Kinetics of the interactions between yeast elongation factors 1A and 1Balpha, guanine nucleotides, and aminoacyl-tRNA

The interactions of elongation factor 1A (eEF1A) from Saccharomyces cerevisiae with elongation factor 1Balpha (eEF1Balpha), guanine nucleotides, and aminoacyl-tRNA were studied kinetically by fluorescence stopped-flow. eEF1A has similar affinities for GDP and GTP, 0.4 and 1.1 microm, respectively. Dissociation of nucleotides from eEF1A in the absence of the guanine nucleotide exchange factor is slow (about 0.1 s(-1)) and is accelerated by eEF1Balpha by 320-fold and 250-fold for GDP and GTP, respectively. The rate constant of eEF1Balpha binding to eEF1A (10(7)-10(8) M (-1) s(-1)) is independent of guanine nucleotides. At the concentrations of nucleotides and factors prevailing in the cell, the overall exchange rate is expected to be in the range of 6 s(-1), which is compatible with the rate of protein synthesis in the cell. eEF1A.GTP binds Phe-tRNA(Phe) with a K(d) of 3 nm, whereas eEF1A.GDP shows no significant binding, indicating that eEF1A has similar tRNA binding properties as its prokaryotic homolog, EF-Tu.     

Automated SNP Genotype Clustering Algorithm to Improve Data Completeness in High-Throughput SNP Genotyping Datasets from Custom Arrays

High-throughput SNP genotyping platforms use automated genotype calling algo- rithms to assign genotypes. While these algorithms work efficiently for individual platforms, they are not compatible with other platforms, and have individual biases that result in missed genotype calls. Here we present data on the use of a second complementary SNP genotype clustering algorithm. The algorithm was originally designed for individual fluorescent SNP genotyping assays, and has been opti- mized to permit the clustering of large datasets generated from custom-designed Affymetrix SNP panels. In an analysis of data from a 3K array genotyped on 1,560 samples, the additional analysis increased the overall number of genotypes by over 45,000, significantly improving the completeness of the experimental data. This analysis suggests that the use of multiple genotype calling algorithms may be ad- visable in high-throughput SNP genotyping experiments. The software is written in Perl and is available from the corresponding author.     

A role for Insulin-like growth factor 2 in specification of the fast skeletal muscle fibre

Background  

Fibre type specification is a poorly understood process beginning in embryogenesis in which skeletal muscle myotubes switch myosin-type to establish fast, slow and mixed fibre muscle groups with distinct function. Growth factors are required to establish slow fibres; it is unknown how fast twitch fibres are specified. Igf-2 is an embryonically expressed growth factor with established in vitro roles in skeletal muscle. Its localisation and role in embryonic muscle differentiation had not been established.     

An essay on the evolution of cognition: Constructing a theoretical conceptual framework

In this essay we provide an interdisciplinary approach to the problem of the evolution of human cognition and suggest the theoretical framework of genetic system theory (GST) for organizing the relevant content of several disciplines. This bio-social-cultural theory is based on the assumption that organisms are dynamic systems which interact with one another and their environment and are themselves composed of dynamic internal relations at several levels. Special emphasis will be placed upon these internal cellular and molecular mechanisms underlying the physiological mechanisms of learning and memory. The human individual organism is emphasized because in its experiential activity over time it is the site of integration for social, and cultural stimuli and because of its unique properties among living things. The primary disciplines for our discussion are drawn from the biological, social, and humanistic sciences and several concrete examples are given from each science.     

Enhanced oligonucleotide-nanoparticle conjugate stability using thioctic acid modified oligonucleotides

Metallic nanoparticles of gold functionalized with oligonucleotides conventionally use a terminal thiol modification and have been used in a wide range of applications. Although readily available, the oligonucleotide–nanoparticle conjugates prepared in this way suffer from a lack of stability when exposed to a variety of small molecules or elevated temperatures. If silver is used in place of gold then this lack of stability is even more pronounced. In this study we report the synthesis of highly stabilized oligonucleotide–nanoparticle conjugates using a simple oligonucleotide modification. A modified solid support was used to generate 3′-thioctic acid modified oligonucleotides by treatment with an N-hydroxysuccimidyl ester of thioctic acid. Unusually, both gold and silver nanoparticles have been investigated in this study and show that these disulphide-modified oligonucleotide probes offer significant improvements in nanoparticle stability when treated with dithiothreitol (DTT) compared with monothiol analogues. This is a significant advance in oligonucleotide–nanoparticle conjugate stability and for the first time allows silver nanoparticles to be prepared that are more stable than standard gold-thiol functionalized nanoparticles. This opens up the possibility of using silver nanoparticles functionalized with oligonucleotides as an alternative to gold.     

Respiratory distress and neonatal lethality in mice lacking Golgi alpha1,2-mannosidase IB involved in N-glycan maturation

There are three mammalian Golgi alpha1,2-mannosidases, encoded by different genes, that form Man5GlcNAc2 from Man(8-9)GlcNAc2 for the biosynthesis of hybrid and complex N-glycans. Northern blot analysis and in situ hybridization indicate that the three paralogs display distinct developmental and tissue-specific expression. The physiological role of Golgi alpha1,2-mannosidase IB was investigated by targeted gene ablation. The null mice have normal gross appearance at birth, but they display respiratory distress and die within a few hours. Histology of fetal lungs the day before birth indicate some delay in development, whereas neonatal lungs show extensive pulmonary hemorrhage in the alveolar region. No significant histopathological changes occur in other tissues. No remarkable ultrastructural differences are detected between wild type and null lungs. The membranes of a subset of bronchiolar epithelial cells are stained with lectins from Phaseolus vulgaris (leukoagglutinin and erythroagglutinin) and Datura stramonium in wild type lungs, but this staining disappears in lungs from null mice. Mass spectrometry of N-glycans from different tissues shows no significant changes in global N-glycans of null mice. Therefore, only a few glycoproteins required for normal lung function depend on alpha1,2-mannosidase IB for maturation. There are no apparent differences in the expression of several lung epithelial cell and endothelial cell markers between null and wild type mice. The alpha1,2-mannosidase IB null phenotype differs from phenotypes caused by ablation of other enzymes in N-glycan biosynthesis and from other mouse gene disruptions that affect pulmonary development and function.     

Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data

Background

Genomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.

Results

We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.

Conclusion

We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.     

Observations on the effects of protease inhibitors on the suppression of experimental allergic encephalomyelitis

Inhibitors of proteolytic enzymes were tested for their ability to suppress the clinical signs and CNS lesions produced by injection of purified myelin in complete Freund's adjuvant into Lewis rats. Pepstatin or a series of neutral protease inhibitors including aprotinin, soybean trypsin inhibitor, leupeptin, antipain, transaminomethyl cyclohexane carboxylic acid (AMCA), -amino caproic acid (EACA) nitrophenyl guanidino benzoate (NPGB),d- andl-polylysine, or a new commercial protease inhibitor, dipropionyl Rhein (DPR) were injected daily beginning on day 7 after immunization of rats with myelin. Aprotinin and soybean trypsin inhibitor exacerbated the symptoms and lesions of experimental allergic encephalomyelitis (EAE), leupeptin and antipain had no effect, and the plasminogen activators AMCA, EACA, NPGB, as well as poly-l- and poly-d-lysine and DPR suppressed various aspects of EAE. The measurement of acid protease as a biochemical method for quantitation of the degree of cellular infiltration into the CNS is proposed, and the results with the various treatments presented. AMCA and NPGB may exert their effects at the site of entrance of the lymphoid cells into the CNS.