Sümer Aras

Regulation and function of central nervous system chemokines.

In this paper, we discuss the potential involvement of a new family of cytokines, termed chemokines, in CNS inflammatory pathology. Chemokines are a family of proinflammatory cytokines which are able to stimulate target‐cell‐specific directional migration of leukocytes. Because of this feature, chemokines may be potent mediators of inflammatory processes. We have previously reported observations indicating that chemokines may be involved in the process of lesion formation during autoimmune inflammation within CNS, and, in particular, are likely participants in the process of influx of inflammatory cells into the CNS parenchyma. We observed also that mechanical injury of brain and subsequent post‐traumatic inflammation may in part be mediated by chemokines. Chemokines undoubtedly co‐operate with cell‐associated adhesion molecules during recruitment of leukocytes from blood to CNS. The sequential expression of soluble and membrane‐bound signals for leukocyte migration is an intricate process that can be interrupted by a variety of strategies. Our data suggest that chemokines may represent a promising target for future therapy of inflammatory conditions, including CNS inflammation resulting from varied insults.

Regulation of human IP-10 gene expression in astrocytoma cells by inflammatory cytokines

Because of its prominent expression in central nervous system inflammatory pathology by astrocytes, we examined the mechanism of human IP‐10 (hIP‐10) gene induction by interferon‐γ (IFN‐γ) and tumor necrosis factor‐alpha (TNF‐α) in astrocytoma cells. When present together, IFN‐γ and TNF‐α induced robust accumulation of hIP‐10 mRNA, but hIP‐10 mRNA was minimally induced when astrocytoma cells were treated with individual cytokines. This pattern of expression resembled that previously described for murine IP‐10 (mIP‐10) gene induction in fibroblasts and in rat astroglia. Nuclear run‐on experiments showed that the synergistic effect of the cytokines resulted from an increased rate of IP‐10 transcriptional initiation. Functional analysis of the hIP‐10 promoter after deletion and substitution mutagenesis indicated that an interferon‐stimulated response element (ISRE) governed both simple response to IFN‐γ and synergy with TNF‐α. Synergistic induction of hIP‐10 also required an ISRE‐proximal nuclear factor kappa‐B (NFκB) binding site. TNF‐α‐induced NFκB binding activity at this site was composed of RelA (p65) homodimers. Our results document that cis‐elements through which cytokines mediate synergistic induction of IP‐10 in mouse and human are strictly conserved despite divergence elsewhere within the proximal 5′‐flanking region. J. Neurosci. Res. 54:169–180, 1998.

Evaluation of copper-induced stress on eggplant (Solanum melongena L.) seedlings at the molecular and population levels by use of various biomarkers

Heavy-metal contamination is an important environmental problem in the world. It is known that high concentrations of heavy metals cause toxic damage to cells and tissues. In this study the effects of copper (Cu(2+)) contamination were determined at the molecular and population levels in eggplant (Solanum melongena L.) seedlings exposed to various concentrations of the metal ion. Inhibition of root growth, reduction in dry weight and total soluble protein content in the roots of eggplant seedlings were observed with increasing Cu(2+) concentrations. In ecotoxicology, analysis by random amplification of polymorphic DNA (RAPD) has been applied as a suitable biomarker assay for plants. For the RAPD analyses, nine RAPD primers were found to produce unique polymorphic band patterns and were subsequently used to produce a total of 80 and 168 bands in the roots of untreated and treated eggplant seedlings, respectively. The changes in RAPD profiles after Cu(2+) contamination were considered as variations, i.e. as gain and/or loss of bands compared with control seedlings. These results suggest that changes in genomic template stability could be detected with RAPD profiles and this result could be compared with the growth, dry weight and total soluble protein content of the seedlings grown at various Cu(2+) concentrations. The measurements of parameters at the molecular and population levels are fundamental to accumulate valuable information and to understand clearly the effect of a contaminant on an organism in ecotoxicology.

Isolation of DNA for RAPD analysis from dry leaf material of someHesperis L. specimens

An improved protocol for the isolation of DNA from dry material of someHesperis specimens is described. The isolated DNA is suitable for random amplification of polymorphic DNA (RAPD) analysis. Different DNA extraction protocols were examined to determine which might yield DNA from dry leaf tissue ofHesperis specimens. The methods examined include the protocols with hexadecyltrimethylammonium bromide (CTAB) described by Doyle and Doyle (1987); sodium dodecyl sulfate (SDS) by Dellaporta et al. (1983); and CTAB and SDS, the modified minipreparation, by Dellaporta et al (1983). None of these procedures yielded DNA of suitable purity for RAPD assay. We established an improved procedure involving CTAB and enzymatic digestion of proteins and RNA. The recovery of DNA with an average yield of 25 mg/g of leaf material was possible with this procedure. RAPD bands, which could be used to distinguish amongHesperis specimens, were generated.

Regulation of monocyte chemoattractant protein (MCP)-1 transcription by interferon-gamma (IFN-γ) in human astrocytoma cells: postinduction refractory state of the gene, governed by its upstream elements

Monocyte chemoattractant protein (MCP)‐1 is expressed by astrocytes in diverse inflammatory states and is a key regulator of monocyte recruitment to the central nervous system (CNS). In the current study, we addressed mechanisms by which transcription of the human MCP‐1 gene (hMCP‐1) was terminated, after induction by interferon (IFN)‐γ. Our results demonstrated that IFN‐γ‐induced transcription of hMCP‐1 was followed by a refractory state, during which hMCP‐1 was resistant to restimulation by either IFN‐γ or heterologous activators such as TNF‐α. This refractory state affected the hMCP‐1 gene selectively, as other IFN‐γ‐inducible genes remained responsive to restimulation. The IFN‐γ‐induced hMCP‐1 refractory state was governed at the transcriptional level and was sensitive to protein synthesis inhibitors, suggesting a requirement for newly expressed components. A minimal 213 base pair hMCP‐1 regulatory element directed both IFN‐γ‐mediated transcription and the subsequent refractory state. We previously demonstrated that IFN‐γ treatment resulted in coordinate protein occupancy in vivo of two hMCP‐1 promoter elements, a gamma‐activated site (GAS) and a GC‐rich element. During the refractory state, IFN‐γ treatment failed to induce protection of either the hMCP‐1 GAS element or the GC box. These results furnish insight into the expression of hMCP‐1 during CNS inflammation and provide the first delineation of an IFN‐γ‐induced transcriptional refractory state.—Zhou, Z.‐H. L., Han, Y., Wei, T., Aras, S., Chaturvedi, P., Tyler, S., Rani, M. R. S., Ransohoff, R. M. Regulation of monocyte chemoattractant protein (MCP)‐1 transcription by interferon‐gamma (IFN‐γ) in human astrocytoma cells: postinduction refractory state of the gene, governed by its upstream elements. FASEB J. 15, 383‐392 (2001)

Assessment of air pollution genotoxicity by RAPD in Evernia prunastri L. Ach. from around iron-steel factory in Karabük, Turkey

Lichens are widely used in biomonitoring studies of air pollution, either as bioindicators of air quality or as bioaccumulators of atmospheric deposition. Over the past decade, several molecular techniques have been developed to provide information on diversity, genotoxicology, genetic relationships, etc. The heavy metal contents of Evernia prunastri samples were determined by atomic absorption spectrometry. The Random Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR) method was used to describe the pattern of DNA band variation in the samples influenced by the environmental pollution. The study was designed to describe the level of pollution in an area contaminated with smoke and waste from an iron-steel factory, and to reveal the level of potential genotoxic agents around this source of pollution. The study also examined the suitability of the lichen samples for the detection of genotoxicity.

Characterization of stress induced by copper and zinc on cucumber (Cucumis sativus L.) seedlings by means of molecular and population parameters.

Contamination of plants with heavy metals could result in damage in DNA, such as mutations and cross-links with proteins. These altered DNA profiles may become visible in changes such as the appearance of a new band, or loss of an existing band, in the random amplified polymorphic DNA (RAPD) assay. In this study, various concentrations of copper and zinc salts were applied to cucumber seedlings during germination. Results displayed abnormalities in germination and also changes in root elongation, dry weight and total soluble protein level. All treatment concentrations (40, 80, 160, 240, 320, and 640mg/L) used in the study caused a decrease/delay in germination of the cucumbers to different extents. Inhibition or activation of root elongation was considered to be the first effect of metal toxicity in the tested plants. Application of the metal salts and the combined solutions on cucumber (Cucumis sativus L.) seedlings revealed similar consequences for total soluble protein level, dry weight and ultimately in inhibitory rates as well. The data obtained from RAPD band-profiles and genomic template stability (GTS) showed results that were consistent with the population parameters. In this regard, we conclude that molecular marker assays can be applied in combination with population parameters to measure genotoxic effects of heavy metals on plants.

Relationships among lipid peroxidation, SOD enzyme activity, and SOD gene expression profile in Lycopersicum esculentum L. exposed to cold stress

The current study was designed to evaluate lipid peroxidation (via malondialdehyde) levels, the superoxide dismutase (SOD) gene expression profile, and SOD enzyme activity in tomato plants (Lycopersicum esculentum L.) subjected to different time periods of cold stress (control, 2, 4, 6, 8, and 10 days). Results revealed that maximum lipid peroxidation occurred in plants exposed to cold stress for 10 days, and SOD enzyme activity gradually increased with increasing exposure to cold stress. The level of mRNA increased within 4 days of cold treatment. After this period, the level tended to decrease and reached a minimum by the eighth day. A complex gene expression profile was determined, which was not statistically significant until the eighth day. At the 10th day of cold treatment, the mRNA level of SOD increased and changes between the 8th and 10th day were found to be statistically significant at the P < 0.05 level. These results suggest that the SOD gene and enzyme play a key role in resistance to cold stress conditions in tomato plants.

Heavy Metal Accumulation in Pseudevernia furfuracea (L.) Zopf from the Karabük Iron-Steel Factory in Karabük, Turkey

Pseudevernia furfuracea (L.) Zopf lichen specimens were collected every 5 km starting from around an iron-steel factory located in the central area of Karabük province, up to Yenice Forest. Zn, Cu, Mn, Fe, Pb, Ni, Cd, Cr contents were analyzed in the samples collected from polluted and unpolluted areas. A Pseudevernia furfuracea (L.) Zopf sample from Yenice Forest was used as a control. The reason for this choise was the abundance of species diversity, and therefore sample collection might cause a very low impact on natural population density. The forest is among the 100 forested areas that must be urgently taken under protection according to WWF (World Wildlife Fund) researches. Results of the current study manifested significant variations among the contents of these elements between stations. As expected, the pollution sources, such as iron-steel factory, roads and railroads, industry, heavy traffic, and waste treatment plants, have major impact on the heavy metal accumulation in P. furfuracea (L.) Zopf, and, in accordance to their location, samples 8 and 10 displayed high element accumulation. Surprisingly, although Yenice Forest is under protection, results of our study showed that the region is becoming polluted by the infl uence of many pollution sources in the area. The present study also confirms the efficient metal accumulation capacity of lichens

Comparative Genotoxicity Analysis of Heavy Metal Contamination in Higher Plants

Heavy metal pollution basically results from natural sources like volcanic eruptions, weathering of rocks and anthropogenic sources like mining. These activities are significantly increased in the past few decades as a result of burning of fossil fuels, industrial activities, automotive emissions, use of metal-enriched materials, mining, farm manures, wastewater irrigation, sewage sludge, pesticide usage, industrial and domestic wastes and many other factors. Heavy metals may enter the food chain as a result of their uptake by edible plants, thus, the determination of heavy metals in environmental samples is very important. For screening and monitoring the impacts of heavy metals, higher plants which provide useful genetic system, have been used as a biomonitor/bioindicator of cytogenetic and mutagenic effects (Constantin & Owens, 1982; Grant, 1994, Kachenko et al. 2004; Alirzaveya et al. 2006).