Proteomics shows Hsp70 does not bind peptide sequences indiscriminately in vivo

Heat shock protein 70 (Hsp70) binds peptide and has several functions that include protein folding, protein trafficking, and involvement with immune function. However, endogenous Hsp70-binding peptides had not previously been identified. Therefore, we eluted and identified several hundred endogenously bound peptides from Hsp70 using liquid chromatography ion trap mass spectrophotometry (LC-ITMS). Our work shows that the peptides are capable of binding Hsp70 as previously described. They are generally 8-26 amino acids in length and correspond to specific regions of many proteins. Through computationally assisted analysis of peptides eluted from Hsp70 we determined variable amino acid sequences, including a 5 amino acid core sequence that Hsp70 favorably binds. We also developed a computer algorithm that predicts Hsp70 binding within proteins. This work helps to define what peptides are bound by Hsp70 in vivo and suggests that Hsp70 facilitates peptide selection by aiding a funneling mechanism that is flexible but allows only a limited number of peptides to be processed.     

Stereochemistry of glutamate receptor agonist efficacy: engineering a dual-specificity AMPA/kainate receptor

Upon agonist binding, the bilobate ligand-binding domains of the ionotropic glutamate receptors (iGluR) undergo a cleft closure whose magnitude correlates broadly with the efficacy of the agonist. AMPA (alpha-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid) and kainate are nonphysiological agonists that distinguish between subsets of iGluR. Kainate acts with low efficacy at AMPA receptors. Here we report that the structure-based mutation L651V converts the GluR4 AMPA receptor into a dual-specificity AMPA/kainate receptor fully activated by both agonists. To probe the stereochemical basis of partial agonism, we have also investigated the correlation between agonist efficacy and a series of vibrational and fluorescence spectroscopic signals of agonist binding to the corresponding wild-type and mutant GluR4 ligand-binding domains. Two signals track the extent of channel activation: the maximal change in intrinsic tryptophan fluorescence and the environment of the single non-disulfide bonded C426, which appears to probe the strength of interactions with the ligand alpha-amino group. Both of these signals arise from functional groups that are poised to detect changes in the extent of channel cleft closure and thus provide additional information about the coupling between conformational changes in the ligand-binding domain and activation of the intact receptor.     

Database resources of the National Center for Biotechnology

In addition to maintaining the GenBank(R) nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval resources for the data in GenBank and other biological data made available through NCBI's Web site. NCBI resources include Entrez, PubMed, PubMed Central (PMC), LocusLink, the NCBITaxonomy Browser, BLAST, BLAST Link (BLink), Electronic PCR (e-PCR), Open Reading Frame (ORF) Finder, References Sequence (RefSeq), UniGene, HomoloGene, ProtEST, Database of Single Nucleotide Polymorphisms (dbSNP), Human/Mouse Homology Map, Cancer Chromosome Aberration Project (CCAP), Entrez Genomes and related tools, the Map Viewer, Model Maker (MM), Evidence Viewer (EV), Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheritance in Man (OMIM), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD), and the Conserved Domain Architecture Retrieval Tool (CDART). Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih.gov.     

Effect of incubation temperature on isolation of Campylobacter jejuni genotypes from foodstuffs enriched in Preston broth

Preston broth and agar incubated at either 37 or 42 degrees C have been widely used to isolate campylobacters from foodstuffs. The consequences of using either incubation temperature were investigated. Retail packs of raw chicken (n = 24) and raw lamb liver (n = 30) were purchased. Samples were incubated in Preston broth at 37 and 42 degrees C and then streaked onto Preston agar and incubated as before. Two Campylobacter isolates per treatment were characterized. Poultry isolates were genotyped by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and flagellin PCR-restriction fragment length polymorphism, and lamb isolates were genotyped by RAPD only. In total, 96% of the poultry and 73% of the lamb samples yielded campylobacters. The lamb isolates were all Campylobacter jejuni, as were 96% of the poultry isolates, with the remainder being Campylobacter lari. The incubation temperature had no significant effect on the number of positive samples or on the species isolated. However, genotyping of the C. jejuni isolates revealed profound differences in the types obtained. Overall (from poultry and lamb), the use of a single incubation temperature, 37 degrees C, gave 56% of the total number of RAPD C. jejuni genotypes, and hence, 44% remained undetected. The effect was especially marked in the poultry samples, where incubation at 37 degrees C gave 47% of the PFGE genotypes but 53% were exclusively recovered after incubation at 42 degrees C. Thus, the incubation temperature of Preston media selects for certain genotypes of C. jejuni, and to detect the widest range, samples should be incubated at both 37 and 42 degrees C. Conversely, genotyping results arising from the use of a single incubation temperature should be interpreted with caution.     

Synergy between angiotensin and aldosterone in evoking sodium appetite in baboons

The synergy between ANG II and aldosterone (Aldo) in the induction of salt appetite, extensively studied in rats, has been tested in baboons. ANG II was infused intracerebroventricularly at 0.5 or 1.0 microg/h; Aldo was infused subcutaneously at 20 microg/h. Separate infusions over 7 days had no significant effect on the daily intake of 300 mM NaCl. Concurrent infusions, however, increased daily NaCl intake approximately 10-fold and daily water intake approximately 2.5-fold. In addition, the combined infusions caused 1) a reduction in daily food intake, 2) changes in blood composition indicative of increased vasopressin release, and 3) changes of urinary excretion rates of cortisol and Aldo indicative of increased ACTH release. Arterial blood pressure, measured in two baboons, rose during concurrent ANG II and Aldo treatment. These results indicate a potent synergy between central ANG II and peripheral Aldo in stimulating salt appetite in baboons. At the same time, other ANG II-specific brain mechanisms concerned with water intake, food intake, vasopressin release, ACTH release, and blood pressure regulation appear to have been activated by the same type of synergy. These central enhancement processes have never been previously demonstrated in primates.     

Bower decorations attract females but provoke other male spotted bowerbirds: bower owners resolve this trade-off

Elaborate secondary sexual traits offset the costs that they impose on their bearer by facilitating reproductive benefits, through increased success in intrasexual contests or increased attractiveness to choosy mates. Some traits enhance both strategies. Conversely, I show that spotted bowerbirds Chlamydera maculata may face a trade-off. The trait that best predicts their mating success, numbers of Solanum berries exhibited on a bower, also provokes increased intrasexual aggression in the form of bower destructions by neighbouring bower owners, which reduce the quality of the male's bower. At natural berry numbers, levels of mating success in the population are skewed, but levels of destruction do not vary with berry number. When berry numbers are artificially exaggerated, increased levels of destructions occur, but mating success does not increase. When offered excess berries, either to add to the bower or artificially placed on the bower, bower owners preferred to use numbers of berries related to the number that they displayed naturally. This decision is made without direct experience of the attendant changes in destruction or mating success. This indicates that bower owners may assess their own social standing in relation to their neighbours and modulate their display accordingly.     

Quantitative high-performance liquid chromatography/electrospray ionization tandem mass spectrometric analysis of 2- and 3-series prostaglandins in cultured tumor cells

This paper describes a rapid and simple technique for the simultaneous quantitative analysis of PGE(2), PGE(3), and other closely related prostaglandins from cultured cells using liquid chromatography/electrospray ionization tandem mass spectrometry. This method permits quantification of selected individual prostaglandins derived either from arachidonic acid (AA) or eicosapentaenoic acid (EPA) from cell extracts without tedious derivatization, lengthy sample preparation, and separation required by GC-MS- or HPLC-UV-based methods. The validation assessment showed that the quantitative determination is linear (r(2)>0.999) for both PGE(2) and PGE(3) in the range tested (1-500 ng/ml, 0.0028-1.4 microM) and a coefficient of variation lower than 10% was obtained for samples analyzed on 3 separate days. The detection limit was 2.5 pg for both PGE(2) and PGE(3). Extraction efficiency of PGE(2) and PGE(3) from cell suspensions ranged from 89.4 to 98.2%. As an application of the method, prostaglandins formed by EPA in human lung cancer A549 cells were determined. A 62% reduction of PGE(2) formation was noted when A549 cells were treated with 10 microM of EPA. Concomitantly, EPA increased formation of PGE(3) by 10-fold in A549 cells. This is the first report that unequivocally demonstrates that EPA can be converted to PGE(3) by cyclooxygenase in human cancer cells.     

Accuracy of mothers' retrospective reports of smoking during pregnancy: comparison with twin sister informant ratings.

Retrospective assessment of maternal smoking or substance use during pregnancy is sometimes unavoidable. The unusually close relationship of twin sister pairs permits comparison of self-report data versus co-twin informant data on substance use during pregnancy. Information about smoking during pregnancy has been gathered from a series of mothers from an Australian volunteer twin panel (576 women reporting on 995 pregnancies), supplemented in many cases by independent ratings of their smoking by twin sister informants (821 pregnancies). Estimates of the proportion of women who had never smoked regularly (56-58%), who had smoked but did not smoke during a particular pregnancy (16-21%), or who smoked throughout the pregnancy (16-18%), were in good agreement whether based on self-report or twin sister informant data. However, informants underreported cases who smoked during the first trimester but then quit (1-3% versus 7-9% by self-report). Women who smoked throughout pregnancy (by informant report) rarely denied a history of regular smoking (< 1%), although a small proportion of apparent false negative cases were identified where they either denied smoking during a pregnancy (9%) or denied smoking beyond the first trimester (10%). We conclude that retrospective smoking data can safely be used to identify potential associations of later child outcomes with maternal smoking during pregnancy.