Enhanced resolution of glycosylphosphatidylinositol-anchored and transmembrane proteins from the lipid-rich myelin membrane by two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis (2-DE) has become a powerful and widely used technique for proteomic analyses. However, the limited ability of 2-DE to resolve transmembrane and glycosylphosphatidylinositol (GPI)-anchored proteins has slowed the identification of proteins from membrane-rich biological samples. Myelin is an unusually lipid-rich membrane with relatively few major proteins but many quantitatively minor proteins, most of which have an unknown identity and/or function. The goal of this study was to identify the optimal conditions of 2-DE for the separation of myelin proteins. We have identified two detergents, the nonionic n-dodecyl beta-D-maltoside and the zwitterionic amidosulfobetaine ASB-14, that are more effective in solubilizing myelin proteins than the commonly used zwitterionic detergent 3-[(3-cholamidopropyl)- dimethylammonio]-1-propanesulfonate (CHAPS). These detergents significantly enhance the solubility of both transmembrane (e.g., the highly hydrophobic and multiply acylated myelin proteolipid protein) and GPI-anchored (e.g., contactin and neuronal cell adhesion molecule) myelin proteins and enable their resolution by 2-DE. We conclude that these detergents are effective tools for the 2-DE analysis of myelin, and that they may be more generally useful for the analysis of membrane-rich biological samples.     

Performance of high-throughput DNA quantification methods

Background

The accuracy and precision of estimates of DNA concentration are critical factors for efficient use of DNA samples in high-throughput genotype and sequence analyses. We evaluated the performance of spectrophotometric (OD) DNA quantification, and compared it to two fluorometric quantification methods, the PicoGreen^® assay (PG), and a novel real-time quantitative genomic PCR assay (QG) specific to a region at the human BRCA1 locus. Twenty-Two lymphoblastoid cell line DNA samples with an initial concentration of ~350 ng/uL were diluted to 20 ng/uL. DNA concentration was estimated by OD and further diluted to 5 ng/uL. The concentrations of multiple aliquots of the final dilution were measured by the OD, QG and PG methods. The effects of manual and robotic laboratory sample handling procedures on the estimates of DNA concentration were assessed using variance components analyses.

Results

The OD method was the DNA quantification method most concordant with the reference sample among the three methods evaluated. A large fraction of the total variance for all three methods (36.0–95.7%) was explained by sample-to-sample variation, whereas the amount of variance attributable to sample handling was small (0.8–17.5%). Residual error (3.2–59.4%), corresponding to un-modelled factors, contributed a greater extent to the total variation than the sample handling procedures.

Conclusion

The application of a specific DNA quantification method to a particular molecular genetic laboratory protocol must take into account the accuracy and precision of the specific method, as well as the requirements of the experimental workflow with respect to sample volumes and throughput. While OD was the most concordant and precise DNA quantification method in this study, the information provided by the quantitative PCR assay regarding the suitability of DNA samples for PCR may be an essential factor for some protocols, despite the decreased concordance and precision of this method.

     

Structure-activity relationships of human urotensin II and related analogues on rat aortic ring contraction

The sequence of human urotensin II (UII) has been recently established as H-Glu-Thr-Pro-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH, and it has been reported that UII is the most potent mammalian vasoconstrictor peptide identified so far. A series of UII analogues was synthesized, and the contractile activity of each compound was studied in vitro using de-endothelialised rat aortic rings. Replacement of each amino acid by an L-alanine or by a D-isomer showed that the N- and C-terminal residues flanking the cyclic region of the amidated peptide were relatively tolerant to substitution. Conversely, replacement of any residue of the cyclic region significantly reduced the contractile activity of the molecule. The octapeptide UII(4-11) was 4 times more potent than UII, indicating that the C-terminal region of the molecule possesses full biological activity. Alanine or D-isomer substitutions in UII(4-11) or in UII(4-11)-NH2, respectively, showed a good correlation with the results obtained for UII-NH2. Disulfide bridge disruption or replacement of the cysteine residues by their D-enantiomers markedly reduced the vasoconstrictor effect of UII and its analogues. In contrast, acetylation of the N-terminal residue of UII and UII-NH2 enhanced the potency of the peptide. Finally, monoiodination of the Tyr6 residue in UII(4-11) increased by 5 fold the potency of the peptide in the aortic ring bioassay. This structure-activity relationship study should provide useful information for the rational design of selective and potent UII receptor agonists and antagonists.     

Synthesis and cytotoxic evaluation of novel thiazolocarbazoles. Part III

Novel thiazolocarbazole derivatives have been synthesized via the corresponding imino-1,2,3-dithiazoles. In vitro antitumor activity of these polyheterocyclic compounds was studied.     

Potential inhibitors of angiogenesis. Part I: 3-(imidazol-4(5)-ylmethylene)indolin-2-ones

The synthesis and pharmacological evaluation of new 3-(imidazol-4(5)-ylmethylene)indolin-2-ones, analogues of SU-5416, are reported. The final compounds 20-51 were obtained by Knoevenagel coupling between the substituted indolin-2-ones 1-15 and either the formylimidazole derivatives 16-18 or 2-formyl-3,5-dimethylpyrrole 19. Methylation at the nitrogen atom of the indolin-2-one and/or imidazole moities was carried out in the presence of the couple NaH/DMF. A Mannich reaction afforded the 1-dimethylaminomethyl derivatives 43 and 48. The antiangiogenic activity of these compounds was evaluated in a three dimensional in vitro rat aortic ring assay. In this test, compound 20 induced a decrease of angiogenesis comparable to that observed with SU-5416; the vascular density indexes at 1 microM were 30 +/- 18 and 22 +/- 4% of control, respectively. The compounds were also evaluated, in an independent manner, as inhibitors of the human EGF-receptor tyrosine kinase activity. As expected, only minor activities were observed with four compounds, out of thirty-one, exerting inhibitory effects in the range of 40-55% at 10 microM concentration.     

Protein Information Resource: a community resource for expert annotation of protein data

The Protein Information Resource, in collaboration with the Munich Information Center for Protein Sequences (MIPS) and the Japan International Protein Information Database (JIPID), produces the most comprehensive and expertly annotated protein sequence database in the public domain, the PIR-International Protein Sequence Database. To provide timely and high quality annotation and promote database interoperability, the PIR-International employs rule-based and classification-driven procedures based on controlled vocabulary and standard nomenclature and includes status tags to distinguish experimentally determined from predicted protein features. The database contains about 200,000 non-redundant protein sequences, which are classified into families and superfamilies and their domains and motifs identified. Entries are extensively cross-referenced to other sequence, classification, genome, structure and activity databases. The PIR web site features search engines that use sequence similarity and database annotation to facilitate the analysis and functional identification of proteins. The PIR-Inter-national databases and search tools are accessible on the PIR web site at http://pir.georgetown.edu/ and at the MIPS web site at http://www.mips.biochem.mpg.de. The PIR-International Protein Sequence Database and other files are also available by FTP.     

Functional morphology of the equine pelvic flexure and its role in disease. A review

The hindgut is the major site in the horse for nutrient digestion and absorption. Most of this activity occurs in the large intestinal compartments, i.e., cecum, right and left ventral colon and left and right dorsal colon. The colonic pelvic flexure is a short and narrow loop connecting the left ventral and left dorsal colon. It is not significant directly in digestive and absorptive processes but plays an important functional role in regulating colonic aboral and retropropulsive transit of digesta through its motility pacemaker activity. The pelvic flexure also contributes to the pathophysiology of colic, the leading cause of death in horses. Its narrow lumen may contribute to colonic impaction, and malfunctions of the pacemaker may contribute to volvuli and colonic displacements. Neuronal and ganglion density of the myenteric plexus is increased at the pelvic flexure and adjacent left dorsal colon pacemaker region. Contractile activity, vasoactive intestinal peptide (VIP) and neurokinins-1 and -3 are all enhanced in the pelvic flexure. The mucosa histologically resembles that of the ventral and dorsal colon, with apically-granulated principal cells and goblet cells lining the luminal surface. Clustered intranuclear inclusions resembling the cytoplasmic granules are also observed by electron microscopy in the principal cells as elsewhere in the horse colon. Further neuroendocrine and morphologic investigation of the pelvic flexure is warranted due to the great importance of this localized region for normal function and pathophysiology.     

Supercomplexes in the respiratory chains of yeast and mammalian mitochondria

Around 30-40 years after the first isolation of the five complexes of oxidative phosphorylation from mammalian mitochondria, we present data that fundamentally change the paradigm of how the yeast and mammalian system of oxidative phosphorylation is organized. The complexes are not randomly distributed within the inner mitochondrial membrane, but assemble into supramolecular structures. We show that all cytochrome c oxidase (complex IV) of Saccharomyces cerevisiae is bound to cytochrome c reductase (complex III), which exists in three forms: the free dimer, and two supercomplexes comprising an additional one or two complex IV monomers. The distribution between these forms varies with growth conditions. In mammalian mitochondria, almost all complex I is assembled into supercomplexes comprising complexes I and III and up to four copies of complex IV, which guided us to present a model for a network of respiratory chain complexes: a 'respirasome'. A fraction of total bovine ATP synthase (complex V) was isolated in dimeric form, suggesting that a dimeric state is not limited to S.cerevisiae, but also exists in mammalian mitochondria.     

Palaeogenetic analysis of (pre)historic artifacts and its significance for anthropology

The possibility of isolating ancient DNA (aDNA) from all kinds of (pre)historic anthropogenetic artifacts opens new perspectives. This study applies palaeogenetic techniques to three anthropological issues: 1. Palaeodiet. DNA sequences from organic residues in vessels identify Precolumbian Aztec diet. 2. (Pre)historic husbandry and economic structures. aDNA data can reveal the species and the genetic evolutionary stage of animals and plants and show the manner and the extent of their growth, cultivation, or domestication. 3. Production techniques, use, and functionality. Identification of the plant or animal source of an archaeological find can reveal the use or the function of the find. Examples from a Celtic "sausage-end" and an Aztec "eye salve" are given.     

On meta-analytic assessment of surrogate outcomes

We discuss the strengths and weaknesses of the meta-analytic approach to estimating the effect of a new treatment on a true clinical outcome measure, T, from the effect of treatment on a surrogate response, S. The meta-analytic approach (see Daniels and Hughes (1997) 16, 1965-1982) uses data from a series of previous studies of interventions similar to the new treatment. The data are used to estimate relationships between summary measures of treatment effects on T and S that can be used to infer the magnitude of the effect of the new treatment on T from its effects on S. We extend the class of models to cover a broad range of applications in which the parameters define features of the marginal distribution of (T, S). We present a new bootstrap procedure to allow for the variability in estimating the distribution that governs the between-study variation. Ignoring this variability can lead to confidence intervals that are much too narrow. The meta-analytic approach relies on quite different data and assumptions than procedures that depend, for example, on the conditional independence, at the individual level, of treatment and T, given S (see Prentice (1989) 8, 431-440). Meta-analytic calculations in this paper can be used to determine whether a new study, based only on S, will yield estimates of the treatment effect on T that are precise enough to be useful. Compared to direct measurement on T, the meta-analytic approach has a number of limitations, including likely serious loss of precision and difficulties in defining the class of previous studies to be used to predict the effects on T for a new intervention.