Negative regulation of cytoplasmic RNA-mediated antiviral signaling

The recent, rapid progress in our understanding of cytoplasmic RNA-mediated antiviral innate immune signaling was initiated by the discovery of retinoic acid-inducible gene I (RIG-I) as a sensor of viral RNA. It is now widely recognized that RIG-I and related RNA helicases, melanoma differentiation-associated gene-5 (MDA5) and laboratory of genetics and physiology-2 (LGP2), can initiate and/or regulate RNA and virus-mediated type I IFN production and antiviral responses. As with other cytokine systems, production of type I IFN is a transient process, and can be hazardous to the host if unregulated, resulting in chronic cellular toxicity or inflammatory and autoimmune diseases. In addition, the RIG-I-like receptor (RLR) system is a fundamental target for virus-encoded immune suppression, with many indirect and direct examples of interference described. In this article, we review the current understanding of endogenous negative regulation in RLR signaling and explore direct inhibition of RLR signaling by viruses as a host immune evasion strategy.     

Engineering custom-designed osteochondral tissue grafts

Tissue engineering is expected to help us outlive the failure of our organs by enabling the creation of tissue substitutes capable of fully restoring the original tissue function. Degenerative joint disease, which affects one-fifth of the US population and is the country's leading cause of disability, drives current research of actively growing, functional tissue grafts for joint repair. Toward this goal, living cells are used in conjunction with biomaterial scaffolds (serving as instructive templates for tissue development) and bioreactors (providing environmental control and molecular and physical regulatory signals). In this review, we discuss the requirements for engineering customized, anatomically-shaped, stratified grafts for joint repair and the challenges of designing these grafts to provide immediate functionality (load bearing, structural support) and long-term regeneration (maturation, integration, remodeling).     

Widespread and ample peptide overlapping between HCV and Homo sapiens proteomes

Alignment of protein sequences is fundamental in analyzing homology, evolutionary events and functional relationships. Searching for the epitopic peptide platform underlying hepatitis C virus (HCV) infection and autoimmune phenomena, we have used sequence-sequence peptide matching to compare the HCV polyprotein sequence to the human proteome. The following results were obtained: (1) pentamers from HCV polyprotein have a widespread and high level of similarity to a large number of human proteins (19,605 human proteins, that is 57.6% of the human proteome); (2) remarkable similarity between the two proteomes persists even using longer peptide motifs as probes for identity scanning; (3) only a limited number of HCV pentameric fragments have no similarity to the human host, so representing molecular sequence signatures of the virus. We conclude that the widespread sharing of numerous perfect exact matches between HCV and human proteomes might explain HCV persistence in humans.     

The Influence of Very Low Illumination on the Postural Sway of Young and Elderly Adults

The purpose of the present study was to evaluate the influence of very low ambient illumination and complete darkness on the postural sway of young and elderly adults. Eighteen healthy young participants aged 23.8±1.5 years and 26 community-dwelling elderly aged 69.8±5.6 years were studied. Each participant performed four tests while standing on a force platform in the following conditions: in normal light (215 lx) with open eyes and with closed eyes, in very low illumination (0.25 lx) with open eyes, and in complete darkness with open eyes. The sequences of the tests in the altered visual conditions were determined by random blocs. Postural sway was assessed by means of the force platform measurements. The centre of pressure variables: the medio-lateral and antero-posterior path lengths, mean velocities, sway areas, and fractal dimensions were analysed. Very low illumination resulted in a statistically significant increase in postural sway in both the young and elderly groups compared to normal light, although the increase was significantly smaller than those observed in the eyes closed and complete darkness condition, and no significant effects of illumination on fractal dimensions were detected. The gains of the sways in the very low or no illumination conditions relative to the normal light condition were significantly larger in the group of young participants than in the group of elderly participants (up to 50% and 25%, respectively). However, the response patterns to changes in illumination were similar in the young and elderly participants, with the exception of the short-range fractal dimension of the medio-lateral sway. In conclusion, very low illumination resulted in increased postural sway compared to normal illumination; however, in the closed eye and complete darkness conditions, postural sway was significantly higher than in the very low illumination condition regardless of the age of the participants.     

The impact of long-term past exposure to elemental mercury on antioxidative capacity and lipid peroxidation in mercury miners

Limited information is available on the effects of chronic mercury exposure in relation to the risk of cardiovascular disease (CVD). It is known from in vitro and in vivo studies that Hg can promote lipid peroxidation through promotion of free radical generation, and interaction with antioxidative enzymes and reduction of bioavailable selenium. The objective of the study was to test the hypothesis that long-term past occupational exposure to elemental Hg (Hg⁰) can modify antioxidative capacity and promote lipid peroxidation in miners.

The study population comprised 54 mercury miners and 58 workers as the control group. The miners were examined in the post-exposure period. We evaluated their previous exposure to Hg⁰, the putative appearance of certain nonspecific symptoms and signs of micromercurialism, as well as the main behavioural and biological risk factors for CVD, and determined: 1) Hg and Se levels in blood and urine, 2) antioxidative enzymes, Cu/Zn superoxide dismutase (CuZn-SOD), catalase (CAT), and selenoenzyme glutathione peroxidase (GSH-Px) activity in erythrocytes as indirect indices of free radical activity, 3) pineal hormone melationin (MEL) in blood and urine, and 4) lipid hydroperoxides (LOOHs) and malondialdehyde (MDA) as lipid peroxidation products.

The mercury miners were intermittently exposed to Hg⁰ for periods of 7 to 31 years. The total number of exposure periods varied from 13 to 119. The cumulative U-Hg peak level varied from 794-11,365 μg/L. The current blood and urine Hg concentrations were practically on the same level in miners and controls. Miners showed some neurotoxic and nephrotoxic sequels of micromercurialism. No significant differences in behavioural and biological risk factors for CVD were found between miners and controls. A weak correlation (r = 0.36, p < 0.01) between systolic blood pressure and average past exposure U-Hg level was found. The mean P-Se in miners (71.4 μg/L) was significantly lower (p < 0.05) than in the controls (77.3 μg/L), while the mean U-Se tended to be higher (p < 0.05) in miners (16.5 μg/g creatinine) than in the controls (14.0 μg/g creatinine). Among antioxidative enzyme activities, only CAT in erythrocytes was significantly higher (p < 0.01) in miners (3.14 MU/g Hb) than in the controls (2.65 MU/g Hb). The mean concentration of B-MEL in miners (44.3 ng/L) was significantly higher (p < 0.01) than in the controls (14.9 ng/L). The mean value of U-MEL sulphate (31.8 μg/L) in miners was significantly lower (p < 0.01) than in the control group (46.9 μg/L). Among the observed lipid peroxidative products, the mean concentration of U-MDA was statistically higher (p < 0.01) in miners (0.21 μmol/mmol creatinine) than in the controls (0.17 μmol/mmol creatinine).

In the group of miners with high mercury accumulation and the presence of some nonspecific symptoms and signs of micromercurialism, the results of our study partly support the assumption that long-term occupational exposure to Hg⁰ enhances the formation of free radicals even several years after termination of occupational exposure. Therefore, long-term occupational exposure to Hg⁰ could be one of the risk factors for increased lipid peroxidation and increased mortality due to ischaemic heart disease (ICH) found among the mercury miners of the Idrija Mine.     

Saponins in tissue culture of <Emphasis Type="Italic">Primula veris</Emphasis> L.

Roots of Primula veris L. contain considerable amounts of triterpene saponins, which are used in medicine as expectorants. P. veris is in many places an endangered plant, and its production in the field is laborious and a low yielding process. Plant tissue culture provides an alternative means for producing secondary metabolites. Shoot apex, callus, suspension, and root cultures of P. veris were developed for saponin production. In these cultures, the content of triterpene saponins, with focus on primula acid I, the most dominant saponin in Primula species, was determined and compared to that in soil-grown plants. The highest content of primula acid I was observed in root cultures, on average 29.5 mg/g dry weight. Some culture lines contained higher amounts of primula acid I (62.6 mg/g dry weight) than the roots of plants grown in soil.     

Photochemical Properties of Camptothecin in the Presence of Copper(II) Ions: The Role of Radicals as Prospective Species in Photodynamic Therapy

Camptothecin (CPT) is an anticancer drug that inhibits topoisomerase I (Topo I) by forming a ternary DNA-CPT-Topo I complex. However, it has also been shown that UVA-irradiated CPT in the absence of Topo I produces significant DNA damage to cancer cells. In this work, we explored and identified free radicals generated in these processes. From the low-temperature EPR spectrum of Cu(II)-CPT complex, a proximity between Cu(II) ion and 20-hydroxy group of lactone E ring of CPT is proposed. Upon irradiation (λ = 365 nm) of the Cu(II)-CPT complex in de-oxygenated dimethylsulfoxide (DMSO), the EPR signal of Cu(II) measured in situ at room temperature shows formal first-order exponential decay with a formal half-life of 11 min. By the use of a specific Cu(I) chelating agent, neocuproine, it was shown that, during this process, Cu(II) is reduced to Cu(I). The loss in EPR signal intensity of the Cu(II)-CPT complex upon irradiation is accompanied by the appearance of a new EPR signal at g ≈ 2.0022. Application of the spin trap nitrosodurene (ND) revealed that the main radical product formed upon continuous irradiation of CPT in DMSO solutions is the hydroxyl radical (trapped in DMSO as the ^•CH₃ adduct) and superoxide radical. Application of 2,2,6,6-tetramethyl-4-piperidinol has revealed that irradiation of CPT in aerated DMSO solution also leads to formation of singlet oxygen (¹O₂). Our spectroscopic experiments indicate that CPT is a promising photosensitizer and that radicals and singlet oxygen generated upon illumination play a central role in DNA cleavage and in the induction of apoptosis in cancer cells.     

Heterogeneity in expression of the <Emphasis Type="Italic">Escherichia coli</Emphasis> colicin K activity gene <Emphasis Type="Italic">cka</Emphasis> is controlled by the SOS system and stochastic factors

Phenotypic diversity provides populations of prokaryotic and eukaryotic organisms with the flexibility required to adapt to and/or survive environmental perturbations. Consequently, there is much interest in unraveling the molecular mechanisms of heterogeneity. A classical example of heterogeneity in Escherichia coli is the subset (3%) of the population that expresses the colicin K activity gene (cka) upon nutrient starvation. Here, we report on the mechanism underlying this variable response. As colicin synthesis is regulated by the LexA protein, the central regulator of the SOS response, we focused on the role of LexA and the SOS system in the variable cka expression. Real-time RT-PCR showed that the SOS system, without exogenous DNA damage, induces moderate levels of cka expression. The use of cka-gfp fusions demonstrated that modification of the conserved LexA boxes in the cka promoter region affected LexA binding affinity and the percentage of cka-gfp expressing cells in the population. A lexA-gfp fusion showed that the lexA gene is highly expressed in a subset of bacteria. Furthermore, cka-gfp fusions cloned into higher copy plasmid vectors increased the percentage of cka-gfp positive bacteria. Together, these results indicate that the bistability in cka expression in the bacterial population is determined by (1) basal SOS activity, (2) stochastic factors and possibly (3) the interplay of LexA dimers at cka operator. Other LexA regulated processes could exhibit similar regulation.