Terpene chemodiversity of relict conifers Picea omorika, Pinus heldreichii, and Pinus peuce, endemic to Balkan

Terpenes are often used as ecological and chemotaxonomic markers of plant species, as well as for estimation of geographic variability. Essential oils of relic and Balkan endemic/subendemic conifers, Picea omorika, Pinus heldreichii, and P. peuce, in central part of Balkan Peninsula (Serbia and Montenegro), on the level of terpene classes and common terpene compounds were investigated. In finding terpene combinations, which could show the best diversity between species and their natural populations, several statistical methods were applied. Apart from the content of different terpene classes (P. omorika has the most abundant O-containing monoterpenes and sesquiterpenes; P. heldreichii and P. peuce have the largest abundance of sesquiterpene and monoterpene hydrocarbons, resp.), the species are clearly separated according to terpene profile with 22 common compounds. But, divergences in their populations were established only in combination of several compounds (specific for each species), and they were found to be the results of geomorphologic, climatic, and genetic factors. We found similarities between investigated species and some taxa from literature with respect to terpene composition, possibly due to hybridization and phylogenetic relations. Obtained results are also important regarding to chemotaxonomy, biogeography, phylogeny, and evolution of these taxa.     

Strawberry polyphenols attenuate ethanol-induced gastric lesions in rats by activation of antioxidant enzymes and attenuation of MDA increase

Background and Aim

Free radicals are implicated in the aetiology of gastrointestinal disorders such as gastric ulcer, colorectal cancer and inflammatory bowel disease. Strawberries are common and important fruit due to their high content of essential nutrient and beneficial phytochemicals which seem to have relevant biological activity on human health. In the present study we investigated the antioxidant and protective effects of three strawberry extracts against ethanol-induced gastric mucosa damage in an experimental in vivo model and to test whether strawberry extracts affect antioxidant enzyme activities in gastric mucosa.

Methods/Principal Findings

Strawberry extracts were obtained from Adria, Sveva and Alba cultivars. Total antioxidant capacity and radical scavenging capacity were performed by TEAC, ORAC and electron paramagnetic resonance assays. Identification and quantification of anthocyanins was carried out by HPLC-DAD-MS analyses. Different groups of animals received 40 mg/day/kg body weight of strawberry crude extracts for 10 days. Gastric damage was induced by ethanol. The ulcer index was calculated together with the determination of catalase and SOD activities and MDA contents. Strawberry extracts are rich in anthocyanins and present important antioxidant capacity. Ethanol caused severe gastric damage and strawberry consumption protected against its deleterious role. Antioxidant enzyme activities increased significantly after strawberry extract intake and a concomitantly decrease in gastric lipid peroxidation was found. A significant correlation between total anthocyanin content and percent of inhibition of ulcer index was also found.

Conclusions

Strawberry extracts prevented exogenous ethanol-induced damage to rats'' gastric mucosa. These effects seem to be associated with the antioxidant activity and phenolic content in the extract as well as with the capacity of promoting the action of antioxidant enzymes. A diet rich in strawberries might exert a beneficial effect in the prevention of gastric diseases related to generation of reactive oxygen species.     

Distribution of Heavy Metals and Arsenic in Soils of Belgrade (Serbia and Montenegro) 1

Soils of the urban and suburban area of Belgrade have been hardly studied, especially concerning their concentrations of potentially toxic metals. The present paper is aimed at determining the possible pollution in soils. The total acid soluble concentrations of heavy metals and As in the samples were determined. It was found that they were arranged in the order Zn > Ni > Pb > Cr > Cu > As > Hg > Cd in samples collected in the examined area (the order of the elements is based on their arithmetic mean concentrations). In all the samples collected at 0–10 and 40–50 cm depths from 46 selected sites, the contents of Pb and Zn were lower at the depth 40-50 cm. Using target values given by the Dutch Ministry of Housing, Spatial Planning and Environment, it may be concluded that Belgrade soil can, for the most part, be regarded as unpolluted. Traffic seems to be one of the main sources of these metals, but the influence of other factors cannot be excluded.     

Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide

Apoptosis is a highly regulated and programmed cell breakdown process characterized by numerous changes. Since it is implicated in many pathological as well as physiological processes, it is vital to have reliable methods for detecting cell death. In this study, we compared several methods for detecting apoptosis and necrosis in human leukocytes. Apoptosis was induced either by incubating the cells with various doses of cycloheximide (CHX) or by 312 nm UVB irradiation. The methods used for detecting apoptosis were light microscopy (May Grunwald-Giemsa and trypan blue staining), fluorescence microscopy (acridin orange/ethidium bromide and annexin V/propidium iodide staining) and agarose gel electrophoresis of fragmented genomic DNA. Our study showed that CHX-induced apoptosis in cultured peripheral blood mononuclear cells but had no effect on apoptosis in polymorphonuclear cells, so its effect depends on cell type. Evaluation and comparison of the methods for detecting apoptosis showed the following. A Giemsa-stained cytospin allows the main morphological characteristics of necrotic and apoptotic death to be recognized. Trypan blue staining, widely used for estimating cell viability, is valueless for detecting apoptosis. Both fluorescence methods provided reliable and reproducible results and distinguished clearly between subpopulations of apoptotic cells, and were closely intercorrelated. Although applicable to a wide spectrum of cell types, agar electrophoresis of extracted DNA cannot be applied to all cell types and apoptotic conditions. Generally, microscopic examination of acridin orange/ethidium bromide stained cells can be recommended as the most reliable of the methods tested.     

High Diversity of Indigenous Populations of Dalmatian Sage (Salvia officinalis L.) in Essential‐Oil Composition

Essential oils of 25 indigenous populations of Dalmatian sage (Salvia officinalis L.) that represent nearly half of native distribution area of the species were analyzed. Plantlets collected from wild populations were grown in the same field under the same environmental conditions and then sampled for essential‐oil analysis. The yield of essential oil ranged from 1.93 to 3.70% with average of 2.83%. Among the 62 compounds detected, eight (cis‐thujone, camphor, trans‐thujone, 1,8‐cineole, β‐pinene, camphene, borneol, and bornyl acetate) formed 78.13–87.33% of essential oils of individual populations. Strong positive correlations were observed between camphor and β‐pinene, β‐pinene and borneol, as well as between borneol and bornyl acetate. The strongest negative correlation was detected between camphor and trans‐thujone. Principal component analysis (PCA) on the basis of eight main compounds showed that first main component separated populations with high thujone content, from those rich in camphor, while the second component separated populations rich in cis‐thujone from those rich in trans‐thujone. Cluster analysis (CA) led to the identification of three chemotypes of S. officinalis populations: cis‐thujone; trans‐tujone, and camphor/β‐pinene/borneol/bornyl acetate. We propose that differences in essential oils of 25 populations are mostly genetically controlled, since potential environmental factors were controlled in this study.     

Environment-related variations of the composition of the essential oils of rosemary (Rosmarinus officinalis L.) in the Balkan Penninsula

Composition of the essential oils of Rosmarinus officinalis of ten populations from the Balkan Peninsula were determined by GC/FID and GC/MS. The main constituents were 1,8-cineole, camphor, α-pinene, and borneol. Multivariate statistical analysis (UPGMA cluster analysis and principal-component analysis (PCA)) revealed two major types of rosemary oil, i.e., 1,8-cineole and camphor-type, and two intermediate types, i.e., camphor/1,8-cineole/borneol type and 1,8-cineole/camphor type. The regression analyses (simple linear regression and stepwise multiple regression) have shown that, with respect to basic geographic, orographic, and 19 bioclimatic characteristics of each population, bioclimatic factor temperature of habitat represented the dominant abiogenetic factor, which, in chemical sense, led to differentiation of populations in the studied region. Also, the regression analysis have shown that some constituents of essential oils are independent of any single bioclimatic factors. However, some constituents display statistically significant correlations with some abiotic factors.     

Somatostatin-14 influences pituitary–ovarian axis in peripubertal rats

The effects of multiple somatostatin (SRIH-14) administration on the pituitary-ovarian axis were examined in peripubertal rats. Female Wistar rats received subcutaneously, two daily doses of 20 mug SRIH-14 per 100 g body weight (b.w.) for five consecutive days (from the 33rd to the 37th day of life). Follicle-stimulating (FSH), luteinizing (LH) and somatotropic (GH) cells were examined by the peroxidase-anti-peroxidase immunocytochemical method. Changes in cell volumes, volume densities and number per unit area (mm(2)) of FSH-, LH- and GH-immunoreactive cells were evaluated by stereology and morphometry. Serum FSH and LH levels were determined by RIA. Ovaries were analyzed by simple point counting of follicles. The volumes and volume densities of FSH-, LH- and GH-immunoreactive cells were significantly decreased while their numbers per mm(2) remained unchanged. SRIH-14 induced a significant decrease in serum FSH and LH levels. In the ovary, SRIH-14 induced an increase in the number of primordial follicles, followed by a reduction in the number of small healthy growing follicles and absence of preovulatory follicles. The number of atretic follicles was unchanged. We concluded that treatment with SRIH-14 during the peripubertal period markedly inhibited pituitary FSH, LH and GH cells. In the ovary, SRIH-14 acted by inhibiting folliculogenesis without affecting atretic processes.     

Cerebellum Lipids in Rats After Chronic Ethanol Treatment

Eighteen male Wistar rats weighing approximately 200 g were divided into three groups of six animals each. The experimental animals were maintained on nutritionally complete diets in which ethanol comprised 45% of the available energy. Control animals were pair-fed an equivalent diet in which sucrose was substituted isocalorically for ethanol. An additional control group received unlimited access to standard pelleted laboratory food and water. The investigations were carried out over 24 weeks. The effects on phospholipid, monogalactosyl glycolipid, and ganglioside composition after 24 weeks of feeding 43% alcohol were studied. There is abundant evidence that the changes in the cerebellum membrane phospholipids (phosphatidylethanolamine and phosphatidylcholine), gangliosides (GT1b), and myelin lipids (phosphatidylserine, sphingophospholipid, phosphatidylinositol, cerebrosides with hydroxy fatty acids, sulfoglycolipids, and monosialoganglioside GM1) occur as a result of chronic ethanol treatment.     

The impact of autophagy on cell death modalities in CRL-5876 lung adenocarcinoma cells after their exposure to γ-rays and/or erlotinib

In most patients with lung cancer radiation treatment is used either as single agent or in combination with radiosensitizing drugs. However, the mechanisms underlying combined therapy and its impact on different modes of cell death have not yet been fully elucidated. We aimed to examine effects of single and combined treatments with γ-rays and erlotinib on radioresistant CRL-5876 human lung adenocarcinoma cells with particular emphasis on cell death. CRL-5876 cells were treated with γ-rays and/or erlotinib and changes in cell cycle, DNA repair dynamics, ultrastructure, nuclear morphology and protein expression were monitored at different time points. To reveal the relationship between types of cell death that arise after these treatments, autophagy was blocked with chloroquine. We found that higher dose of γ-rays causes G2/M arrest while adding of erlotinib to this treatment decreases the number of cells in S phase. Impact of erlotinib on kinetics of disappearance of irradiation-induced DNA double strand breaks is reflected in the increase of residual γ-H2AX foci after 24 h. γ-rays provoke cytoprotective autophagy which precedes development of senescence. Erlotinib predominantly induces apoptosis and enlarges the number of apoptotic cells in the irradiated CRL-5876 cells. Chloroquine improved cytotoxicity induced by radiation and erlotinib, increased apoptosis and decreased senescence in the CRL-5876 cells. The results obtained on CRL-5876 cells indicate significant radiosensitizing effect of erlotinib and suggest that chloroquine in the combination with the above treatments may have an additional antitumor effect in lung adenocarcinoma.