beta c) chain of the receptors for GM-CSF, interleukin(IL)-3 and IL-5 (beta c-null mice) display normal steady state hemopoiesis and develop lung disease similar to the human condition, alveolar proteinosis, due to a lack of signaling by GM-CSF. We therefore expected to observe a heightened sensitivity to Leishmania major in the beta c-null mice. Surprisingly, the beta c-null mice were more resistant to cutaneous infection than wild-type (wt) mice. Upon intradermal injection of L. major promastigotes, fewer beta c-null mice developed cutaneous lesions than wt mice and these lesions were smaller and healed more rapidly than in wt mice. This resistance to disease was associated with a reduced percentage of in vitro infected beta c-null macrophages. Macrophages from beta c-null mice displayed a more activated phenotype and produced increased amounts of nitric oxide following infection with L. major, both in vivo and in vitro. Paradoxically, however, the parasite burden in the draining lymph nodes was similar in both beta c-null and wt mice, suggesting that at least a subpopulation of cells was susceptible to the parasite. The mechanism preventing normal lesion development remains to be elucidated.     

Research in the exercise sciences: where do we go from here?

The goal of this article is to provide a perspective on how research involving the acute and chronic effects of exercise (referred to as "exercise sciences") on the structure and function of organs systems will evolve in the next century. Within the last 30 years, exercise-related research has rapidly transitioned from an organ to a subcellular/molecular focus. Thus future research will continue to be heavily influenced by molecular biology tools, fueled by both emerging technologies (e.g., "gene-chip microarrays") designed to dissect gene function on a macro scale as well as by the completion of the human genome project in which the approximately 80,000 genes comprising humans will be completely sequenced. These successes will drive the emerging fields of functional genomics (the dissecting of a gene's identity and function) and proteomics (the study of the properties of proteins). Funding levels at the National Institutes of Health will likely increase in order to expand these emerging fields as well as provide avenues for translating fundamental knowledge into solving the complexities of a number of degenerative diseases influenced heavily by activity/inactivity factors such as cardiopulmonary disease, diabetes, obesity, and the debilitating disorders associated with aging. Thus there are many challenges facing future exercise scientists who must harness the new technologies and take an aggressive stance in bringing this important field to the forefront.