Role of SLC11A1 (formerly NRAMP1) in regulation of signal transduction induced by Toll-like receptor 7 ligands

Modulation of immune responses using Toll-like receptor (TLR) ligands is fast becoming one of the main new approaches for the treatment of infectious and allergic diseases. Characterizing the role of genetic factors in modulating responses to these ligands will be crucial in determining the efficacy of a particular treatment. Our previous findings have shown that treatment of Mycobacterium bovis BCG infection with a synthetic TLR7 ligand resulted in a reduction of the splenic bacterial load only in mice carrying a wild-type allele of Nramp1. To understand further how natural resistance-associated macrophage protein 1 (NRAMP1) modulates responses to TLR7 ligands, we have analysed various important TLR7 signal transduction events in macrophage cell lines derived from B10.ANramp1r and B10.ANramp1-/- mice. The Nramp1 genotype did not affect TLR7 receptor expression, ligand uptake or intracellular processing. Following TLR7 ligand stimulation, p38 mitogen-activated protein kinase (MAPK) activation was significantly reduced in B10A.Nramp1-/- macrophages compared with B10A.Nramp1r cells. Interestingly, levels of protein kinase C zeta (PKCzeta) activation were also found to be lower in B10A.Nramp1-/- macrophages and inhibition of this kinase in B10A.Nramp1r cells led to a reduction in cytokine production. Taken together, the data demonstrate a role for NRAMP1 in modulating p38 MAPK and PKCzeta activity, which leads to reduced cytokine induction by TLR7 ligands.     

Genome evolution in Arabidopsis/Brassica: conservation and divergence of ancient rearranged segments and their breakpoints

Since the tetraploidization of the Arabidopsis thaliana ancestor 30-35 million years ago (Mya), a wave of chromosomal rearrangements have modified its genome architecture. The dynamics of this process is unknown, as it has so far been impossible to date individual rearrangement events. In this paper, we present evidence demonstrating that the majority of rearrangements occurred before the Arabidopsis-Brassica split 20-24 Mya, and that the segmental architecture of the A. thaliana genome is predominantly conserved in Brassica. This finding is based on the conservation of four rearrangement breakpoints analysed by fluorescence in situ hybridization (FISH) and RFLP mapping of three A. thaliana chromosomal regions. For this purpose, 95 Arabidopsis bacterial artificial chromosomes (BACs) spanning a total of 8.25 Mb and 81 genetic loci for 36 marker genes were studied in the Brassica oleracea genome. All the regions under study were triplicated in the B. oleracea genome, confirming the hypothesis of Brassica ancestral genome triplication. However, whilst one of the breakpoints was conserved at one locus, it was not at the two others. Further comparison of their organization may indicate that the evolution of the hexaploid Brassica progenitor proceeded by several events, separated in time. Genetic mapping and reprobing with rDNA allowed assignment of the regions to particular Brassica chromosomes. Based on this study of regional organization and evolution, a new insight into polyploidization/diploidization cycles is proposed.     

Investigating the activity of 2-substituted alkyl-6-(2,5-dioxopyrrolidin-1-yl)hexanoates as skin penetration enhancers

Skin penetration enhancers are used in the formulation of transdermal delivery systems for drugs that are otherwise not sufficiently skin-permeable. We generated two series of esters by multi-step synthesis with substituted 6-aminohexanoic acid as potential transdermal penetration enhancers by multi-step synthesis. The synthesis of all newly prepared compounds is presented here. Structure confirmation of all generated compounds was accomplished by (1)H NMR, (13)C NMR, IR and MS spectroscopy. All the prepared compounds were analyzed using RP-HPLC and their lipophilicity (logk) was determined. The hydrophobicity (logP/ClogP) of the studied compounds was also calculated using two commercially available programs and 3D structures of the selected compounds were investigated by means of ab initio calculations of geometry and molecular dynamic simulations. All the synthesized esters were tested for their in vitro transdermal penetration-enhancing activity and showed higher enhancement ratios than oleic acid. The highest enhancement ratios were exhibited by compound 5f (C((2)) substituted with piperidine-2-one, C(11) ester chain) and 5a (C((2)) substituted with piperidine-2-one, C(6) ester chain). The series with a ω-lactam ring (piperidin-2-one; 5a-g), showed slightly higher activities than those with morpholine (6a-6g). All of the agents showed minimal anti-proliferative activity (IC(50) >6.25μM), indicating they would have low cytotoxicity when administered as chemical penetration enhancers. The relationships between the lipophilicity and the chemical structure of the studied compounds, as well as the correlation between their chemical structure and transdermal penetration-enhancing activity, are discussed.     

A false expression of CD8 antigens on CD4+ T cells in a routine flow cytometry analysis

The two-colour flow cytometry method applied in a routine enumeration of peripheral blood T lymphocyte subsets reveals that in some patients the entire population of CD4+ lymphocytes seems to express CD8 determinants as well. However, expression of the CD8 antigens on the cell surface is much lower in comparison with typical CD8+ cells. Moreover, in one-colour staining with an anti-CD8 antibody, cells with weak CD8 expression are not observed and only one typical population of CD8+ lymphocytes is seen. Investigating this phenomenon, we showed that after washing patient cells in RPMI before CD4/CD8 staining, the CD4+ T cell population did not show CD8 "co-expression". These results suggest that CD4+ lymphocytes, which seem to co-express CD8 antigen, in fact do not have this antigen on their surface. Moreover, after the addition of patient plasma to healthy donor cells prior to CD4/CD8 staining, a weak CD8 expression on normal CD4+ cells was noticed. Therefore we can assume that the agent(s) causing this phenomenon is/are present in the plasma of some patients. Altogether, these observations suggest that this phenomenon is nonspecific and probably results from cross-linking of anti-CD8 mAbs with anti-CD4 mAbs caused by factor(s) present in plasma of some patient. However, identification of that/these factor(s) requires further research.     

Quantitative analysis of the oxidative DNA lesion, 2,2-diamino-4-(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (oxazolone), in vitro and in vivo by isotope dilution-capillary HPLC-ESI-MS/MS

A major DNA oxidation product, 2,2-diamino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (oxazolone), can be generated either directly by oxidation of dG or as a secondary oxidation product with an intermediate of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). Site-specific mutagenesis studies indicate that oxazolone is a strongly mispairing lesion, inducing approximately 10-fold more mutations than 8-oxo-dG. While 8-oxo-dG undergoes facile further oxidation, oxazolone appears to be a stable final product of guanine oxidation, and, if formed in vivo, can potentially serve as a biomarker of DNA damage induced by oxidative stress. In this study, capillary liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) methods were developed to enable quantitative analysis of both 8-oxo-dG and oxazolone in DNA from biological sources. Sensitive and specific detection of 8-oxo-dG and oxazolone in enzymatic DNA hydrolysates was achieved by isotope dilution with the corresponding 15N-labeled internal standards. Both nucleobase adducts were formed in a dose-dependent manner in calf thymus DNA subjected to photooxidation in the presence of riboflavin. While the amounts of oxazolone continued to increase with the duration of irradiation, those of 8-oxo-dG reached a maximum at 20 min, suggesting that 8-oxo-dG is converted to secondary oxidation products. Both lesions were found in rat liver DNA isolated under carefully monitored conditions to minimize artifactual oxidation. Liver DNA of diabetic and control rats maintained on a diet high in animal fat contained 2-6 molecules of oxazolone per 10(7) guanines, while 8-oxo-dG amounts in the same samples were between 3 and 8 adducts per 10(6) guanines. The formation of oxazolone lesions in rat liver DNA, their relative stability in the presence of oxidants and their potent mispairing characteristics suggest that oxazolone may play a role in oxidative stress-mediated mutagenesis.     

Mononuclear copper(II) complexes of alloferons 1 and 2: a combined potentiometric and spectroscopic studies

Mononuclear copper(II) complexes of the alloferon 1 His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly, alloferon 2 Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly, Ac-alloferon 1 Ac-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly and Ac-alloferon 2 Ac-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly have been studied by potentiometric, UV-vis, CD and EPR spectroscopic methods. The potentiometric and spectroscopic data shows that acetylation of the amino terminal group induces significant changes in the coordination properties of the Ac-alloferons 1 and 2 compared to the alloferons 1 and 2, respectively. The presence of four (Ac-alloferon 1) or three (Ac-alloferon 2) histidyl residues provides a high possibility for the formation of macrochelates via the exclusive binding of imidazole-N donor atoms. The macrochelation suppresses, but cannot preclude the deprotonation and metal ion coordination of amide functions and the CuH₋₃L species with {N_Im, 3N⁻} bonding mode at pH above 8 are formed. The N-terminal amino group of the alloferons 1 and 2 takes part in the coordination of the metal ion and the 4N complex with {NH₂, 3N_Im} coordination mode dominates at physiological pH 7.4 for alloferon 1 and the 3N {NH₂, CO, 2N_Im} binding mode for alloferon 2. However, at higher pH values sequential amide nitrogens are deprotonated and coordinated to copper(II) ions.     

Releasing primary dormancy in <Emphasis Type="Italic">Avena fatua</Emphasis> L. caryopses by smoke-derived butenolide

The effect of smoke and smoke-derived butenolide in releasing dormancy of caryopses (referred to as seeds) of the economically important weed Avena fatua L. was studied. Seeds of A. fatua are dormant after harvest. Both smoke-water and butenolide, applied continuously, removed dormancy in darkness at 15, 20 and 25°C and slightly at 30°C. Butenolide was very active at a concentration of 10⁻⁸ M. Butenolide at 10⁻⁸ M was also able to remove dormancy at 20°C when applied for 12 or 24 h at 4°C or for 3 to 24 h at 20°C. Sensitivity to butenolide decreased with longer preincubation times in water. This compound was less effective in releasing dormancy in the light than in darkness. Dormancy release by butenolide involves induction of cell-cycle activity just before coleorhiza protrusion. Stimulatory effects of smoke-water and butenolide were also observed in respect of seedling growth and vigor.     

Degradation of gasoline aromatic hydrocarbons by two N2-fixing soil bacteria

Two bacterial strains, 3A and 5A, isolated from soil, were selected for their ability to degrade gasoline aromatic compounds and to fix N₂. Strains 3A and 5A have been ascribed to the genera Agrobacterium and Alcaligenes, respectively. Using gasoline as the sole carbon source these strains were as effective at degrading benzene, toluene and xylene as Pseudomonas putida ATCC12236, a reference biodegrading strain.