The evaluation of the genotoxicity of two commonly used food colors: Quinoline Yellow (E 104) and Brilliant Black BN (E 151)

Additives, especially colors, are in widespread use in the food industry. With the exception of the quinolines, food colors are relatively weak mutagens and are certified as safe additives despite reports that some people have allergic reactions to them. The number of food additives is still on the increase, and research on their potential mutagenic/carcinogenic activity in vivo is very expensive. Using two different cellular model systems, human lymphocytes in vitro and Vicia faba root tip meristems of in vivo, we evaluated the potential cytological and genotoxic effects of two dyes: Quinoline Yellow (E 104) and Brilliant Black BN (E 151). Two relatively new, very sensitive and rapid tests - the micronucleus and Comet assays - were used in this study. The data provided in this paper showed the genotoxic effects of the two analyzed food colors, and confirmed the diagnostic value of the MN and Comet assays for screening potentially genotoxic substances.     

Mode of transfer of spermatozoa in Orthoptera Tettigoniidae

A morphological and ultrastructural study was carried out on the spermatophore and spermatodoses of some species of Orthoptera Tettigoniidae. From the results concerning the spermatophore it emerged that this structure has a morphological and ultrastructural organization represented by a dilated ampulla and a peduncle or neck. From the examination of freshly deposited spermatophores and those at various time intervals thereafter, it was seen that these structures other than allowing gamete transfer, represent the site where spermatodesms, organized in the male genital tracts, undergo reorganization to acquire their definitive morphological and structural characteristics as found in the female genital tracts. The spermatodoses, in the same way as the spermatophore, represent capsules containing spermatodesms, which are originated in the spermatheca, their specific morphology seems to diversify according to the species considered. As regards their role, it is hypothesized that these structures represent a long-term conservation mechanism for spermatozoa inside the seminal receptacle.     

Optimization of apolipoprotein B mRNA editing by APOBEC1 apoenzyme and the role of its auxiliary factor, ACF

Expression and purification to homogeneity of the apolipoprotein B mRNA editing subunit, APOBEC1, has allowed the demonstration that this apoenzyme has considerable residual enzymatic activity on a minimal apoB mRNA substrate, even in the absence of any auxiliary factors. Assay of this activity as a function of various experimental conditions has led to substantial optimization of assay conditions through the use of incomplete factorial and response surface experiments. Surprisingly, the apoenzyme is thermostable, and has a temperature optimum near 45 degrees C. We have used these optimized conditions, to assess steady-state kinetic parameters for APOBEC1 mRNA editing activity with and without the auxiliary factor, ACF. An important effect of the auxiliary factor is to broaden the temperature range of APOBEC1 activity, lowering the optimal temperature and enabling it to function optimally at lower temperatures. A model consistent with this observation is that at lower temperatures ACF promotes a conformational transition in the RNA substrate that occurs spontaneously at higher temperature. Notably, the substantial RNA editing activity of APOBEC1 alone may be responsible for the "hyperediting" observed upon overexpression of APOBEC1 in transgenic mice.     

Combined Vitamins B12b and C Induce the Glutathione Depletion and the Death of Epidermoid Human Larynx Carcinoma Cells HEp-2

The combination of hydroxocobalamin (vitamin B_12b) and ascorbicacid (vitamin C) can cause the death of tumor cells at the concentrationsof the components at which they are nontoxic when administeredseparately. This cytotoxic action on epidermoid human larynx carcinomacells HEp-2 in vitro is shown to be due to the hydrogen peroxidegenerated by the combination of vitamins B_12b and C. The drop inthe glutathione level preceding cell death was found to be the result ofcombined action of the vitamins. It is supposed that the induction of celldeath by combined action of vitamins B_12b and C is connected to the damageof the cell redox system.     

Electrochromic properties of mixed valence binuclear ruthenium complexes adsorbed on nanocrystalline SnO2 films

The electrochromic properties of two new mixed valence ruthenium complexes: K[(NC₅H₄CH₂PO₃H₂)Ru^III(NH₃)₄(NC)Ru^II(CN)₅] and K[(NC₅H₄PO₃H₂)Ru^III(NH₃)₄(NC)Ru^II(CN)₅], where phosphonic acid groups have been introduced at the pyridine ligand, have been studied in homogeneous solution and adsorbed on transparent nanocrystalline SnO₂ electrodes. These species exhibit a superior stability with respect to the previously studied, K[(NC₅H₄CO₂H)Ru^III(NH₃)₄NCRu^II(CN)₅] complex, showing negligible optical density changes after cycling 20 000 times the electrodes between −0.5 and 0.5 V versus SCE.     

Characterization of cellular and humoral immune responses to alkaline phosphatase from fertile hydatid cysts in the human peripheral blood

Hydatid cyst, the larval stage of the tapeworm Echinococcus granulosus and a causative agent of cystic echinococcosis, possesses a vast number of antigenic peptides that are constantly presented in the host immune system during infection. Here, we sought to provide more information about the cellular/humoral components engaged in the peripheral immune reactions to the fertile-cyst-derived Echinococcus alkaline phosphatase (E.ALP) in human hosts. Lymphoproliferative and cytokine responses after recall of E.ALP suggested the presence of specific immune reactions against the antigen. Interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin (IL)-10 had the highest fold increase over the spontaneous levels in response to hydatid crude antigen (HCA). Recall of E.ALP, as well as its encounter, boosted IFN-γ, TNF-α, IL-2, and IL-6 responses in peripheral blood mononuclear cells cultures (PBMCs). The HCA-driven levels of all the cytokines in the culture supernatants of normal PBMCs were higher than those measured after E.ALP encounter. Immunoglobulin G (IgG)-profile in response to HCA showed the dominance of specific IgG1, IgG2, and IgG4 antibodies, but relatively lower affinity of IgG3 to this antigen. IgG1 and IgG3 were the only isotypes detected in serum responses to E.ALP. Our findings suggested that E.ALP contributes to the early phase of immune responses to the parasite, likely by induction of proinflammatory profiles and clonal expansion of high-affinity IgG1- and IgG3-secreting plasma cells, suggesting the value of E.ALP as a candidate to develop novel therapeutic and immunization strategies.     

Intractable Headaches,Ischemic Stroke,and Seizures Are Linked to the Presence of Anti-β2GPI Antibodies in Patients with Systemic Lupus Erythematosus

Background

Neuropsychiatric systemic lupus erythematosus (NPSLE) is a common and potentially fatal manifestation of SLE. Antiphospholipid antibodies (aPL) such as lupus anticoagulant (LA), anticardiolipin (aCL) and antibodies to β2glycoprotein I (anti-β2GPI), the most important aPL antigen, are thought to play a role in some forms of NPSLE. As of yet, their specific roles in NPSLE manifestations remain to be elucidated.

Methodology/Principal Findings

57 SLE patients (53 women) were assessed for LA, aCL and anti-β₂GPI twice, to determine persistent positivity. All patients were examined by neurology and psychiatry specialists. 69 healthy subjects were assessed as controls. NPSLE was diagnosed in 74% of patients. Headaches were the most prevalent manifestation of NPSLE (39%), followed by cerebrovascular disease (CVD) (23%), depressive disorders (19.0%), and seizures (14%). NPSLE and non-NPSLE patients showed comparable SLE activity and corticosteroid use. In 65% of patients neuropsychiatric manifestations preceded SLE diagnosis. aPL profiles of NPSLE patients and non-NPSLE patients were similar. Headaches and ischemic stroke were independently associated with anti-β₂GPI-IgM (OR=5.6; p<0.05), and seizures were linked to anti-β₂GPI-IgG (OR=11.3; p=0.01).

Conclusions

In SLE patients, neuropsychiatric manifestations occur frequently and early, often before the disease is diagnosed. Autoantibodies to β2GPI are linked to non-specific headaches, ischemic stroke and seizures, and show a better predictive value than aCL and LA. These findings may help to improve the diagnosis of NPSLE and should prompt further studies to characterize the role of anti-β2GPI in the pathogenesis of this condition.     

A conformational transition state accompanies tryptophan activation by B. stearothermophilus tryptophanyl-tRNA synthetase

B. stearothermophilus tryptophanyl-tRNA synthetase catalysis proceeds via high-energy protein conformations. Unliganded MD trajectories of the pretransition-state complex with Mg(2+)ATP and the (post) transition-state analog complex with adenosine tetraphosphate relax rapidly in opposite directions, the former regressing, the latter progressing along the structural reaction coordinate. The two crystal structures (rmsd 0.7 A) therefore lie on opposite sides of a conformational free-energy maximum as the chemical transition state forms. SNAPP analysis illustrates the complexity of the associated long-range conformational coupling. Switching interactions in four nonpolar core regions are locally isoenergetic throughout the transition. Different configurations, however, propagate their effects to unfavorable, longer-range interactions at the molecular surface. Designed mutation shows that switching interactions enhance the rate, perhaps by destabilizing the ground state immediately before the transition state and limiting nonproductive diffusion before and after the chemical transition state, thereby reducing the activation entropy. This paradigm may apply broadly to energy-transducing enzymes.     

Dynamic changes in the localization of MAPK cascade components controlling pathogenesis-related (PR) gene expression during innate immunity in parsley

The activation of mitogen-activated protein kinase (MAPK) cascades is an important mechanism for stress adaptation through the control of gene expression in mammals, yeast, and plants. MAPK activation has emerged as a common mechanism by which plants trigger pathogen defense responses following innate immune recognition of potential microbial pathogens. We are studying the non-host plant defense response of parsley to attempted infection by Phytophthora species using an experimental system of cultured parsley cells and the Phytophthora-derived Pep-13 peptide elicitor. Following receptor-mediated recognition of this peptide, parsley cells trigger a multifaceted innate immune response, involving the activation of three MAPKs that have been shown to function in the oxidative burst-independent activation of defense gene expression. Using this same experimental model we now report the identification of a MAPK kinase (MAPKK) that functions upstream in this pathway. This kinase, referred to as PcMKK5 based on sequence similarity to Arabidopsis thaliana AtMKK5, is activated in parsley cells following Pep-13 treatment and functions as an in vivo activator of all three MAPKs previously shown to be involved in this response. Gain- and loss-of-function mutant versions of PcMKK5, when used in protoplast co-transfection assays, demonstrated that kinase activity of PcMKK5 is required for PR gene promoter activation following Pep-13 treatment. Furthermore, using specific antibodies and immunofluorescent labeling, we demonstrate that activation of MAPKs in parsley cells correlates with an increase in their nuclear localization, which is not detectable for activated PcMKK5. These results suggest that activation of gene expression through MAPK cascades during innate immune responses in plants involves dynamic changes in the localization of the proteins involved, which may reflect the distribution of key protein substrates for the activated MAPKs.     

Elemental sulfur and acetate can support life of a novel strictly anaerobic haloarchaeon

Archaea domain is comprised of many versatile taxa that often colonize extreme habitats. Here, we report the discovery of strictly anaerobic extremely halophilic euryarchaeon, capable of obtaining energy by dissimilatory reduction of elemental sulfur using acetate as the only electron donor and forming sulfide and CO₂ as the only products. This type of respiration has never been observed in hypersaline anoxic habitats and is the first example of such metabolic capability in the entire Archaea domain. We isolated and cultivated these unusual organisms, selecting one representative strain, HSR2, for detailed characterization. Our studies including physiological tests, genome sequencing, gene expression, metabolomics and [¹⁴C]-bicarbonate assimilation assays revealed that HSR2 oxidized acetate completely via the tricarboxylic acid cycle. Anabolic assimilation of acetate occurred via activated glyoxylate bypass and anaplerotic carboxylation. HSR2 possessed sulfurtransferase and an array of membrane-bound polysulfide reductase genes, all of which were expressed during the growth. Our findings suggest the biogeochemical contribution of haloarchaea in hypersaline anoxic environments must be reconsidered.