Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Extramedullary hematopoiesis in a case of benign mixed mammary tumor in a female dog: cytological and histopathological assessment

Backgroud  

Extramedullary hematopoiesis (EMH) is defined as the presence of hematopoietic stem cells such as erythroid and myeloid lineage plus megakaryocytes in extramedullary sites like liver, spleen and lymph nodes and is usually associated with either bone marrow or hematological disorders. Mammary EMH is a rare condition either in human and veterinary medicine and can be associated with benign mixed mammary tumors, similarly to that described in this case.     

Stem/progenitor and intermediate cell types and the origin of human prostate cancer

Theories of cell lineage in human prostatic epithelium, based on protein expression, propose that basal and luminal cells: 1) are either independently capable of self-renewal or 2) arise from stem cells expressing a full spectrum of proteins (p63, cytokeratins CK5/14, CK8/18, and glutathione-S-transferase-pi [GST-pi]) similar to cells of the embryonic urogenital sinus (UGS). Such embryonic-like stem cells are thought to give rise to mature basal cells and secretory luminal cells. By single cell cloning of an immortalized, normal human prostate-derived, non-tumorigenic RWPE-1 cell line, we isolated and characterized two epithelial cell types, WPE-stem and WPE-int. WPE-stem cells show: i) strong, sixfold greater nuclear expression of p63; ii) nearly twofold greater expression of CK14; iii) threefold less CK18, and iv) low androgen receptor (AR) expression as compared with WPE-int cells. WPE-stem cells are androgen-independent for growth and survival. WPE-int cells express very low p63 and CK5/14, and high CK18. WPE-int cells are androgen-independent for growth and survival but are highly responsive as shown by androgen induction of AR and prostate specific antigen (PSA). Compared with WPE-int cells, WPE-stem cells are smaller and show more rapid growth. WPE-stem cells can grow in an anchorage-independent manner in agar with 4.5-fold greater cloning efficiency and as free floating "prostaspheres" in liquid medium; and express over 40-fold higher matrix metalloproteinase-2 activity. These results indicate that WPE-stem cells express several features characteristic of stem/progenitor cells present in the UGS and in adult prostatic epithelium. In contrast, WPE-int cells have an intermediate, committed phenotype on the pathway to luminal cell differentiation. We propose that in normal prostatic epithelium, cells exist at many stages in a continuum of differentiation progressing from stem cells to definitive basal and luminal cells. Establishment and characterization of clones of human prostatic epithelial cells provide novel models for determining cell lineages, the origin of prostate cancer, and for developing new strategies for tumor prevention and treatment.     

Agglomerates of aberrant DNA methylation are associated with toxicant-induced malignant transformation

Epigenetic dysfunction is a known contributor in carcinogenesis, and is emerging as a mechanism involved in toxicant-induced malignant transformation for environmental carcinogens such as arsenicals or cadmium. In addition to aberrant DNA methylation of single genes, another manifestation of epigenetic dysfunction in cancer is agglomerative DNA methylation, which can participate in long-range epigenetic silencing that targets many neighboring genes and has been shown to occur in several types of clinical cancers. Using in vitro model systems of toxicant-induced malignant transformation, we found hundreds of aberrant DNA methylation events that emerge during malignant transformation, some of which occur in an agglomerative fashion. In an arsenite-transformed prostate epithelial cell line, the protocadherin (PCDH), HOXC and HOXD gene family clusters are targeted for agglomerative DNA methylation. The agglomerative DNA methylation changes induced by arsenicals appear to be common and clinically relevant events, since they occur in other human cancer cell lines and models of malignant transformation, as well as clinical cancer specimens. Aberrant DNA methylation in general occurred more often within histone H3 lysine-27 trimethylation stem cell domains. We found a striking association between enrichment of histone H3 lysine-9 trimethylation stem cell domains and toxicant-induced agglomerative DNA methylation, suggesting these epigenetic modifications may become aberrantly linked during malignant transformation. In summary, we found an association between toxicant-induced malignant transformation and agglomerative DNA methylation, which lends further support to the hypothesis that epigenetic dysfunction plays an important role in toxicant-induced malignant transformation.     

Simultaneous determination of flavonoids and phenylethanoids in the flowers of Verbascum densiflorum and V. phlomoides by high‐performance liquid chromatography

Introduction – Mullein (Verbascum) flowers are highly valued herbal drugs used in the treatment of inflammation, asthma, spasmodic coughs and other respiratory tract diseases. Their phenolic constituents are considered to be responsible for the anti‐inflammatory and antimicrobial activity of the herb. However, knowledge about the contents of phenolics in flowers is limited and no HPLC method for their analysis is available. Objective – To develop and validate an RP‐HPLC‐UV method for the simultaneous determination of eight flavonoids and two phenylethanoids in the flowers of Verbascum densiflorum and V. phlomoides. Methodology – HPLC separation was accomplished on a C₁₈ Lichrosphere 100 column (5 µm, 250 mm × 4.6 mm, i.d.) with an acetonitrile gradient elution using aqueous 0.5% (w/v) orthophosphoric acid solution containing 1% (v/v) tetrahydrofurane. Results – All the calibration curves showed good linear correlation coefficients (r > 0.997) over the wide test ranges. The relative standard deviation of the method was less than 3.4% for intra‐ and inter‐day assays, and the average recoveries were between 93.5 and 101.9%. High sensitivity was demonstrated with detection limits of 0.062–0.083 µg/mL for flavonoid aglycones, 0.156–0.336 µg/mL for flavonoid glycosides and 0.390–0.555 µg/mL for phenylethanoids. The flower samples of V. phlomoides were found to contain high levels of diosmin and tamarixetin 7‐rutinoside (2.327–2.392% of dry weight), whereas verbascoside (0.688–0.742% of dry weight) and luteolin 7‐glucoside (0.204–0.279% of dry weight) dominated in the V. densiflorum flower. Conclusion – The HPLC method established is appropriate for the quality assurance and the differentiation of V. phlomoides and V. densiflorum samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Cardiomyocyte Ca2+ handling and structure is regulated by degree and duration of mechanical load variation

Cardiac transverse (t)‐tubules are altered during disease and may be regulated by stretch‐sensitive molecules. The relationship between variations in the degree and duration of load and t‐tubule structure remains unknown, as well as its implications for local Ca²⁺‐induced Ca²⁺ release (CICR). Rat hearts were studied after 4 or 8 weeks of moderate mechanical unloading [using heterotopic abdominal heart–lung transplantation (HAHLT)] and 6 or 10 weeks of pressure overloading using thoracic aortic constriction. CICR, cell and t‐tubule structure were assessed using confocal‐microscopy, patch‐clamping and scanning ion conductance microscopy. Moderate unloading was compared with severe unloading [using heart‐only transplantation (HAHT)]. Mechanical unloading reduced cardiomyocyte volume in a time‐dependent manner. Ca²⁺ release synchronicity was reduced at 8 weeks moderate unloading only. Ca²⁺ sparks increased in frequency and duration at 8 weeks of moderate unloading, which also induced t‐tubule disorganization. Overloading increased cardiomyocyte volume and disrupted t‐tubule morphology at 10 weeks but not 6 weeks. Moderate mechanical unloading for 4 weeks had milder effects compared with severe mechanical unloading (37% reduction in cell volume at 4 weeks compared to 56% reduction after severe mechanical unloading) and did not cause depression and delay of the Ca²⁺ transient, increased Ca²⁺ spark frequency or impaired t‐tubule and cell surface structure. These data suggest that variations in chronic mechanical load influence local CICR and t‐tubule structure in a time‐ and degree‐dependent manner, and that physiological states of increased and reduced cell size, without pathological changes are possible.     

The Mathematical Model of the Bcl-2 Family Mediated MOMP Regulation Can Perform a Non-Trivial Pattern Recognition

Interactions between individual members of the B-cell lymphoma 2 (Bcl-2) family of proteins form a regulatory network governing mitochondrial outer membrane permeabilization (MOMP). Bcl-2 family initiated MOMP causes release of the inter-membrane pro-apoptotic proteins to cytosol and creates a cytosolic environment suitable for the executionary phase of apoptosis. We designed the mathematical model of this regulatory network where the synthesis rates of the Bcl-2 family members served as the independent inputs. Using computational simulations, we have then analyzed the response of the model to up-/downregulation of the Bcl-2 proteins. Under several assumptions, and using estimated reaction parameters, a non-linear stimulus-response emerged, whose characteristics are associated with bistability and switch-like behavior. Interestingly, using the principal component analysis (PCA) we have shown that the given model of the Bcl-2 family interactions classifies the random combinations of inputs into two distinct classes, and responds to these by one of the two qualitatively distinct outputs. As we showed, the emergence of this behavior requires specific organization of the interactions between particular Bcl-2 proteins.     

Effects of anticonvulsant agents and ethanol on the complexes of epileptic foci created by applications of bicuculline, strychnine, and penicillin in the rat brain cortex

Anticonvulsive effects of direct agonists of the GABA receptor system, medinal and phenazepam, as well as ethanol, on the complexes of epileptic foci created in the rat brain cortex by applications of various convulsant agents (bicuculline, strychnine, or penicillin) were studied. Ethanol, as well as phenazepam and medinal, were shown to possess high anti-epileptic activity in relation to the complexes. It was shown, using an i.v. infusion technique, that threshold doses of strychnine and bicuculline depend, in a linear and a nonlinear fashion, respectively, on the alcohol doses administered. A specific feature was found in the pharmacolgoical effect of ethanol: anticonvulsive activity of ethanol was pronounced within the postinjection interval from 5 min to 4 h, while within the following interval, from 4 to 24 h, the animals turned into a seizure-ready state. The subseizure phase was observed under the conditions when the penicillin complex was induced, and also following i.v. injections of strychnine and bicuculline.     

Radiation-induced DNA damage as a function of hydration. I. Release of unaltered bases.

The release of unaltered bases from irradiated DNA, hydrated between 2.5 and 32.7 mol of water per mole of nucleotide (gamma), was investigated using HPLC. The objective of this study was to elucidate the yield of the four DNA bases as a function of dose, extent of hydration, and the presence or absence of oxygen. The increase in the yield of radiation-induced free bases was linear with dose up to 90 kGy, except for the DNA with gamma = 2.5, for which the increase was linear only to 10 kGy. The yield of free bases as a function of gamma was not constant in either the absence or the presence of oxygen over the range of hydration examined. For DNA with gamma between 2.5 and 15, the yield of free bases was nearly constant under nitrogen, but decreased under oxygen. However, for DNA with gamma greater than 15, the yield increased rapidly under both nitrogen and oxygen. The yield of free bases was described by a model that depended on two factors: 1) a change in the DNA conformation from a mixture of the A and C conformers in vacuum-dried DNA to predominantly the B conformer in the fully hydrated DNA, and 2) the proximity of the water molecules to the DNA. Irradiation of the inner water molecules (gamma less than 15) was less efficient than irradiation of the outer water molecules (gamma greater than 15), by a factor of approximately 3.3, in forming DNA lesions that resulted in the release of an unaltered base. This factor is similar to the previously published relative efficiency of 2.8 with which hydroxyl radicals and base cations induce DNA strand breaks. Our irradiation results are consistent with the hypothesis that the G value for the first 12-15 water molecules of the DNA hydration layer is the same as the G value for the form of DNA to which it is bound (i.e., the pseudo-C or the B form). Thus we suggest that the release of bases originating from irradiation of the hydration water is obtained predominantly: (1) by charge transfer from the direct ionization of the first 12-15 water molecules of the primary hydration layer and (2) by the attack of hydroxyl radicals generated in the outer, more loosely bound water molecules.     

Structural analogues of diosgenyl saponins: Synthesis and anticancer activity

Saponins display various biological activities including anti-tumor activity. Recently intensive research has been focused on developing saponins for tumor therapies. The diosgenyl saponin dioscin is one of the most common steroidal saponins and exhibits potent anticancer activity in several human cancer cells through apoptosis-inducing pathways. In this paper, we describe the synthesis of several diosgenyl saponin analogues containing either a 2-amino-2-deoxy-β-d-glucopyranosyl residue or an α-l-rhamnopyranosyl-(1→4)-2-amino-2-deoxy-β-d-glucopyranosyl residue with different acyl substituents on the amino group. The cytotoxic activity of these compounds was evaluated in MCF-7 breast cancer cells and HeLa cervical cancer cells. Structure–activity relationship studies show that the disaccharide saponin analogues are in general less active than their corresponding monosaccharide analogues. The incorporation of an aromatic nitro functionality into these saponin analogues does not exhibit significant effect on their cytotoxic activity.