Natural selection in the <Emphasis Type="Italic">TLR-</Emphasis>related genes in the course of primate evolution

The innate immune system constitutes the front line of host defense against pathogens. Toll-like receptors (TLRs) recognize molecules derived from pathogens and play crucial roles in the innate immune system. Here, we provide evidence that the TLR-related genes have come under natural selection pressure in the course of primate evolution. We compared the nucleotide sequences of 16 TLR-related genes, including TLRs (TLR1–10), MYD88, TILAP, TICAM1, TICAM2, MD2, and CD14, among seven primate species. Analysis of the non-synonymous/synonymous substitution ratio revealed the presence of both strictly conserved and rapidly evolving regions in the TLR-related genes. The genomic segments encoding the intracellular Toll/interleukin 1 receptor domains, which exhibited lower rates of non-synonymous substitution, have undergone purifying selection. In contrast, TLR4, which carried a high proportion of non-synonymous substitutions in the part of extracellular domain spanning 200 amino acids, was found to have been the suggestive target of positive Darwinian selection in primate evolution. However, sequence analyses from 25 primate species, including eight hominoids, six Old World monkeys, eight New World monkeys, and three prosimians, showed no evidence that the pressure of positive Darwinian selection has shaped the pattern of sequence variations in TLR4 among New World monkeys and prosimians. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biophysics of risk aversion based on neurotransmitter receptor theory

Decision under risk and uncertainty has been attracting attention in neuroeconomics and neuroendocrinology of decision-making. This paper demonstrated that the neurotransmitter receptor theory-based value (utility) function can account for human and animal risk-taking behavior. The theory predicts that (i) when dopaminergic neuronal response is efficiently coupled to the formation of ligand-receptor complex, subjects are risk-aversive (irrespective of their satisfaction level) and (ii) when the coupling is inefficient, subjects are risk-seeking at low satisfaction levels, consistent with risk-sensitive foraging theory in ecology. It is further suggested that some anomalies in decision under risk are due to inefficiency of the coupling between dopamine receptor activation and neuronal response. Future directions in the application of the model to studies in neuroeconomics of addiction and neuroendocrine modulation of risk-taking behavior are discussed.     

Branched-chain amino acids and arginine suppress MaFbx/atrogin-1 mRNA expression via mTOR pathway in C2C12 cell line

The effect of amino acid on muscle protein degradation remains unclear. Recent studies have elucidated that proteolysis in catabolic conditions occurs through ubiquitin-proteasome proteolysis pathway and that muscle-specific ubiquitin ligases (atrogin-1 and MuRF1) play an important role in protein degradation. In the present study, we examined the direct effect of 5 mM amino acids (leucine, isoleucine, valine, glutamine and arginine) on atrogin-1 and MuRF1 levels in C2C12 muscle cells and the involved intracellular signal transduction pathway. Leucine, isoleucine and valine suppressed atrogin-1 and MuRF1 mRNA levels (approximately equal to 50%) at 6 and 24 h stimulations. Arginine showed a similar effect except at 24 h-treatment for atrogin-1 mRNA. However, glutamine failed to reduce atrogin-1 and MuRF1 mRNA levels. The inhibitory effect of leucine, isoleucine or arginine on atrogin-1 mRNA level was reversed by rapamycin, although wortmannin did not reverse the effect. PD98059 and HA89 reduced basal atrogin-1 level without influencing the inhibitory effects of those amino acids. The inhibitory effect of leucine, isoleucine or arginine on MuRF1 mRNA levels was not reversed by rapamycin. Taken together, these findings indicated that leucine, isoleucine and arginine decreased atrogin-1 mRNA levels via mTOR and that different pathways were involved in the effect of those amino acids on MuRF1 mRNA levels.     

Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease

Marble brain disease (MBD) also known as Guibaud-Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded beta-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619-633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order: wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. In conclusion: (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.     

Benzo[a]pyrene-induced toxicity: paradoxical protection in Cyp1a1(-/-) knockout mice having increased hepatic BaP-DNA adduct levels.

Previous studies have shown that cytochrome P450 1A1 (CYP1A1), CYP1B1, and prostaglandin-endoperoxide synthase (PTGS2) are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), and all three metabolize BaP to reactive DNA-binding intermediates and excreted products. Because these three enzymes show differing patterns of basal levels, inducibility, and tissue-specific expression, animal studies are necessary to delineate the role of CYP1A1 in BaP-mediated toxicity. In mice receiving large daily doses of BaP (500 mg/kg i.p.), Cyp1a1(-/-) knockout mice are protected by surviving longer than Cyp1a1(+/-) heterozygotes. We found that a single 500 mg/kg dose of BaP induces hepatic CYP1A1 mRNA, protein, and enzyme activity in Cyp1a1(+/-) but not in Cyp1a1(-/-) mice; TCDD pretreatment increases further the CYP1A1 in Cyp1a1(+/-) but not Cyp1a1(-/-) mice. Although a single 500 mg/kg dose of BaP was toxic to Cyp1a1(+/-) mice (serum liver enzyme elevated about 2-fold above control levels at 48 h), Cyp1a1(-/-) mice displayed no hepatotoxicity. Unexpectedly, we found 4-fold higher BaP-DNA adduct levels in Cyp1a1(-/-) than in Cyp1a1(+/-) mice; TCDD pretreatment lowered the levels of BaP-DNA adducts in both genotypes, suggesting the involvement of other TCDD-inducible detoxification enzymes. BaP was cleared from the blood much faster in Cyp1a1(+/-) than Cyp1a1(-/-) mice. Our results suggest that absence of the CYP1A1 enzyme protects the intact animal from BaP-mediated liver toxicity and death, by decreasing the formation of large amounts of toxic metabolites, whereas much slower metabolic clearance of BaP in Cyp1a1(-/-) mice leads to greater formation of BaP-DNA adducts.     

Prospectively Isolated Cancer-Associated CD10+ Fibroblasts Have Stronger Interactions with CD133+ Colon Cancer Cells than with CD133? Cancer Cells

Although CD133 has been reported to be a promising colon cancer stem cell marker, the biological functions of CD133⁺ colon cancer cells remain controversial. In the present study, we investigated the biological differences between CD133⁺ and CD133⁻ colon cancer cells, with a particular focus on their interactions with cancer-associated fibroblasts, especially CD10⁺ fibroblasts. We used 19 primary colon cancer tissues, 30 primary cultures of fibroblasts derived from colon cancer tissues and 6 colon cancer cell lines. We isolated CD133⁺ and CD133⁻ subpopulations from the colon cancer tissues and cultured cells. In vitro analyses revealed that the two populations showed similar biological behaviors in their proliferation and chemosensitivity. In vivo analyses revealed that CD133⁺ cells showed significantly greater tumor growth than CD133⁻ cells (P = 0.007). Moreover, in cocultures with primary fibroblasts derived from colon cancer tissues, CD133⁺ cells exhibited significantly more invasive behaviors than CD133⁻ cells (P<0.001), especially in cocultures with CD10⁺ fibroblasts (P<0.0001). Further in vivo analyses revealed that CD10⁺ fibroblasts enhanced the tumor growth of CD133⁺ cells significantly more than CD10⁻ fibroblasts (P<0.05). These data demonstrate that the in vitro invasive properties and in vivo tumor growth of CD133⁺ colon cancer cells are enhanced in the presence of specific cancer-associated fibroblasts, CD10⁺ fibroblasts, suggesting that the interactions between these specific cell populations have important roles in cancer progression. Therefore, these specific interactions may be promising targets for new colon cancer therapies.     

Isolation and characterization of the adenylate cyclase structural gene of Neurospora crassa.

A single gene (nac) encoding an adenylate cyclase was cloned from the genomic DNA library of Neurospora crassa, using the DNA fragment encoding the catalytic domain of adenylate cyclase of Saccharomyces cerevisiae as a probe. The open reading frame of this gene (6900 base pairs) was interrupted three time by introns. The protein encoded consists of 2300 amino acids and has adenylate cyclase activity. N. crassa adenylate cyclase has a high degree of homology with the catalytic domains of yeast and bovine brain adenylate cyclases.     

Control of adenosine 3',5'-monophosphate level and protein phosphorylation by depolarizing agents in Coprinus macrorhizus

The treatment of mycelial cells with membrane-active antibiotics, uncouplers of oxidative phosphorylation and KCl leads to a transient increase in adenosine 3',-5'-monophosphate (cyclic AMP) levels in Coprinus macrorhizus. The maximal values and duration of increase in the cyclic AMP level depended on the kind and amount of these drugs. The treatment with these drugs simultaneously resulted in a rapid increase in the phosphorylation of three cellular proteins. The levels and time course of phosphorylation of these proteins were paralleled with the increase of cyclic AMP level in response to the drugs used. Thus, the treatment of these drugs causes the transient increase of cyclic AMP level and cyclic AMP stimulates the phosphorylation of particular proteins by activating protein kinases.     

Ultrastructures of leuco-adsorption on monolayers infected with influenza virus

Leuco-adsorption occurring in influenza virus infected-cell cultures was studied morphologically to clarify the mechanisms of adsorption of leukocytes. Among the various types of chicken leukocytes studied, such as lymphocytes, monocytes, granulocytes, and thrombocytes, all were found to adhere to the virus-infected cells. The adsorption seems to occur through at least two processes, one is mediated by microvilli (microvillus-attachment), and the other is direct adherence of both cells (cell-to-cell-attachment). In the former, the leukocytes are bound to the microvilli protruding from the infected MDCK cells and in the latter both cell membranes attach directly. In the cell-to-cell-attachment, there was an electron-lucent gap of about 12 nm in width in the intermembranous space of the junctional regions. This region was similar morphologically to the gap junction. As a result of leucoadsorption no cytolytic effects occurred in the MDCK cells under the experimental conditions.