Phytochemical and flow cytometric analyses of Devil’s claw cell cultures

A cell suspension culture of Devil’s claw (Harpagophytum procumbens), a South African plant with high medicinal value, cultivated under submerged conditions showed stable growth and accumulated high amounts of biomass (18.2 g l⁻¹). Flow cytometry analyses of the suspension’s cell cycle kinetics showed that proportions of cells in G₀/G₁ and S phases varied insignificantly (between 69–76% and 9–13%, respectively) during the cultivation, while the proportion of G₂/M-phase cells increased until day 8 of cultivation, when the exponential phase of cell growth ended. Metabolite production in the culture was studied through simultaneous determination of three bioactive phenylethanoid glycosides (verbascoside, β-OH-verbascoside and leucosceptoside A) by high performance liquid chromatography. It was found that suspended Devil’s claw cells accumulated mainly verbascoside (517.3 mg l⁻¹), followed by leucosceptoside A (107.1 mg l⁻¹) and β-OH-verbascoside (80.3 mg l⁻¹). In addition, several fatty acids and β-sitosterol were identified in the cell suspension by gas chromatographic-mass spectrometry analysis. Comparison of the results with previously acquired data for Harpagophytum procumbens transformed roots indicate that cell suspensions cultures are more promising as potential commercial sources of metabolites such as phenylethanoid glycosides.     

Recruitment of human cytomegalovirus immediate-early 2 protein onto parental viral genomes in association with ND10 in live-infected cells

The human cytomegalovirus (HCMV) immediate-early 2 (IE2) transactivator has previously been shown to form intranuclear, dot-like accumulations in association with subnuclear structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. We recently observed that IE2 can form dot-like structures even after infection of PML knockdown cells, which lack genuine ND10. To further analyze the determinants of IE2 subnuclear localization, a recombinant HCMV expressing IE2 fused to the enhanced green fluorescent protein was constructed. We infected primary human fibroblasts expressing Sp100 fused to the autofluorescent protein mCherry while performing live-cell imaging experiments. These experiments revealed a very dynamic association of IE2 dots with ND10 structures during the first hours postinfection: juxtaposed structures rapidly fused to precise co-localizations, followed by segregation, and finally, the dispersal of ND10 accumulations. Furthermore, by infecting PML knockdown cells we determined that the number of IE2 accumulations was dependent on the multiplicity of infection. Since time-lapse microscopy in live-infected cells revealed that IE2 foci developed into viral replication compartments, we hypothesized that viral DNA could act as a determinant of IE2 accumulations. Direct evidence that IE2 molecules are associated with viral DNA early after HCMV infection was obtained using fluorescence in situ hybridization. Finally, a DNA-binding-deficient IE2 mutant could no longer be recruited into viral replication centers, suggesting that the association of IE2 with viral DNA is mediated by a direct DNA contact. Thus, we identified viral DNA as an important determinant of IE2 subnuclear localization, which suggests that the formation of a virus-induced nucleoprotein complex and its spatial organization is likely to be critical at the early stages of a lytic infection.     

Targeted Deposition of Antibodies on a Multiplex CMOS Microarray and Optimization of a Sensitive Immunoassay Using Electrochemical Detection

Background

The CombiMatrix ElectraSense® microarray is a highly multiplex, complementary metal oxide semiconductor with 12,544 electrodes that are individually addressable. This platform is commercially available as a custom DNA microarray; and, in this configuration, it has also been used to tether antibodies (Abs) specifically on electrodes using complementary DNA sequences conjugated to the Abs.

Methodology/Principal Findings

An empirical method is described for developing and optimizing immunoassays on the CombiMatrix ElectraSense® microarray based upon targeted deposition of polypyrrole (Ppy) and capture Ab. This process was automated using instrumentation that can selectively apply a potential or current to individual electrodes and also measure current generated at the electrodes by an enzyme-enhanced electrochemical (ECD) reaction. By designating groups of electrodes on the array for different Ppy deposition conditions, we determined that the sensitivity and specificity of a sandwich immunoassay for staphylococcal enterotoxin B (SEB) is influenced by the application of different voltages or currents and the application time. The sandwich immunoassay used a capture Ab adsorbed to the Ppy and a reporter Ab labeled for fluorescence detection or ECD, and results from these methods of detection were different.

Conclusions/Significance

Using Ppy deposition conditions for optimum results, the lower limit of detection for SEB using the ECD assay was between 0.003 and 0.01 pg/ml, which represents an order of magnitude improvement over a conventional enzyme-linked immunosorbant assay. In the absence of understanding the variables and complexities that affect assay performance, this highly multiplexed electrode array provided a rapid, high throughput, and empirical approach for developing a sensitive immunoassay.     

Body mass index, waist circumference and waist-to-hip ratio: which anthropometric indicator is better predictor for the hypertension development in women population of the island Cres

The aim of this research was to investigate the prevalence of obesity and high blood pressure and to prove which of three anthropometric indicators of obesity - waist circumference, body mass index (BMI) waist-to-hip ratio - is better predictor for the development of hypertension in women population of the island of Cres. We approached separately groups of women with measured high blood pressure and with previously diagnosed. The research was preformed within the research project "Genetic and biomedical characteristics of the population of the island of Cres". This was the cross sectional study and data were obtained on the sample of 247 females over 18 years old that voluntarily participated in this study. In our study group the prevalence of overweight was 39.0%, obesity 27.5%, increased waist circumference was present in 69.4% while increased blood pressure was found in 53.0% examinees. Our results indicate that age, BMI, impaired glucose concentration and serum cholesterol could be considered as predictors for the development of arterial hypertension, whether measured or previously diagnosed.     

Plasmacytoid dendritic cells limit viral replication, pulmonary inflammation, and airway hyperresponsiveness in respiratory syncytial virus infection

Plasmacytoid dendritic cells (pDC), as major producers of IFN-alpha, are thought not only to be pivotal in antiviral immunity, but also to limit allergic inflammation. In this study, we delineate the role of pDC in a mouse model of respiratory syncytial virus (RSV)-induced airway inflammation. Bone marrow-derived pDC generated high levels of IFN-alpha upon RSV infection, and the percentage of pDC expressing MHC class II and maturation-associated costimulatory molecules was increased. However, their weak Ag-presenting capacity was not enhanced. Furthermore, pDC induced marked levels of IL-10 in T cell cultures irrespective of infection. In vivo, numbers of pDC in the lung increased early after RSV infection and remained elevated throughout the inflammatory phase and the resolution phase of infection. Depletion of pDC resulted in increases in peak RSV titers, pulmonary inflammation, and airway hyperresponsiveness. In contrast, adoptive transfer of activated pDC to the airways reduced RSV copy numbers. In conclusion, RSV infection induces activation of murine pDC with robust IFN-alpha production, limiting replication and accelerating elimination of RSV. In addition to this innate response, pDC also may play an immune regulatory role in reducing pulmonary inflammation and inhibiting the development of airway hyperresponsiveness.     

Intraspecific genetic diversity of Pyrenophora tritici-repentis (Died.) Drechs. (Drechslera tritici-repentis[Died.] Shoem.) detected by random amplified polymorphic DNA assays

Abstract

Random amplified polymorphic DNAs (RAPDs) were used to study the genetic variation of Pyrenophora tritici-repentis isolates causing wheat tan spot. Two independent experiments were conducted in 2002 – 2003. In 2002, 40 isolates collected in Russia (Krasnodar region, Bashkiria), Germany, and the Czech Republic were studied and 35 unique RAPD genotypes were identified. Most of the genetic variation (72%) was observed within populations and 28% between them. In 2003, 69 new isolates from Russia (Dagestan, North Osetia, Bashkiria), Germany, and the Czech Republic were studied and 47 unique RAPD genotypes were identified. As in 2002, most of the genetic variation (75%) was observed within populations and 25% between them. Total gene diversity in each group ranged from 0.67 – 1.00 for 2002 and was 1.00 for 2003. The average gene diversity was estimated between 0.13 and 0.20 in 2002 and between 0.07 and 0.18 in 2003. A dendrogramme based on genetic distances between isolates illustrates that the variation is distributed on a small scale (0.3 – 4.0%). Estimated F_ST values and clustering of isolates on dendrogrammes suggest that groups of isolates from Bashkiria and groups of isolates from Dagestan and North Osetia are separated from others and may be considered as different geographical populations. No clear differentiation between isolates from other sites was revealed.     

Cytotoxic activities of Hypericum perforatum L. extracts against 2D and 3D cancer cell models

Cytotechnology - Six extracts were obtained from plant species Hypericum perforatum L., collected at Samsun in Turkey. The aim of this study was to examine the mechanisms of the anticancer activity...