Functional proteome analysis of the banana plant (Musa spp.) using de novo sequence analysis of derivatized peptides

We report the use of chemical derivatization with MALDI-MS/MS analysis for de novo sequence analysis. Using three frequently used homology-based search algorithms, we were able to identify more than 40 proteins from banana, a nonmodel plant with unsequenced genome. Furthermore, this approach allowed the identification of different isoforms. We also observed that the identification score obtained varied according to the position of the peptide sequences in the query using the MS-Blast algorithm.     

Phosphatidic acid regulates tyrosine phosphorylating activity in human neutrophils: enhancement of Fgr activity

In human neutrophils, the activation of phospholipase D and the Tyr phosphorylation of proteins are early signaling events upon cell stimulation. We found that the pretreatment of neutrophils with ethanol (0.8%) or 1-butanol (0.3%), which results in the accumulation of phosphatidylalcohol at the expense of phosphatidic acid (PA), decreased the phorbol myristate acetate-stimulated Tyr phosphorylation of endogenous proteins (42, 115 kDa). When neutrophil cytosol was incubated in the presence or absence of PA, these and other endogenous proteins became Tyr-phosphorylated in a PA-dependent manner. In contrast, phosphatidylalcohols exhibited only 25% (phosphatidylethanol) or 5% (phosphatidylbutanol) of the ability of PA to stimulate Tyr phosphorylation in the cell-free assay. Similarly, other phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, polyphosphoinositides, and sphingosine 1-phosphate) showed little ability to stimulate Tyr phosphorylation. These data suggest that PA can function as an intracellular regulator of Tyr phosphorylating activity. Gel filtration chromatography of leukocyte cytosol revealed a peak of PA-dependent Tyr phosphorylating activity distinct from a previously described PA-dependent phosphorylating activity (Waite, K. A., Wallin, R., Qualliotine-Mann, D., and McPhail, L. C. (1997) J. Biol. Chem. 272, 15569-15578). Among the protein Tyr kinases expressed in neutrophils, only Fgr eluted exclusively in the peak of PA-dependent Tyr phosphorylating activity. Importantly, Fgr isolated from unstimulated neutrophil lysates showed increased activity in the presence of PA but not phosphatidylbutanol. Moreover, the pretreatment of neutrophils with 1-butanol decreased Fgr activity in cells stimulated with formyl-methionyl-leucyl phenylalanine plus dihydrocytochalasin B. Together, these results suggest a new second messenger role for PA in the regulation of Tyr phosphorylation.     

Protein kinases C translocation responses to low concentrations of arachidonic acid

Arachidonic acid (AA) directly activates protein kinases C (PKC) and may thereby serve as a regulatory signal during cell stimulation. The effect, however, requires a > or =20 microm concentration of the fatty acid. We find that human polymorphonuclear neutrophils (PMN) equilibrated with a ligand for the diacylglycerol receptor on PKC, [(3)H]phorbol dibutyrate (PDB), increased binding of [(3)H]PDB within 15 s of exposure to > or =10-30 nm AA. Other unsaturated fatty acids, but not a saturated fatty acid, likewise stimulated PDB binding. These responses, similar to those caused by chemotactic factors, resulted from a rise in the number of diacylglycerol receptors that were plasma membrane-associated and therefore accessible to PDB. Unlike chemotactic factors, however, AA was fully active on cells overloaded with Ca(2+) chelators. The major metabolites of AA made by PMN, leukotriene B(4) and 5-hydroxyicosatetraenoate, did not mimic AA, and an AA antimetabolite did not block responses to AA. AA also induced PMN to translocate cytosolic PKCalpha, beta(II), and delta to membranes. This response paralleled PDB binding with respect to dose requirements, time, Ca(2+)-independence, resistance to an AA antimetabolite, and induction by another unsaturated fatty acid but not by a saturated fatty acid. Finally, HEK 293 cells transfected with vectors encoding PKCbeta(I) or PKCdelta fused to the reporter enhanced green fluorescent protein (EGFP) were studied. AA caused EGFP-PKCbeta translocation from cytosol to plasma membrane at > or =0.5 microm, and EGFP-PKCdelta translocation from cytosol to nuclear and, to a lesser extent, plasma membrane at as little as 30 nm. We conclude that AA induces PKC translocations to specific membrane targets at concentrations 2-4 orders of magnitude below those activating the enzymes. These responses, at least as they occur in PMN, do not require changes in cell Ca(2+) or oxygenation of the fatty acid. AA seems more suited for signaling the movement than activation of PKC.     

Neurofibrillary degeneration in progressive supranuclear palsy and corticobasal degeneration: tau pathologies with exclusively "exon 10" isoforms

Pathological tau proteins that constitute the basic matrix of neuronal inclusions observed in numerous neurodegenerative disorders are disease specific. This is mainly the consequence of the aggregation of specific sets of tau isoforms according to the diseases, i.e., six isoforms in Alzheimer's disease (AD) and exclusively the three tau isoforms lacking the corresponding sequence of exon 10 (E10-) in Pick's disease (PiD). By using antibodies specific to the different tau isoforms and one- and two-dimensional gel electrophoresis followed by western blots, we demonstrate herein a third group of neurodegenerative disorders characterized by intraneuronal inclusions exclusively constituted of tau isoforms containing the sequence corresponding to exon 10, progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Together, tau isoforms with exon 10 clearly differentiate three groups of neurodegenerative diseases: AD, PiD, and PSP/CBD. For each group, the neuropathological and clinical phenotypes are most likely related to specific sets of tau isoforms expressed by the vulnerable neuronal populations. The recently described mutations of the tau gene responsible for familial frontotemporal dementias also support this hypothesis.     

A novel nuclear export signal and a REF interaction domain both promote mRNA export by the Epstein-Barr virus EB2 protein

A striking characteristic of mRNA export factors is that they shuttle continuously between the cytoplasm and the nucleus. This shuttling is mediated by specific factors interacting with peptide motifs called nuclear export signals (NES) and nuclear localization signals. We have identified a novel CRM-1-independent transferable NES and two nuclear localization signals in the Epstein-Barr virus mRNA export factor EB2 (also called BMLF1, Mta, or SM) localized at the N terminus of the protein between amino acids 61 and 146. We have also found that a previously described double NES (amino acids 213-236) does not mediate the nuclear shuttling of EB2, but is an interaction domain with the cellular export factor REF in vitro. This newly characterized REF interaction domain is essential for EB2-mediated mRNA export. Accordingly, in vivo, EB2 is found in complexes containing REF as well as the cellular factor TAP. However, these interactions are RNase-sensitive, suggesting that the RNA is an essential component of these complexes.     

Sequence analysis of insecticidal genes from Xenorhabdus nematophilus PMFI296

Three strains of Xenorhabdus nematophilus showed insecticidal activity when fed to Pieris brassicae (cabbage white butterfly) larvae. From one of these strains (X. nematophilus PMFI296) a cosmid genome library was prepared in Escherichia coli and screened for oral insecticidal activity. Two overlapping cosmid clones were shown to encode insecticidal proteins, which had activity when expressed in E. coli (50% lethal concentration [LC(50)] of 2 to 6 microg of total protein/g of diet). The complete sequence of one cosmid (cHRIM1) was obtained. On cHRIM1, five genes (xptA1, -A2, -B1, -C1, and -D1) showed homology with up to 49% identity to insecticidal toxins identified in Photorhabdus luminescens, and also a smaller gene (chi) showed homology to a putative chitinase gene (38% identity). Transposon mutagenesis of the cosmid insert indicated that the genes xptA2, xptD1, and chi were not important for the expression of insecticidal activity toward P. brassicae. One gene (xptA1) was found to be central for the expression of activity, and the genes xptB1 and xptC1 were needed for full activity. The location of these genes together on the chromosome and therefore present on a single cosmid insert probably accounted for the detection of insecticidal activity in this E. coli clone. Although multiple genes may be needed for full activity, E. coli cells expressing the xptA1 gene from the bacteriophage lambda P(L) promoter were shown to have insecticidal activity (LC(50) of 112 microg of total protein/g of diet). This is contrary to the toxin genes identified in P. luminescens, which were not insecticidal when expressed individually in E. coli. High-level gene expression and the use of a sensitive insect may have aided in the detection of insecticidal activity in the E. coli clone expressing xptA1. The location of these toxin genes and the chitinase gene and the presence of mobile elements (insertion sequence) and tRNA genes on cHRIM1 indicates that this region of DNA represents a pathogenicity island on the genome of X. nematophilus PMFI296.     

Epstein-Barr virus mRNA export factor EB2 is essential for production of infectious virus

The splicing machinery which positions a protein export complex near the exon-exon junction mediates nuclear export of mRNAs generated from intron-containing genes. Many Epstein-Barr virus (EBV) early and late genes are intronless, and an alternative pathway, independent of splicing, must export the corresponding mRNAs. Since the EBV EB2 protein induces the cytoplasmic accumulation of intronless mRNA, it is tempting to speculate that EB2 is a viral adapter involved in the export of intronless viral mRNA. If this is true, then the EB2 protein is essential for the production of EBV infectious virions. To test this hypothesis, we generated an EBV mutant in which the BMLF1 gene, encoding the EB2 protein, has been deleted (EBV(BMLF1-KO)). Our studies show that EB2 is necessary for the production of infectious EBV and that its function cannot be transcomplemented by a cellular factor. In the EBV(BMLF1-KO) 293 cells, oriLyt-dependent DNA replication was greatly enhanced by EB2. Accordingly, EB2 induced the cytoplasmic accumulation of a subset of EBV early mRNAs coding for essential proteins implicated in EBV DNA replication during the productive cycle. Two herpesvirus homologs of the EB2 protein, the herpes simplex virus type 1 protein ICP27 and, the human cytomegalovirus protein UL69, only partly rescued the phenotype of the EBV(BMLF1-KO) mutant, indicating that some EB2 functions in virus production cannot be transcomplemented by ICP27 and UL69.     

Inter-familial relationships of the shorebirds (Aves: Charadriiformes) based on nuclear DNA sequence data

Background  

Phylogenetic hypotheses of higher-level relationships in the order Charadriiformes based on morphological data, partly disagree with those based on DNA-DNA hybridisation data. So far, these relationships have not been tested by analysis of DNA sequence data. Herein we utilize 1692 bp of aligned, nuclear DNA sequences obtained from 23 charadriiform species, representing 15 families. We also test earlier suggestions that bustards and sandgrouses may be nested with the charadriiforms. The data is analysed with methods based on the parsimony and maximum-likelihood criteria.     

Acute metal stress in Populus tremula×P. alba (717‐1B4 genotype): Leaf and cambial proteome changes induced by cadmium2+

The comprehension of metal homeostasis in plants requires the identification of molecular markers linked to stress tolerance. Proteomic changes in leaves and cambial zone of Populus tremula×P. alba (717‐1B4 genotype) were analyzed after 61 days of exposure to cadmium (Cd) 360 mg/kg soil dry weight in pot‐soil cultures. The treatment led to an acute Cd stress with a reduction of growth and photosynthesis. Cd stress induced changes in the display of 120 spots for leaf tissue and 153 spots for the cambial zone. It involved a reduced photosynthesis, resulting in a profound reorganisation of carbon and carbohydrate metabolisms in both tissues. Cambial cells underwent stress from the Cd actually present inside the tissue but also a deprivation of photosynthates caused by leaf stress. An important tissue specificity of the response was observed, according to the differences in cell structures and functions.