Comparison of some antioxidant properties of plant extracts from Origanum vulgare,Salvia officinalis,Eleutherococcus senticosus and Stevia rebaudiana

Phenolic compounds from plants are known for their antioxidant properties and have been proposed as therapeutic agents to counteract oxidative stress. However, under normal circumstances, the body only receives a very small amount of these substances in the diet. We have investigated the effect of extracts from known and frequently used plants as part of diet, food seasoning, medicinal tea, and sweetener at different concentrations on the ability to scavenge free radicals, to affect antioxidant enzymes, and finally in the survival of cancer cell lines. We found extract concentrations of about 100 μg.ml^?1 more indicative in the assessment of all parameters investigated. Ginseng possessed a very good ability to scavenge superoxide and hydroxyl radicals, while stevia also manifested significant effects against hydroxyl radicals. Both extracts also showed NO decomposition ability. The antioxidant defense system against the excessive production of radicals in mitochondria was sufficient. In contrast, the range of operating concentrations for sage and oregano mainly presented no significant effects against reactive oxygen and nitrogen species. Taken together with the significantly reduced activity of glutathione peroxidase, this led to the depletion of glutathione. The demonstrated modulation of redox state capability was sufficient to affect the viability of all tested cancer cell lines, but especially A-549, CEM and HeLa by oregano extract. Results support the promising role of the tested extracts as a source of compounds for further in vivo studies with the ability to powerfully interfere with or modify the redox state of cells according to the type of disease, which is expected to be associated with oxidative stress.     

Improvement of gamma-amino butyric acid production by an overexpression of glutamate decarboxylase from Pyrococcus horikoshii in Escherichia coli

In this study, the effect of glutamate decarboxylase from Pyrococcus horikoshii on gamma-aminobutyric acid (GABA) production was investigated in Escherichia coli for the first time. E. coli with overexpressed P. horikoshii glutamate decarboxylase was cultured at various pH levels and temperatures to determine the optimum conditions for GABA production. The highest final GABA concentration, 5.07 g/L, was obtained from 10 g/L of monosodium glutamate (MSG) with a GABA yield of 83% at 30°C and pH 3.5. When P. horikoshii glutamate decarboxylase was introduced into a GABA aminotransferase knock-out E. coli XBT strains, 5.69 g/L of GABA was produced with a GABA yield of 93%.     

Dysregulation of Wnt inhibitory factor 1 (Wif1) expression resulted in aberrant Wnt-β-catenin signaling and cell death of the cloaca endoderm,and anorectal malformations

In mammalian urorectal development, the urorectal septum (urs) descends from the ventral body wall to the cloaca membrane (cm) to partition the cloaca into urogenital sinus and rectum. Defective urs growth results in human congenital anorectal malformations (ARMs), and their pathogenic mechanisms are unclear. Recent studies only focused on the importance of urs mesenchyme proliferation, which is induced by endoderm-derived Sonic Hedgehog (Shh). Here, we showed that the programmed cell death of the apical urs and proximal cm endoderm is particularly crucial for the growth of urs during septation. The apoptotic endoderm was closely associated with the tempo-spatial expression of Wnt inhibitory factor 1 (Wif1), which is an inhibitor of Wnt-β-catenin signaling. In Wif1^lacZ/lacZ mutant mice and cultured urorectum with exogenous Wif1, cloaca septation was defective with undescended urs and hypospadias-like phenotypes, and such septation defects were also observed in Shh^−/− mutants and in endodermal β-catenin gain-of-function (GOF) mutants. In addition, Wif1 and Shh were expressed in a complementary manner in the cloaca endoderm, and Wif1 was ectopically expressed in the urs and cm associated with excessive endodermal apoptosis and septation defects in Shh^−/− mutants. Furthermore, apoptotic cells were markedly reduced in the endodermal β-catenin GOF mutant embryos, which counteracted the inhibitory effects of Wif1. Taken altogether, these data suggest that regulated expression of Wif1 is critical for the growth of the urs during cloaca septation. Hence, Wif1 governs cell apoptosis of urs endoderm by repressing β-catenin signal, which may facilitate the protrusion of the underlying proliferating mesenchymal cells towards the cm for cloaca septation. Dysregulation of this endodermal Shh-Wif1-β-catenin signaling axis contributes to ARM pathogenesis.Urorectal development starts at gestational week-4 in humans and at E10.5 in mice, and is divided into three phases: genital tubercle (GT) outgrowth, cloaca septation and urethra tubularization. GT outgrowth is initiated with a paired genital swellings on either side of the cloaca (caudal end of the rectum), which fuse medially to form the future external genitalia. Cloaca septation is the partitioning of the cloaca by urorectal septum (urs) into the rectum and urogenital sinuses (ugs). Urorectal development is indistinguishable in male and female embryos before urethra tubularization. Defective urorectal development results in a number of congenital anomalies frequently accompany with deficient excretory and copulatory functions, greatly influencing the patients'' quality of life. In particular, the anorectal malformations (ARMs) are the most common urorectal defects and affect boys and girls with an incidence of 2–5 in 10 000 live births,^{1, 2} but its pathogenic mechanisms are poorly understood. ARM phenotypes are also partly included in several diseases, such as Currarino syndromes, Townes Brocks Syndromes and VACTERL complex, and these patients display other anomalies including anal fistula, sacral malformations and renal malformations.^{3, 4, 5, 6}During cloaca septation, the urs, an endoderm-lined mesenchyme, elongates from the ventral body wall to the cloaca membrane (cm) (Figure 1a). The proximal cm is regressing when the apical urs endoderm approximates to the cm leading to the formation of the urethral duct (ud) and the anal opening, which signifies the completion of cloaca septation (Figure 1b). After septation, the cm regression associated with the proximal-to-distal urs elongation coupled with fusion of the preputial fold mesoderm along the ventral midline of the tubercle leads to the formation of urethral opening at the distal end of the GT (Figure 1c).Open in a separate windowFigure 1Descent of the urorectal septum and regression of the cloaca membrane during cloaca septation. Schematic diagram of cloaca development showing the descent of urs and regression of proximal cm leads to anal opening after cloaca septation (a–c). Normal cloaca development in rat embryos from E14.5 to E16.5 (a′–c′). Rat treated with ethylenethiourea displayed persistent cloaca at E14.5 (d; n=12), hypospadias-like phenotypes with incomplete cloaca septation at E15.5 (e; n=15) and E16.5 (f); n=9). Scale bar: 50 μmDeletion of Shh, Wnt5a or Bmp7 in mouse embryos resulted in reduced proliferation of the urs mesenchyme, incomplete urs elongation and septation defects.^{7, 8, 9, 10} In addition, the cm was either disintegrated or remained intact in these null embryos, giving rise to hypospadias-like or persistent cloaca phenotypes (Figure 1). Nonetheless, deletion of these genes not only leads to a decrease in cell proliferation but also affect cell apoptosis.^{11, 12}Shh and Wnt signaling are indispensable for the septation process. Shh is expressed in the cloaca endoderm and mediates the proliferation of urs mesenchyme through Gli2. Shh and Gli2 mutants displayed persistent cloaca. However, deletion of Shh eradicated the cm, exposing the unseptated cloaca exteriorly, whereas the cm remained intact in the loss of Gli2.^{7, 13} Wnt5a is crucial for urorectal development and regulates the outgrowth of GT.^{8, 10} Studies suggest that Wnt-β-catenin signaling activity is tightly regulated in urorectal development.^{14, 15} Furthermore, β-catenin expression was downregulated in Shh mutants.¹⁶ Constitutive activated β-catenin in Shh null background can partially rescue defective development of the GT and the cm.^{11, 16} All these indicated that Wnt-β-catenin signaling functions downstream of Shh. How Wnt signaling is regulated during cloaca septation has not been investigated.Wif1 (Wnt inhibitory factor 1) is a secreted Wnt inhibitor, which exerts its inhibitory effect on Wnt signaling by binding directly to Wnt ligands and by preventing the ligands from binding to their cell surface receptors. The presence of Wif1 leads to β-catenin degradation, thereby turning off Wnt-β-catenin signaling.¹⁷ Most of the recent studies focused on the impact of epigenetic silencing of Wif1 in various carcinomas,¹⁸ and restoration of Wif1 activity in cancer cells induced apoptosis of malignant cancers.¹⁹ By contrast, reports on the regulatory functions of Wif1 in embryonic development are limited. Previous study suggested that Wif1 has high affinity to Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt9a and Wnt11;²⁰ and Wif1 regulated chondrogenesis in cartilage-mesenchyme interfaces via the inhibition of Wnt3a-mediated mesenchyme growth in embryos.²¹ A recent study revealed that expression of Wif1 is androgen responsive in prostate bud formation. Loss of Wif1 in prostate glands induced ectopic expression of other secretory Wnt inhibitors to compensate for the loss of Wif1 activity in these mutants.²² Furthermore, Smad1 directly targets the Wif1 promoter and controls Wif1 gene expression in lung epithelial cell development.²³ Taken all these indicated that Wnt-β-catenin signaling is precisely regulated in a number of embryonic developmental processes, and Wif1 modulates the Wnt-β-catenin signaling activity.In this study, we discovered that the finely regulated expression of Wif1 is crucial for cloaca septation, and dysregulated Wif1 expression caused septation defects. Shh and Wif1 were expressed in a complementary manner at the cloaca endoderm, and deletion of Shh induced ectopic Wif1 expression, associated with excessive endoderm apoptosis and septation defects. Comparable septation defects were observed in Wif1^lacZ/lacZ mutant mice, in cultured urorectum with exogenous Wif1, in Shh^−/− mutants and in endodermal β-catenin GOF mutants. In conclusion, this study suggests that Wif1 regulates endodermal cell apoptosis by mediating and regulating Shh-Wnt-β-catenin signaling, thus having an essential role during urorectal development.     

Mathematical modelling of arsenic transport,distribution and detoxification processes in yeast

Arsenic has a dual role as causative and curative agent of human disease. Therefore, there is considerable interest in elucidating arsenic toxicity and detoxification mechanisms. By an ensemble modelling approach, we identified a best parsimonious mathematical model which recapitulates and predicts intracellular arsenic dynamics for different conditions and mutants, thereby providing novel insights into arsenic toxicity and detoxification mechanisms in yeast, which could partly be confirmed experimentally by dedicated experiments. Specifically, our analyses suggest that: (i) arsenic is mainly protein‐bound during short‐term (acute) exposure, whereas glutathione‐conjugated arsenic dominates during long‐term (chronic) exposure, (ii) arsenic is not stably retained, but can leave the vacuole via an export mechanism, and (iii) Fps1 is controlled by Hog1‐dependent and Hog1‐independent mechanisms during arsenite stress. Our results challenge glutathione depletion as a key mechanism for arsenic toxicity and instead suggest that (iv) increased glutathione biosynthesis protects the proteome against the damaging effects of arsenic and that (v) widespread protein inactivation contributes to the toxicity of this metalloid. Our work in yeast may prove useful to elucidate similar mechanisms in higher eukaryotes and have implications for the use of arsenic in medical therapy.     

Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic N‐terminal mutations

The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C‐terminal HNH motif. Interestingly, the deletion of four N‐terminal residues (446–449 NColE7 = KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuclease activity, but a detailed analysis of the role of the highly positive and flexible N‐terminus is still missing. Here, we present the study of four mutants, with a decreased activity in the following order: NColE7  >> KGNK > KGNG ～ GGNK > GGNG. At the same time, the folding, the metal‐ion, and the DNA‐binding affinity were unaffected by the mutations as revealed by linear and circular dichroism spectroscopy, isothermal calorimetric titrations, and gel mobility shift experiments. Semiempirical quantum chemical calculations and molecular dynamics simulations revealed that K446, K449, and/or the N‐terminal amino group are able to approach the active centre in the absence of the other positively charged residues. The results suggested a complex role of the N‐terminus in the catalytic process that could be exploited in the design of a controlled nuclease.     

Pyrrolobenzodiazepines (PBDs) Do Not Bind to DNA G-Quadruplexes

The pyrrolo[2,1-c][1,4] benzodiazepines (PBDs) are a family of sequence-selective, minor-groove binding DNA-interactive agents that covalently attach to guanine residues. A recent publication in this journal (Raju et al, PloS One, 2012, 7, 4, e35920) reported that two PBD molecules were observed to bind with high affinity to the telomeric quadruplex of Tetrahymena glaucoma based on Electrospray Ionisation Mass Spectrometry (ESI-MS), Circular Dichroism, UV-Visible and Fluorescence spectroscopy data. This was a surprising result given the close 3-dimensional shape match between the structure of all PBD molecules and the minor groove of duplex DNA, and the completely different 3-dimensional structure of quadruplex DNA. Therefore, we evaluated the interaction of eight PBD molecules of diverse structure with a range of parallel, antiparallel and mixed DNA quadruplexes using DNA Thermal Denaturation, Circular Dichroism and Molecular Dynamics Simulations. Those PBD molecules without large C8-substitutents had an insignificant affinity for the eight quadruplex types, although those with large π-system-containing C8-substituents (as with the compounds evaluated by Raju and co-workers) were found to interact to some extent. Our molecular dynamics simulations support the likelihood that molecules of this type, including those examined by Raju and co-workers, interact with quadruplex DNA through their C8-substituents rather than the PBD moiety itself. It is important for the literature to be clear on this matter, as the mechanism of action of these agents will be under close scrutiny in the near future due to the growing number of PBD-based agents entering the clinic as both single-agents and as components of antibody-drug conjugates (ADCs).     

Quality of Life and Costs in Parkinson's Disease: A Cross Sectional Study in Hungary

Background

Patient reported outcomes and costs of illness are useful to capture some of the multiple effects of a disease and its treatments. Our aim was to assess quality of life (QoL) and costs of Parkinson''s disease (PD) in Hungary, and to analyze their associations.

Methods

A cross-sectional questionnaire survey was conducted in one neurology university clinic. Clinical characteristics, PD related resource utilizations and productivity loss in the past 12 months were recorded; the Hoehn&Yahr (HY) scale, PDQ-39 and EQ-5D questionnaires were applied. Cost calculation was performed from the societal perspective.

Results

110 patients (34.5% female) were involved with mean age of 63.3 (SD = 11.3) and disease duration of 8.2 (SD = 5.8) years. PDQ-39 summary score was 48.1 (SD = 13.4). The average EQ-5D score was 0.59 (SD = 0.28), and was significantly lower than the population norm in age-groups 45–74. The correlation was significant between EQ-5D and PDQ-39 (−0.47, p = 0.000), the HY scale and EQ-5D (−0.3416, p = 0.0008) and PDQ-39 (0.3419, p = 0.0006) scores. The total mean cost was €6030.2 (SD = 6163.0)/patient/year (direct medical 35.7%, direct non-medical 29.4%, indirect cost 34.9%). A one year increase in disease duration and 0.1 decrease of the EQ-5D utility score increase the yearly costs by 8 to 10%, and 7.8%, respectively. The effect of the PDQ-39 score on total cost was not significant.

Conclusions

Disease severity and public health importance of PD are clearly demonstrated by the magnitude of QoL loss. PD-related costs are substantial, but are much lower in Hungary than in Western European countries. Disease duration and EQ-5D score are significant proxy of costs.