Functional divergence between evolutionary-related LuxG and Fre oxidoreductases of luminous bacteria

In luminous bacteria NAD(P)H:flavin-oxidoreductases LuxG and Fre, there are homologous enzymes that could provide a luciferase with reduced flavin. Although Fre functions as a housekeeping enzyme, LuxG appears to be a source of reduced flavin for bioluminescence as it is transcribed together with luciferase. This study is aimed at providing the basic conception of Fre and LuxG evolution and revealing the peculiarities of the active site structure resulted from a functional variation within the oxidoreductase family. A phylogenetic analysis has demonstrated that Fre and LuxG oxidoreductases have evolved separately after the gene duplication event, and consequently, they have acquired changes in the conservation of functionally related sites. Namely, different evolutionary rates have been observed at the site responsible for specificity to flavin substrate (Arg 46). Also, Tyr 72 forming a part of a mobile loop involved in FAD binding has been found to be conserved among Fre in contrast to LuxG oxidoreductases. The conservation of different amino acid types in NAD(P)H binding site has been defined for Fre (arginine) and LuxG (proline) oxidoreductases.     

Ecophysiological properties of cultivable heterotrophic bacteria and yeasts dominating in phytocenoses of Galindez Island,maritime Antarctica

Antarctic plants are stable specific microenvironments for microbial colonization that are still less explored. In this study, we investigated cultivable heterotrophic bacteria and yeasts dominating in plant samples collected from different terrestrial biotopes near Ukrainian Antarctic Base on Galindez Island, maritime Antarctica. Phylogenetic analysis revealed affiliation of the bacterial isolates to genera Pseudomonas, Stenotrophomonas, Brevundimonas, Sporosarcina, Dermacoccus, Microbacterium, Rothia and Frondihabitans, and the yeast isolates to genera Rhodosporidium, Cryptococcus, Leucosporidiella, Candida and Exophiala. Some ecophysiological properties of isolated strains were determined that are important in response to different stresses such as psychro- and halotolerance, UV-resistance and production of hydrolytic enzymes. The majority of isolates (88 %) was found to be psychrotolerant; all are halotolerant. Significant differences in survival subsequent to UV-C radiation were observed among the isolates, as measured by culturable counts. For the bacterial isolates, lethal doses in the range 80–600 J m^?2 were determined, and for the yeast isolates—in the range 300–1,000 J m^?2. Dermacoccus profundi U9 and Candida davisiana U6 were found as most UV resistant among the bacterial and yeast isolates, respectively. Producers of caseinase, gelatinase, β-glucosidase, and cellulase were detected. To the best of our knowledge, this is the first report on isolation of UV resistant strain D. profundi, and Frondihabitans strain from Antarctica, and on detection of cellulase activity in Antarctic yeast strain C. davisiana. The results obtained contribute to clarifying adaptation strategies of Antarctic microbiota and its possible role in functional stability of Antarctic biocenoses. Stress tolerant strains were detected that are valuable for ecological and applied studies.     

Seed bank composition and above-ground vegetation in response to grazing in sub-Mediterranean oak forests (NW Greece)

We investigate the persistent soil seed bank composition and its relation to the above-ground flora of grazed and non-grazed sub-Mediterranean deciduous oak forests of NW Greece. Twenty-eight taxa were recorded in the soil seed bank and 83 taxa (70 taxa in plots of seed bank sampling) in the above-ground vegetation. The dominant tree species and many woodland species found in the above-ground vegetation were absent from the soil seed bank. Similarity between the soil seed bank and the above-ground vegetation decreased with grazing, and grazing led to a decrease of species richness in above-ground vegetation and soil seed bank. Beta diversity of vegetation among grazed and among non-grazed plots did not differ, but was significantly higher between grazed and non-grazed areas. Beta diversity of the soil seed bank declined with grazing. When applying classification tree and logistic regression analyses, non-grazed forest sites are clearly differentiated by the presence of Phillyrea latifolia, Euphorbia amygdaloides and Brachypodium sylvaticum. PCA ordination of above-ground species composition reflected a gradient from sites grazed by ruminants to non-grazed sites, but no clear structure was detected in the seed bank.     

Effects of human exogenous DNA on production of perforin-containing CD8+ cytotoxic lymphocytes in laboratory setting and clinical practice

We investigated the influence of Panagen DNA preparations on laboratory animals and IFN-induced human dendritic cells, as well as analyzed the data from a phase II clinical trial in the therapy of breast cancer. It was shown that this treatment resulted in increased number of CD8+/perforin+ T cells in peripheral lymphoid organs of experimental animals, in mixed lymphocyte culture population and in peripheral blood of breast cancer patients. Moreover, we demonstrated that when Panagen DNA preparations are used in combination with the standard FAC-based breast cancer therapies, non-specific immune response activity remains at the same levels as observed prior to therapy, whereas in FAC-placebo patients, non-specific immunity is greatly diminished.     

A Crucial Role for Kupffer Cell-Derived Galectin-9 in Regulation of T Cell Immunity in Hepatitis C Infection

Approximately 200 million people throughout the world are infected with hepatitis C virus (HCV). One of the most striking features of HCV infection is its high propensity to establish persistence (∼70–80%) and progressive liver injury. Galectins are evolutionarily conserved glycan-binding proteins with diverse roles in innate and adaptive immune responses. Here, we demonstrate that galectin-9, the natural ligand for the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), circulates at very high levels in the serum and its hepatic expression (particularly on Kupffer cells) is significantly increased in patients with chronic HCV as compared to normal controls. Galectin-9 production from monocytes and macrophages is induced by IFN-γ, which has been shown to be elevated in chronic HCV infection. In turn, galectin-9 induces pro-inflammatory cytokines in liver-derived and peripheral mononuclear cells; galectin-9 also induces anti-inflammatory cytokines from peripheral but not hepatic mononuclear cells. Galectin-9 results in expansion of CD4⁺CD25⁺FoxP3⁺CD127^low regulatory T cells, contraction of CD4⁺ effector T cells, and apoptosis of HCV-specific CTLs. In conclusion, galectin-9 production by Kupffer cells links the innate and adaptive immune response, providing a potential novel immunotherapeutic target in this common viral infection.     

Cerebrovascular diseases in nuclear workers first employed at the Mayak PA in 1948–1972

Incidence and mortality from cerebrovascular diseases (CVD) (430–438 ICD-9 codes) have been studied in a cohort of 18,763 workers first employed at the Mayak Production Association (Mayak PA) in 1948–1972 and followed up to the end of 2005. Some of the workers were exposed to external gamma-rays only while others were exposed to a mixture of external gamma-rays and internal alpha-particle radiation due to incorporated ²³⁹Pu. After adjusting for non-radiation factors, there were significantly increasing trends in CVD incidence with total absorbed dose from external gamma-rays and total absorbed dose to liver from internal alpha radiation. The CVD incidence was statistically significantly higher among workers with total absorbed external gamma-ray doses greater than 0.20 Gy compared to those exposed to lower doses; the data were consistent with a linear trend in risk with external dose. The CVD incidence was statistically significantly higher among workers with total absorbed internal alpha-radiation doses to liver from incorporated ²³⁹Pu greater than 0.025 Gy compared to those exposed to lower doses. There was no statistically significant trend in CVD mortality risk with either external gamma-ray dose or internal alpha-radiation dose to liver. The risk estimates obtained are generally compatible with those from other large occupational studies, although the incidence data point to higher risk estimates compared to those from the Japanese A-bomb survivors. Further studies of the unique cohort of Mayak workers chronically exposed to external and internal radiation will allow improving the reliability and validating the radiation safety standards for occupational and public exposure.     

Synthesis and cytotoxic potency of novel tris(1-alkylindol-3-yl)methylium salts: Role of N-alkyl substituents

Novel derivatives of tris(indol-3-yl)methane and tris(indol-3-yl)methylium salts with the alkyl substituents at the N-atoms of the indole rings were synthesized. An easy substitution of indole rings in trisindolylmethanes for other indoles under the action of acids is demonstrated, and the mechanism of substitution is discussed. To obtain trisindolylmethylium salts, the environmentally safe method of oxidation of trisindolylmethanes with air oxygen in acidic conditions was developed. Tris(1-alkylindol-3-yl)methanes and tris(1-alkylindol-3-yl)methylium salts represent three-bladed molecular propellers whose physico-chemical and biological properties strongly depend on the N-alkyl substituent. The cytotoxicity of novel compounds increased with the number of C atoms in the alkyl chains, with optimal number n = 3–5 whereas the derivatives with longer side chains were less cytotoxic. The most potent novel compounds killed human tumor cells at nanomolar-to-submicromolar concentrations, being one order of magnitude more potent than the prototype antibiotic turbomycin A [tris(indol-3-yl)methylium salt]. Apoptosis in HCT116 colon carcinoma cell line induced by tris(1-pentyl-1H-indol-3-yl)methylium methanesulfonate was detectable at concentrations tolerable by normal blood lymphocytes. Thus, N-alkyl substituted tris(1-alkylindol-3-yl)methylium salts emerge as perspective anticancer drug candidates.     

Production of IFN-β during Listeria monocytogenes infection is restricted to monocyte/macrophage lineage

The family of type I interferons (IFN), which consists of several IFN-α and one IFN-β, are produced not only after stimulation by viruses, but also after infection with non-viral pathogens. In the course of bacterial infections, these cytokines could be beneficial or detrimental. IFN-β is the primary member of type I IFN that initiates a cascade of IFN-α production. Here we addressed the question which cells are responsible for IFN-β expression after infection with the intracellular pathogen Listeria monocytogenes by using a genetic approach. By means of newly established reporter mice, maximum of IFN-β expression was observed at 24 hours post infection in spleen and, surprisingly, 48 hours post infection in colonized cervical and inguinal lymph nodes. Colonization of lymph nodes was independent of the type I IFN signaling, as well as bacterial dose and strain. Using cell specific reporter function and conditional deletions we could define cells expressing LysM as the major IFN-β producers, with cells formerly defined as Tip-DCs being the highest. Neutrophilic granulocytes, dendritic cells and plasmacytoid dendritic cells did not significantly contribute to type I IFN production.     

Identification of Biomarkers of Human Skin Ageing in Both Genders. Wnt Signalling - A Label of Skin Ageing?

The goal of our work has been to investigate the mechanisms of gender-independent human skin ageing and examine the hypothesis of skin being an adequate model of global ageing. For this purpose, whole genome gene profiling was employed in sun-protected skin obtained from European Caucasian young and elderly females (mean age 26.7±4 years [n1 = 7] and 70.75±3.3 years [n2 = 4], respectively) and males (mean age 25.8±5.2 years [n3 = 6] and 76±3.8 years [n4 = 7], respectively) using the Illumina array platform. Confirmation of gene regulation was performed by real-time RT-PCR and immunohistochemistry. 523 genes were significantly regulated in female skin and 401 genes in male skin for the chosen criteria. Of these, 183 genes exhibited increased and 340 decreased expression in females whereas 210 genes showed increased and 191 decreased expression in males with age. In total, 39 genes were common in the target lists of significant regulated genes in males and females. 35 of these genes showed increased (16) or decreased (19) expression independent of gender. Only 4 overlapping genes (OR52N2, F6FR1OP2, TUBAL3 and STK40) showed differential regulation with age. Interestingly, Wnt signalling pathway showed to be significantly downregulated in aged skin with decreased gene and protein expression for males and females, accordingly. In addition, several genes involved in central nervous system (CNS) ageing (f.i. APP, TAU) showed to be expressed in human skin and were significanlty regulated with age. In conclusion, our study provides biomarkers of endogenous human skin ageing in both genders and highlight the role of Wnt signalling in this process. Furthermore, our data give evidence that skin could be used as a good alternative to understand ageing of different tissues such as CNS.