Genetic mapping of the dominant albino locus in rainbow trout (Oncorhynchus mykiss)

Albinism in animals is generally a recessive trait, but in Japan a dominant oculocutaneous albino (OCA) mutant strain has been isolated in rainbow trout (Oncorhyncus mykiss). After confirming that this trait is not due to a tyrosinase gene mutation that causes OCA1 (tyrosinase-negative OCA), we combined the amplified fragment length polymorphism (AFLP) technique with bulked segregant analysis (BSA) to map the gene involved in dominant oculocutaneous albinism. Four AFLP markers tightly linked to the dominant albino locus were identified. One of these markers was codominant and we have it converted into a GGAGT-repeat microsatellite marker, OmyD-AlbnTUF. Using this pentanucleotide-repeat DNA marker, the dominant albino locus has been mapped on linkage group G of a reference linkage map of rainbow trout. The markers identified here will facilitate cloning of the dominant albino gene in rainbow trout and contribute to a better understanding of tyrosinase-negative OCA in animals.     

(-)-Epigallocatechin gallate reduces transforming growth factor beta-stimulated HSP27 induction through the suppression of stress-activated protein kinase/c-Jun N-terminal kinase in osteoblasts

We previously reported that transforming growth factor-beta (TGF-beta) stimulates heat shock protein 27 (HSP27) induction through p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase 1/2 (ERK1/2) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether (-)-epigallocatechin gallate (EGCG), the major polyphenol found in green tea, affects the TGF-beta-stimulated induction of HSP27 in these cells, and its underlying mechanism. EGCG significantly suppressed the HSP27 induction stimulated by TGF-beta in a dose-dependent manner between 10 and 30 microM without affecting the HSP70 levels. TGF-beta with or without EGCG did not affect the advanced oxidation protein products. The TGF-beta-induced phosphorylation of p38 MAP kinase and ERK1/2 was not affected by EGCG. SP600125, a specific inhibitor of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), markedly reduced the HSP27 expression induced by TGF-beta. EGCG significantly suppressed the TGF-beta-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of Smad2. EGCG attenuated the phosphorylation of both MKK4 and TAK1 induced by TGF-beta. These results strongly suggest that EGCG suppresses the TGF-beta-stimulated induction of HSP27 via the attenuation of the SAPK/JNK pathway in osteoblasts, and that this effect is exerted at a point upstream from TAK1.     

A pronounced light-induced zeaxanthin formation accompanied by an unusually slight increase in non-photochemical quenching: a study with barley leaves treated with methyl viologen at moderate light

Light-induced deepoxidation of violaxanthin to antheraxanthin and zeaxanthin in plants is associated with the induction of pronounced xanthophyll-dependent non-photochemical quenching (NPQ). To date, a misbalance between a high amount of zeaxanthin in thylakoid membranes and low NPQ has been explained by an absence of lumen acidification (e.g. when NPQ is measured in the dark after high light stress). In this study, we report that this misbalance can also be observed under moderate light. We found this result (deepoxidation state, DEPS, above 55% and NPQ0.9) in barley leaves treated with 10 μM methyl viologen (MV) under white light (100 μmol photons m⁻² s⁻¹, photosynthetically active radiation (PAR), growth irradiance). The addition of MV at this moderate light did not accelerate electron transport in thylakoid membranes, and induced only slight oxidative stress (no lipid peroxidation, almost unchanged maximum yield of photosystem II photochemistry, a decrease in activity of ascorbate peroxidase, and an increase in that of glutathion reductase). We suggest that, in leaves treated under the conditions used here, the lumen acidification induced by light-limited electron transport in thylakoid membranes was high enough to activate violaxanthin deepoxidase, but not sufficiently high to form the expected number of zeaxanthin-dependent quenching centers in photosystem II antennae.     

Functional characterization of a vitamin B12-dependent methylmalonyl pathway in Mycobacterium tuberculosis: implications for propionate metabolism during growth on fatty acids

Mycobacterium tuberculosis is predicted to subsist on alternative carbon sources during persistence within the human host. Catabolism of odd- and branched-chain fatty acids, branched-chain amino acids, and cholesterol generates propionyl-coenzyme A (CoA) as a terminal, three-carbon (C(3)) product. Propionate constitutes a key precursor in lipid biosynthesis but is toxic if accumulated, potentially implicating its metabolism in M. tuberculosis pathogenesis. In addition to the well-characterized methylcitrate cycle, the M. tuberculosis genome contains a complete methylmalonyl pathway, including a mutAB-encoded methylmalonyl-CoA mutase (MCM) that requires a vitamin B(12)-derived cofactor for activity. Here, we demonstrate the ability of M. tuberculosis to utilize propionate as the sole carbon source in the absence of a functional methylcitrate cycle, provided that vitamin B(12) is supplied exogenously. We show that this ability is dependent on mutAB and, furthermore, that an active methylmalonyl pathway allows the bypass of the glyoxylate cycle during growth on propionate in vitro. Importantly, although the glyoxylate and methylcitrate cycles supported robust growth of M. tuberculosis on the C(17) fatty acid heptadecanoate, growth on valerate (C(5)) was significantly enhanced through vitamin B(12) supplementation. Moreover, both wild-type and methylcitrate cycle mutant strains grew on B(12)-supplemented valerate in the presence of 3-nitropropionate, an inhibitor of the glyoxylate cycle enzyme isocitrate lyase, indicating an anaplerotic role for the methylmalonyl pathway. The demonstrated functionality of MCM reinforces the potential relevance of vitamin B(12) to mycobacterial pathogenesis and suggests that vitamin B(12) availability in vivo might resolve the paradoxical dispensability of the methylcitrate cycle for the growth and persistence of M. tuberculosis in mice.     

Involvement of mitogen-activating protein kinase and intracellular pH in the duration of the metaphase I (MI) pause of starfish oocytes after spawning

The metaphase I (MI) arrest of starfish oocytes is released after spawning. In this study using starfish Asterina pectinifera , the duration of MI after spawning was ~20 min and ~30 min in fertilized and unfertilized oocytes, respectively. This prolongation of MI in unfertilized oocytes, referred to as the MI pause, was maintained by mitogen-activating protein kinase (MAPK) as well as low intracellular pH (~7.0). Contrary to previous reports, MI arrest was not maintained by MAPK, since it was inactive in the oocytes arrested at MI in the ovary and activated immediately after spawning. Also, cyclin B was not degraded at pH 6.7 in the cell-free preparation without MAPK activity, whereas it was degraded at pH 7.0, suggesting that MI arrest was solely maintained by lower pH (< 7.0). Normal development occurred when the spawned oocytes were fertilized before the first polar body formation, whereas fertilization after the first polar body formation increased the rate of abnormal development. Thus, due to MI pause and MI arrest, the probability for fertilization before the polar body formation might be increased, leading to normal development.     

Structural analysis of the human Rad51 protein-DNA complex filament by tryptophan fluorescence scanning analysis: transmission of allosteric effects between ATP binding and DNA binding

Human Rad51 (HsRad51) catalyzes the strand exchange reaction, a crucial step in homologous recombination, by forming a filamentous complex with DNA. The structure of this filament is modified by ATP, which is required and hydrolyzed for the reaction. We analyzed the structure and the ATP-promoted conformational change of this filament. We systematically replaced aromatic residues in the protein, one at a time, with tryptophan, a fluorescent probe, and examined its effect on the activities (DNA binding, ATPase, ATP-promoted conformational change, and strand exchange reaction) and the fluorescence changes upon binding of ATP and DNA. Some residues were also replaced with alanine. We thus obtained structural information about various positions of the protein in solution. All the proteins conserved, at least partially, their activities. However, the replacement of histidine at position 294 (H294) and phenylalanine at 129 (F129) affected the ATP-induced conformational change of the DNA-HsRad51 filament, although it did not prevent DNA binding. F129 is considered to be close to the ATP-binding site and to H294 of a neighboring subunit. ATP probably modifies the structure around F129 and affects the subunit/subunit contact around H294 and the structure of the DNA-binding site. The replacement also reduced the DNA-dependent ATPase activity, suggesting that these residues are also involved in the transmission of the allosteric effect of DNA to the ATP-binding site, which is required for the stimulation of ATPase activity by DNA. The fluorescence analyses supported the structural change of the DNA-binding site by ATP and that of the ATP-binding site by DNA. This information will be useful to build a molecular model of the Rad51-DNA complex and to understand the mechanism of activation of Rad51 by ATP and that of the Rad51-promoted strand exchange reaction.     

Citrus auraptene acts as an agonist for PPARs and enhances adiponectin production and MCP-1 reduction in 3T3-L1 adipocytes

Citrus fruit compounds have many health-enhancing effects. In this study, using a luciferase ligand assay system, we showed that citrus auraptene activates peroxisome proliferator-activated receptor (PPAR)-α and PPARγ. Auraptene induced up-regulation of adiponectin expression and increased the ratio of the amount of high-molecular-weight multimers of adiponectin to the total adiponectin. In contrast, auraptene suppressed monocyte chemoattractant protein (MCP)-1 expression in 3T3-L1 adipocytes. Experiments using PPARγ antagonist demonstrated that these effects on regulation of adiponectin and MCP-1 expression were caused by PPARγ activations. The results indicate that auraptene activates PPARγ in adipocytes to control adipocytekines such as adiponectin and MCP-1 and suggest that the consumption of citrus fruits, which contain auraptene can lead to a partial prevention of lipid and glucose metabolism abnormalities.     

Attempts at a peptide vaccine against paracoccidioidomycosis, adjuvant to chemotherapy

Chemotherapy is the basis of treatment of paracoccidioidomycosis in its various forms. Depending on the Paracoccidioides brasiliensis virulence, the status of host immunity, the degree of tissue involvement and fungal dissemination, treatment can be extended for long periods with an alarming frequency of relapses. Association of chemotherapy with a vaccine to boost the cellular immune response seemed a relevant project not only to reduce the time of treatment but also to prevent relapses and improve the prognosis of anergic cases. The candidate immunogen is the gp43 major diagnostic antigen of P.␣brasiliensis and more specifically its derived peptide P10, carrying the CD4⁺ T-cell epitope. Both gp43 and P10 protected Balb/c mice against intratracheal infections with virulent P. brasiliensis strain. P10 as single peptide or in a multiple-antigen-peptide (MAP) tetravalent construction was protective without adjuvant either by preimmunization and intratracheal challenge or as a therapeutic agent in mice with installed infection. P10 showed additive protective effects in drug-treated mice stimulating a Th-1 type immune response with high IFN-γ and IL-12. P10 and few other peptides in the gp43 were selected by Tepitope algorithm and actually shown to promiscuously bind several prominent HLA-DR molecules suggesting that a peptide vaccine could be devised for a genetically heterogenous population. P10 was protective in animals turned anergic, was effective in a DNA minigene vaccine, and increased the protection by monoclonal antibodies in Balb/c mice. DNA vaccines and peptide vaccines are promising therapeutic tools to be explored in the control of systemic mycoses.