Estimating changes in mutational mechanisms of evolution

By considering three DNA sequences simultaneously there is sufficient information to recover a full Markov model with three transition matrices from the root to each of the sequences. It is necessary to have relatively long sequences because, for nucleotides, the full model requires 39 parameters that are estimated from 63 observable values. This triplet Markov method is evaluated for the protein coding genes of mammalian vertebrate mitochondrial genomes, and, in addition, version for two-state-characters (such as R/Y coding) is implemented. A key finding is that some changes in mutational mechanism differentially affect the mutation rate between pairs of nucleotides: there does not appear to be a universal change in "rate" of evolution. It remains to be explored whether detecting changes in certain nucleotide interchanges can be localized to a particular part of the DNA replication/repair system. In order to estimate divergence dates it may eventually be advantageous to use the nucleotide interchanges that show little rate change.     

An in silico exploration of the neutral network in protein sequence space

Designating amino-acid sequences that fold into a common main-chain structure as "neutral sequences" for the structure, regardless of their function or stability, we investigated the distribution of neutral sequences in protein sequence space. For four distinct target structures (alpha, beta,alpha/beta and alpha+beta types) with the same chain length of 108, we generated the respective neutral sequences by using the inverse folding technique with a knowledge-based potential function. We assumed that neutral sequences for a protein structure have Z scores higher than or equal to fixed thresholds, where thresholds are defined as the Z score for the corresponding native sequence (case 1) or much greater Z score (case 2). An exploring walk simulation suggested that the neutral sequences mapped into the sequence space were connected with each other through straight neutral paths and formed an inherent neutral network over the sequence space. Through another exploring walk simulation, we investigated contiguous regions between or among the neutral networks for the distinct protein structures and obtained the following results. The closest approach distance between the two neutral networks ranged from 5 to 29 on the Hamming distance scale, showing a linear increase against the threshold values. The sequences located at the "interchange" regions between the two neutral networks have intermediate sequence-profile-scores for both corresponding structures. Introducing a "ball" in the sequence space that contains at least one neutral sequence for each of the four structures, we found that the minimal radius of the ball that is centered at an arbitrary position ranged from 35 to 50, while the minimal radius of the ball that is centered at a certain special position ranged from 20 to 30, in the Hamming distance scale. The relatively small Hamming distances (5-30) may support an evolution mechanism by transferring from a network for a structure to another network for a more beneficial structure via the interchange regions.     

An association of 27- and 40-kDa molecules with glycolipids that bind A-B bacterial enterotoxins to cultured cells

It is well recognized that the Shiga-like toxins (Stxs) preferentially bind to Gb3 glycolipids and the cholera toxin (CT) and heat-labile enterotoxin (LTp) bind to GM1 gangliosides. After binding to the cell surface, A-B bacterial enterotoxins have to be internalized by endocytosis. The transport of the toxin-glycolipid complex has been documented in several manners but the actual mechanisms are yet to be clarified. We applied a heterobifunctional cross-linker, sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropionate (SASD), to detect the membrane proteins involved in the binding and the transport of A-B bacterial enterotoxins in cultured cells. Both Stx1 and Stx2 bound to the detergent-insoluble microdomain (DIM) of Vero cells and Caco-2 cells, which were susceptible to the toxin, but neither was bound to insusceptible CHO-K1 cells. Both CT and LTp bound to the DIM of Vero cells, Caco-2 cells, and CHO-K1 cells. In a cross-linking experiment, Stx1 cross-linked only with a 27-kDa molecule, while Stx2, which was more potently toxic than Stx1, cross-linked with 27- and 40-kDa molecules of Vero cells as well as of Caco-2 cells; moreover, no molecules were cross-linked with the insusceptible CHO-K1 cells. LTp was cross-linked only to the 27-kDa molecule of these three cell types but the CT, which was more toxic than LTp, was also cross-linked with 27- and 40-kDa molecules of Vero cells, Caco-2 cells, and CHO-K1 cells. The 27- and the 40-kDa molecules might play a role in the endocytosis and retrograde transport of A-B bacterial enterotoxins.     

Amiloride inhibition of the proton-translocating NADH-quinone oxidoreductase of mammals and bacteria

The proton-translocating NADH-quinone oxidoreductase in mitochondria (complex I) and bacteria (NDH-1) was shown to be inhibited by amiloride derivatives that are known as specific inhibitors for Na(+)/H(+) exchangers. In bovine submitochondrial particles, the effective concentrations were about the same as those for the Na(+)/H(+) exchangers, whereas in bacterial membranes the inhibitory potencies were lower. These results together with our earlier observation that the amiloride analogues prevent labeling of the ND5 subunit of complex I with a fenpyroximate analogue suggest the involvement of ND5 in H(+) (Na(+)) translocation and no direct involvement of electron carriers in H(+) (Na(+)) translocation.     

The effect of dietary polyunsaturated fatty acid on insulin sensitivity and lipid metabolism in Otsuka Long-Evans Tokushima Fatty (OLETF) rats

The effects of insulin sensitivity and lipid metabolism of dietary lard, eicosapentaenoic acid-rich oil (EPA oil) or arachidonic acid oil (AA oil) in Otsuka Long-Evans Tokushima Fatty (OLETF) rats were examined. Blood glucose was not different in each group at 30, 60, 120 min on an oral glucose tolerance test. Fasting blood glucose levels were lower in lard and AA oil groups than in controls. Hepatic triglyceride concentration and liver histochemistry revealed that the fat content was higher in the lard group and the AA oil group than in controls. The EPA oil group showed TG levels as high as the control group. Serum total cholesterol in the EPA oil group was lower, while the level in the AA oil group was higher than in the lard and control groups. HDL cholesterol was 1.5-fold higher in the AA oil group than in controls. Dietary EPA oil or AA oil supplementation showed different effects on lipid metabolism in this model.     

A systematic investigation identifies a significant number of probable pseudogenes in the Escherichia coli genome

Pseudogenes are open reading frames (ORFs) encoding dysfunctional proteins with high homology to known protein-coding genes. Although pseudogenes were reported to exist in the genomes of many eukaryotes and bacteria, no systematic search for pseudogenes in the Escherichia coli genome has been carried out. Genome comparisons of E. coli strains K-12 and O157 revealed that many protein-coding sequences have prematurely terminated orthologs encoding unstable proteins. To systematically screen for pseudogenes, we selected ORFs generated by premature termination of the orthologous protein-coding genes and subsequently excluded those possibly arising from sequence errors. Lastly we eliminated those with close homologs in this and other species, as these shortened ORFs may actually have functions. The process produced 95 and 101 pseudogene candidates in K-12 and O157, respectively. The assigned three-dimensional structures suggest that most of the encoded proteins cannot fold properly and thus are dysfunctional, indicating that they are probably pseudogenes. Therefore, the existence of a significant number of probable pseudogenes in E. coli is predicted, awaiting experimental verification. Most of them were found to be genes with paralogs or horizontally transferred genes or both. We suggest that pseudogenes constitute a small fraction of the genomes of free-living bacteria in general, reflecting the faster elimination than production of pseudogenes.     

Sulfite induces adherence of polymorphonuclear neutrophils to immobilized fibrinogen through activation of Mac-1 beta2-integrin (CD11b/CD18)

Sulfite is a major air pollutant which can cause respiratory tract inflammation characterized by an influx of polymorphonuclear neutrophils (PMN). We have previously shown that human PMN can produce sulfite either spontaneously or in response to stimulation with lipopolysaccharide. We now demonstrate that sulfite activates PMN to adhere to immobilized fibrinogen via the beta2-integrin Mac-1 (CD11b/CD18). Mac-1 expression is not altered by treatment with this agent. Although unaffected by pertussis toxin, sulfite-triggered PMN adhesion was abrogated by pretreating cells with the membrane-impermeant sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a modifier of thiol groups on the cell surface. These results suggest that sulfite-induced PMN adhesion is dependent on a modification of thiols at the cell surface. Given its potent antioxidant and antimicrobial activities, sulfite may act as an endogenous mediator in host defense and/or inflammation.     

Larger genetic differences within africans than between Africans and Eurasians

The worldwide pattern of single nucleotide polymorphism (SNP) variation is of great interest to human geneticists, population geneticists, and evolutionists, but remains incompletely understood. We studied the pattern in noncoding regions, because they are less affected by natural selection than are coding regions. Thus, it can reflect better the history of human evolution and can serve as a baseline for understanding the maintenance of SNPs in human populations. We sequenced 50 noncoding DNA segments each approximately 500 bp long in 10 Africans, 10 Europeans, and 10 Asians. An analysis of the data suggests that the sampling scheme is adequate for our purpose. The average nucleotide diversity (pi) for the 50 segments is only 0.061% +/- 0.010% among Asians and 0.064% +/- 0.011% among Europeans but almost twice as high (0.115% +/- 0.016%) among Africans. The African diversity estimate is even higher than that between Africans and Eurasians (0.096% +/- 0.012%). From available data for noncoding autosomal regions (total length = 47,038 bp) and X-linked regions (47,421 bp), we estimated the pi-values for autosomal regions to be 0.105, 0.070, 0.069, and 0.097% for Africans, Asians, Europeans, and between Africans and Eurasians, and the corresponding values for X-linked regions to be 0.088, 0.042, 0.053, and 0.082%. Thus, Africans differ from one another slightly more than from Eurasians, and the genetic diversity in Eurasians is largely a subset of that in Africans, supporting the out of Africa model of human evolution. Clearly, one must specify the geographic origins of the individuals sampled when studying pi or SNP density.