The effect of oligonucleotide microarray data pre-processing on the analysis of patient-cohort studies

Background  

Intensity values measured by Affymetrix microarrays have to be both normalized, to be able to compare different microarrays by removing non-biological variation, and summarized, generating the final probe set expression values. Various pre-processing techniques, such as dChip, GCRMA, RMA and MAS have been developed for this purpose. This study assesses the effect of applying different pre-processing methods on the results of analyses of large Affymetrix datasets. By focusing on practical applications of microarray-based research, this study provides insight into the relevance of pre-processing procedures to biology-oriented researchers.     

Morphological and immunohistochemical study of testicular capsule and peritubular tissue of emu (<Emphasis Type="Italic">Dromaius novaehollandiae)</Emphasis> and ostrich (<Emphasis Type="Italic">Struthio camelus</Emphasis>)

The testicular capsule and peritubular boundary tissue of the emu and ostrich, as typical representatives of ratite birds, were studied in sexually mature and active birds. The testicular capsule was much thicker (578.1±73.4 μm for the free surface of the ostrich testis, and 176.2±57.5 μm for the emu) than those of members of the Galloanserae. The cellular composition of both testicular capsule and peritubular tissue was similar generally to that of members of the previously studied Galloanserae and of mammals. The tunica albuginea of the testicular capsule mainly comprised smooth-muscle-like or myoid cells mostly running in one direction and occurring in one main mass. Unlike the Galloanserae, the tunica albuginea contained more collagen fibres than smooth muscle cells, especially in the ostrich. Peritubular tissue was similarly composed of smooth-muscle-like cells distributed in several layers. Actin microfilaments and desmin and vimentin intermediate filaments were variably immunoexpressed in these two tissue types in both birds, with a clear dichotomy in the peritubular tissue. Thus, taken together with studies of some members of the Galloanserae, avian testes clearly contain a morphological mechanism that is represented partly by the smooth muscle cells of the testicular capsule and peritubular tissue for transporting the testicular fluid, which is usually copious in birds, and its cellular content from the testis into the excurrent duct system; this mechanism is similar to that found in mammals. The authors are grateful for a University of Pretoria research grant, which aided this work. Dr Peter Ozegbe of the Department of Veterinary Anatomy, University of Ibadan is a recipient of the University of Pretoria Foreign Post-Doctoral Fellowship.     

Activated epidermal growth factor receptor (ErbB1) protects the heart against stress-induced injury in mice

Acute, high-intensity stress induces necrotic lesions in the heart. We found that restraint-and-cold (4 degrees C) exposure (RCE) raises plasma lactate dehydrogenase (LDH), creatine kinase (CK), and transaminase activity in a time-dependent manner, with a peak value 7 h after stimulus cessation. At 24 h, signs of necrotic lesions were observed in paraffin sections stained with hematoxylineosin: focal accumulation of mononuclear cells in subendocardial areas of the left ventricle wall and focal hemorrhage in papillary muscles. In contrast, intermale fighting (IF) did not increase plasma CK activity, although LDH and transaminase activities did increase. In IF, no histological evidence of heart injury was observed. Because IF, but not RCE, increased plasma epidermal growth factor (EGF) concentration by approximately 1,000-fold, we hypothesized that EGF receptor (ErbB1) activation may protect the heart against stress-induced injury. To examine this hypothesis, we injected the ErbB1 tyrosine kinase inhibitor tyrphostin AG-1478 (25 mg/kg ip) immediately before mice were exposed to IF. After 3 h, plasma activities of LDH-1 and CK increased. Plasma enzyme activities were as low in control mice (injected with vehicle alone) as in nonfighting mice. In the last experiment, we injected EGF (0.25 mg/kg ip) 20 min before exposing mice to RCE. After 7 h, plasma LDH-1 and CK activities were significantly lower in these animals than in mice injected with vehicle. The effect required ErbB1 activation, because simultaneous administration of AG-1478 completely abolished the effect of exogenous EGF. We conclude that activated ErbB1, by endogenous or exogenous ligands, may protect the heart against stress-induced injury.     

Repair of DNA-protein cross-links in mammalian cells

DNA-protein cross-links are generated by both endogenous and exogenous DNA damaging agents, as intermediates during normal DNA metabolism, and during abortive base excision repair. Cross-links are relatively common lesions that are lethal when they block progression of DNA polymerases. DNA-protein cross-links may be broadly categorized into four groups by the DNA and protein chemistries near the cross-link and by the source of the cross-link: DNA-protein cross-links may be found (1) in nicked DNA at the 3' end of one strand (topo I), (2) in nicked DNA at the 5' end of one strand (pol beta), (3) at the 5' ends of both strands adjacent to nicks in close proximity (topo II; Spo 11), and (4) in one strand of duplex DNA (UV irradiation; bifunctional carcinogens and chemotherapeutic agents). Repair mechanisms are reasonably well-defined for groups 1 and 3, and suggested for groups 2 and 4. Our work is focused on the recognition and removal of DNA-protein cross-links in duplex DNA (group 4).     

Effects of nucleotide sequence alignment on phylogeny estimation: a case study of 18S rDNAs of apicomplexa

The reconstruction of phylogenetic history is predicated on being able to accurately establish hypotheses of character homology, which involves sequence alignment for studies based on molecular sequence data. In an empirical study investigating nucleotide sequence alignment, we inferred phylogenetic trees for 43 species of the Apicomplexa and 3 of Dinozoa based on complete small-subunit rDNA sequences, using six different multiple-alignment procedures: manual alignment based on the secondary structure of the 18S rRNA molecule, and automated similarity-based alignment algorithms using the PileUp, ClustalW, TreeAlign, MALIGN, and SAM computer programs. Trees were constructed using neighboring-joining, weighted-parsimony, and maximum- likelihood methods. All of the multiple sequence alignment procedures yielded the same basic structure for the estimate of the phylogenetic relationship among the taxa, which presumably represents the underlying phylogenetic signal. However, the placement of many of the taxa was sensitive to the alignment procedure used; and the different alignments produced trees that were on average more dissimilar from each other than did the different tree-building methods used. The multiple alignments from the different procedures varied greatly in length, but aligned sequence length was not a good predictor of the similarity of the resulting phylogenetic trees. We also systematically varied the gap weights (the relative cost of inserting a new gap into a sequence or extending an already-existing gap) for the ClustalW program, and this produced alignments that were at least as different from each other as those produced by the different alignment algorithms. Furthermore, there was no combination of gap weights that produced the same tree as that from the structure alignment, in spite of the fact that many of the alignments were similar in length to the structure alignment. We also investigated the phylogenetic information content of the helical and nonhelical regions of the rDNA, and conclude that the helical regions are the most informative. We therefore conclude that many of the literature disagreements concerning the phylogeny of the Apicomplexa are probably based on differences in sequence alignment strategies rather than differences in data or tree-building methods.      

Short-term insulin infused through the portal vein enhances liver gluconeogenesis and glycogenesis from [3-14C]pyruvate in the starved rat

Insulin infusion through the portal vein immediately after a pulse of [3-14C]pyruvate in 24 hr starved rats enhanced the appearance of [14C]glucose at 2, 5 and 10 min and glucose specific activity at 1, 2 and 20 min in blood collected from the cava vein at the level of the suprahepatic veins. Insulin infusion for 5 min decreased liver pyruvate concentration and enhanced both liver and plasma lactate/pyruvate ratio, and it decreased the plasma concentration of all amino acids. When insulin was infused together with glucose, [14C]glucose levels and glucose specific activity decreased in blood but there was a marked increase in liver [14C]glycogen, glycogen specific activity and glycogen concentration, and an increase in liver lactate/pyruvate ratio. The effect of insulin plus glucose infusion on plasma amino acids concentration was smaller than that found with insulin alone. It is proposed that insulin effect enhancing liver gluconeogenesis is secondary to its effect either enhancing liver glycolysis which modifies the liver's cytoplasmic oxidoreduction state to its more reduced form, increasing liver amino acids consumption or both. In the presence of glucose, products of gluconeogenesis enhanced by insulin are diverted into glycogen synthesis rather than circulating glucose. This together with results of the preceding paper (Soley et al., 1985), indicates that glucose enhances liver glycogen synthesis from C3 units in the starved rat, the process being further enhanced in the presence of insulin.     

Cross-hybridizing snake satellite, Drosophila, and mouse DNA sequences may have arisen independently

Previous reports have interpreted hybridization between snake satellite DNA and DNA clones from a variety of distant taxonomic groups as evidence for evolutionary conservation, which implies common ancestry (homology) and/or convergence (analogy) to produce the cross- hybridizing sequences. We have isolated 11 clones from a genomic library of Drosophila melanogaster, using a cloned 2.5-kb snake satellite probe of known nucleotide sequence. We have also analysed published sequence data from snakes, mice, and Drosophila. These data show that (1) all of the cross-hybridization between the snake, fly, and mouse clones can be accounted for by the presence of either of two tandem repeats, [GATA]n and [GACA]n and (2) these tandem repeats are organized differently among the different species. We find no evidence that these sequences are homologous apart from the existence of the simple repeat itself, although their divergence from a common ancestral sequence cannot be ruled out. The sequences contain a variety of homogeneous clusters of tandem repeats of CATA, GA, TA, and CA, as well as GATA and GACA. We suggest that these motifs may have arisen by a self-accelerating process involving slipped-strand mispairing of DNA. Homogeneity of the clusters might simply be the result of a rate of accumulation of tandem repeats that exceeds that of other mutations.      

Structural comparison of fibroblast growth factor-specific heparan sulfates derived from a growing or differentiating neuroepithelial cell line

Heparan sulfate (HS) glycosaminoglycans are essential modulators of fibroblast growth factor (FGF) activity both in vivo and in vitro, and appear to act by cross-linking particular forms of FGF to appropriate FGF receptors. We have recently isolated and characterized two separate HS pools derived from immortalized embryonic day 10 mouse neuroepithelial 2.3D cells: one from cells in log growth phase, which greatly potentiates the activity of FGF-2, and the other from cells undergoing contact-inhibition and differentiation, which preferentially activates FGF-1. These two pools of HS have very similar functional activities to those species isolated from primary neuroepithelial cells at corresponding stages of active proliferation or differentiation. We present here a structural comparison between these cell line HS species to establish the nature of the changes that occur in the biosynthesis of HS. A combination of chemical and enzymatic cleavage, low pressure chromatography and strong anion-exchange HPLC were used to generate full chain models of each species. Overall, the HS pools synthesized in the dividing cell line pools possessed less complex sulfation than those derived from more differentiated, growth arrested cells.      

Distribution of amino acids and amino-acid enzymes in whole kidney and renal cortex. Effect of 24-h starvation

The levels of activity of amino-acid enzymes (alanine-, aspartate- and tyrosine transaminases, serine dehydratase, glutamate dehydrogenase, glutamine synthetase, arginase and adenylate deaminase) in the kidney cortex and whole kidney of control and 24-h starved adult rats have been determined from crude homogenates. The individual amino-acid content of the two kidney fractions indicated has also been studied. Serine dehydratase was found mainly in the cortex, whilst glutamine synthetase presence was mainly limited to the medulla. The distribution of adenylate deaminase and glutamate dehydrogenase is remarkably uniform along the kidney. Starvation induced decreases in arginase and adenylate deaminase activities as well as a decrease in cortical serine dehydratase. Analysis of the variance of the enzyme data showed no significant overall changes with respect to distribution and starvation. The same analysis applied to aminograms indicated only significant changes when the distribution was studied on starved samples as a whole, with changes mainly in the urea cycle and some essential amino acids. The results suggested a remarkable degree of uniformity in kidney composition both with respect to amino acid and enzyme distribution. This uniformity is not markedly affected by starvation despite the important r?le of kidney in the overall amino-acid economy of the Mammal.     

Complex phylogeographic history of central African forest elephants and its implications for taxonomy

Background

The mitochondrial genomes of snakes are characterized by an overall evolutionary rate that appears to be one of the most accelerated among vertebrates. They also possess other unusual features, including short tRNAs and other genes, and a duplicated control region that has been stably maintained since it originated more than 70 million years ago. Here, we provide a detailed analysis of evolutionary dynamics in snake mitochondrial genomes to better understand the basis of these extreme characteristics, and to explore the relationship between mitochondrial genome molecular evolution, genome architecture, and molecular function. We sequenced complete mitochondrial genomes from Slowinski's corn snake (Pantherophis slowinskii) and two cottonmouths (Agkistrodon piscivorus) to complement previously existing mitochondrial genomes, and to provide an improved comparative view of how genome architecture affects molecular evolution at contrasting levels of divergence.

Results

We present a Bayesian genetic approach that suggests that the duplicated control region can function as an additional origin of heavy strand replication. The two control regions also appear to have different intra-specific versus inter-specific evolutionary dynamics that may be associated with complex modes of concerted evolution. We find that different genomic regions have experienced substantial accelerated evolution along early branches in snakes, with different genes having experienced dramatic accelerations along specific branches. Some of these accelerations appear to coincide with, or subsequent to, the shortening of various mitochondrial genes and the duplication of the control region and flanking tRNAs.

Conclusion

Fluctuations in the strength and pattern of selection during snake evolution have had widely varying gene-specific effects on substitution rates, and these rate accelerations may have been functionally related to unusual changes in genomic architecture. The among-lineage and among-gene variation in rate dynamics observed in snakes is the most extreme thus far observed in animal genomes, and provides an important study system for further evaluating the biochemical and physiological basis of evolutionary pressures in vertebrate mitochondria.