Loop movement and catalysis in creatine kinase

Recently the crystal structure of creatine kinase from Torpedocalifornica was determined to 2.1 A. The dimeric structure revealed two different forms in the unit cell: one monomer was bound to a substrate, MgADP, and the other monomer was bound to a transition-state analogue complex composed of MgADP, nitrate and creatine. The most striking difference between the structures is the movement of two loops (comprising residues 60-70 and residues 323-333) into the active site in the transition state structure. This loop movement effectively occludes the active site from solvent, and the loops appear to be locked into place by a salt bridge formed between His66 and Asp326. His66 is of particular interest as it is located within a PGHP motif conserved in all creatine kinases but not found in other guanidino kinases. We have carried out alanine-scanning mutagenesis of each of the residues in the PGHP motif and determined that only the His66 plays a significant role in the creatine kinase reaction. Although neither residue interacts directly with the substrate, the interaction His66 and Asp326 appears to be important in providing the precise alignment of substrates necessary for phosphoryl group transfer. Finally, it is clear that neither His66 nor Asp326 are responsible for the pKs observed in the pH-rate profile for HMCK.     

Phylogenetic context and Basal metazoan model systems

In comparative studies using model organisms, extant taxa areoften referred to as basal. The term suggests that such taxaare descendants of lineages that diverged early in the historyof some larger taxon. By this usage, the basal metazoans comprisejust four phyla (Placozoa, Porifera, Cnidaria, and Ctenophora)and the large clade Bilateria. We advise against this practicebecause basal refers to a region at the base or root of a phylogenetictree. Thus, referring to an extant taxon or species as basal,or as more basal than another, can be misleading. While muchprogress has been made toward understanding some of the phylogeneticrelationships within these groups, the relationships among themare still largely not known with certainty. Thus, sound inferencesfrom comparative studies of model organisms demand continuedillumination of phylogeny. Hypotheses about the mechanisms underlyingmetazoan evolution can be drawn from the study of model organismsin Cnidaria, Ctenophora, Placozoa, and Porifera, but it is clearthat these model organisms are likely to be derived in manyrespects. Therefore, testing these hypotheses requires the studyof yet additional model organisms. The most effective testsare those that investigate model organisms with phylogeneticpositions among two sister groups comprising a larger cladeof interest.     

Immunoaffinity separation of plasma proteins by IgY microbeads: meeting the needs of proteomic sample preparation and analysis

Separation of complex protein mixtures that have a wide dynamic range of concentration, such as plasma or serum, is a challenge for proteomic analysis. Sample preparation to remove high-abundant proteins is essential for proteomics analysis. Immunoglobulin yolk (IgY) antibodies have unique and advantageous features that enable specific protein removal to aid in the detection of low-abundant proteins and biomarker discovery. This report describes the efficiency and effectiveness of IgY microbeads in separating 12 abundant proteins from plasma with an immunoaffinity spin column or LC column. The protein separation and sample preparation process was monitored via SDS-PAGE, 2-DE, LC-MS/MS, or clinical protein assays. The data demonstrate the high specificity of the protein separation, with removal of 95-99.5% of the abundant proteins. IgY microbeads against human proteins can also selectively remove orthologous proteins of other mammals such as mouse, rat, etc. Besides the specificity and reproducibility of the IgY microbeads, the report discusses the factors that may cause potential variations in protein separation such as protein-protein interactions (known as "Interactome"), binding and washing conditions of immunoaffinity reagents, etc. A novel concept of Seppromics is introduced to address methodologies and science of protein separation in a context of proteomics.     

A wide range of protein isoforms in serum and plasma uncovered by a quantitative intact protein analysis system

We have implemented an orthogonal 3-D intact protein analysis system (IPAS) to quantitatively profile protein differences between human serum and plasma. Reference specimens consisting of pooled Caucasian-American serum, citrate-anticoagulated plasma, and EDTA-anticoagulated plasma were each depleted of six highly abundant proteins, concentrated, and labeled with a different Cy dye (Cy5, Cy3, or Cy2). A mixture consisting of each of the labeled samples was subjected to three dimensions of separation based on charge, hydrophobicity, and molecular mass. Differences in the abundance of proteins between each of the three samples were determined. More than 5000 bands were found to have greater than two-fold difference in intensity between any pair of labeled specimens by quantitative imaging. As expected, some of the differences in band intensities between serum and plasma were attributable to proteins related to coagulation. Interestingly, many proteins were identified in multiple fractions, each exhibiting different pI, hydrophobicity, or molecular mass. This is likely reflective of the expression of different protein isoforms or specific protein cleavage products, as illustrated by complement component 3 precursor and clusterin. IPAS provides a high resolution, high sensitivity, and quantitative approach for the analysis of serum and plasma proteins, and allows assessment of PTMs as a potential source of biomarkers.     

Impact of vitamin E or selenium deficiency on nematode-induced alterations in murine intestinal function

The effects of deficiencies in the antioxidant nutrients, vitamin E and selenium, on the host response to gastrointestinal nematode infection are unknown. The aim of the study was to determine the effect of antioxidant deficiencies on nematode-induced alterations in intestinal function in mice. BALB/c mice were fed control diets or diets deficient in selenium or vitamin E and the response to a secondary challenge inoculation with Heligmosomoides polygyrus was determined. Egg and worm counts were assessed to determine host resistance. Sections of jejunum were mounted in Ussing chambers to measure changes in permeability, absorption, and secretion, or suspended in organ baths to determine smooth muscle contraction. Both selenium and vitamin E deficient diets reduced resistance to helminth infection. Vitamin E, but not selenium, deficiency prevented nematode-induced decreases in glucose absorption and hyper-contractility of smooth muscle. Thus, vitamin E status is an important factor in the physiological response to intestinal nematode infection and may contribute to antioxidant-dependent protective mechanisms in the small intestine.     

Effect of teosinte cytoplasmic genomes on maize phenotype

Determining the contribution of organelle genes to plant phenotype is hampered by several factors, including the paucity of variation in the plastid and mitochondrial genomes. To circumvent this problem, evolutionary divergence between maize (Zea mays ssp. mays) and the teosintes, its closest relatives, was utilized as a source of cytoplasmic genetic variation. Maize lines in which the maize organelle genomes were replaced through serial backcrossing by those representing the entire genus, yielding alloplasmic sublines, or cytolines were created. To avoid the confounding effects of segregating nuclear alleles, an inbred maize line was utilized. Cytolines with Z. mays teosinte cytoplasms were generally indistinguishable from maize. However, cytolines with cytoplasm from the more distantly related Z. luxurians, Z. diploperennis, or Z. perennis exhibited a plethora of differences in growth, development, morphology, and function. Significant differences were observed for 56 of the 58 characters studied. Each cytoline was significantly different from the inbred line for most characters. For a given character, variation was often greater among cytolines having cytoplasms from the same species than among those from different species. The characters differed largely independently of each other. These results suggest that the cytoplasm contributes significantly to a large proportion of plant traits and that many of the organelle genes are phenotypically important.     

Persistence time of loss-of-function mutations at nonessential loci affecting eye color in Drosophila melanogaster

Persistence time of a mutant allele, the expected number of generations before its elimination from the population, can be estimated as the ratio of the number of segregating mutations per individual over the mutation rate per generation. We screened two natural populations of Drosophila melanogaster for mutations causing clear-cut eye phenotypes and detected 25 mutant alleles, falling into 19 complementation groups, in 1164 haploid genomes, which implies 0.021 eye mutations/genome. The de novo haploid mutation rate for the same set of loci was estimated as 2 x 10(-4) in a 10-generation mutation-accumulation experiment. Thus, the average persistence time of all mutations causing clear-cut eye phenotypes is approximately 100 generations (95% confidence interval: 61-219). This estimate shows that the strength of selection against phenotypically drastic alleles of nonessential loci is close to that against recessive lethals. In both cases, deleterious alleles are apparently eliminated by selection against heterozygous individuals, which show no visible phenotypic differences from wild type.