Anti-mitotic and anti-genotoxic effects of Plantago lanceolata aqueous extract on Allium cepa root tip meristem cells

Plantago is the most important genus of Plantaginaceae family and is used in traditional medicine around the world for different purposes. Plantago coronopus L., Plantago major L., Plantago media L. and Plantago lanceolata L. are most commonly used species of Plantago in traditional medicine in Turkey. The main goal of this study was to investigate the eventual anti-mitotic and anti-genotoxic effects of P. lanceolata L. leaf aqueous extracts (15 g/L and 30 g/L) on Allium cepa L. root tip meristem cells which were treated with 0.7% hydrogen peroxide. For this purpose, two different experiments were performed under the same conditions. In the first experiment, Allium cepa onion bulbs were treated with 0.7% H₂O₂ for 1 h. After the H₂O₂ treatment, the onion bulbs were treated with two different concentrations (15 g/L and 30 g/L) of P. lanceolata extracts for 24 h. In the second experiment, A. cepa onion bulbs were treated with two different extract concentrations (15 g/L and 30 g/L) for 24 h and then with 0.7% H₂O₂ for 1 h. The test concentrations were determined according to doses which are recommended in alternative medicinal usage by people. As positive and negative control 0.7% H₂O₂ and tap water was used, respectively. As a result, it was determined that aqueous extracts reduced mitotic index and chromosome aberrations in treatment groups in comparison with controls. These results showed that P. lanceolata aqueous extracts have anti-mitotic and anti-genotoxic effects.     

MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube.

The Mesp1 gene encodes the basic HLH protein MesP1 which is expressed in the mesodermal cell lineage during early gastrulation. Disruption of the Mesp1 gene leads to aberrant heart morphogenesis, resulting in cardia bifida. In order to study the defects in Mesp1-expressing cells during gastrulation and in the specification of mesodermal cell lineages, we introduced a (beta)-galactosidase gene (lacZ) under the control of the Mesp1 promoter by homologous recombination. The early expression pattern revealed by (beta)-gal staining in heterozygous embryos was almost identical to that observed by whole mount in situ hybridization. However, the (beta)-gal activity was retained longer than the mRNA signal, which enabled us to follow cell migration during gastrulation. In heterozygous embryos, the Mesp1-expressing cells migrated out from the primitive streak and were incorporated into the head mesenchyme and heart field. In contrast, Mesp1-expressing cells in the homozygous deficient embryos stayed in the primitive streak for a longer period of time before departure. The expression of FLK-1, an early marker of endothelial cell precursors including heart precursors, also accumulated abnormally in the posterior region in Mesp1-deficient embryos. In addition, using the Cre-loxP site-specific recombination system, we could determine the lineage of the Mesp1-expressing cells. The first mesodermal cells that ingressed through the primitive streak were incorporated as the mesodermal component of the amnion, and the next mesodermal population mainly contributed to the myocardium of the heart tube but not to the endocardium. These results strongly suggest that MesP1 is expressed in the heart tube precursor cells and is required for mesodermal cells to depart from the primitive streak and to generate a single heart tube.     

Life Cycle Assessment of Water Supply Plans in Mediterranean Spain

Life cycle assessment (LCA) was used to compare the current water supply planning in Mediterranean Spain, the so‐called AGUA Programme, with its predecessor, the Ebro river water transfer (ERWT). Whereas the ERWT was based on a single interbasin transfer, the AGUA Programme excludes new transfers and focuses instead on different types of resources, including seawater and brackish water desalination and wastewater reuse, among others. The study includes not only water supply but the whole anthropic cycle of water, from water abstraction to wastewater treatment. In addition to standard LCA impact categories, a specific impact category focusing on freshwater resources is included, which takes into account freshwater scarcity in the affected water catchments. In most impact categories the AGUA Programme obtains similar or even lower impact scores than ERWT. Concerning impacts on freshwater resources, the AGUA Programme obtains an impact score 49% lower than the ERWT. Although the current water planning appears to perform better in many impact categories than its predecessor, this study shows that water supply in Spanish Mediterranean regions is substantially increasing its energy intensity and that Mediterranean basins suffer a very high level of water stress due to increasing demand and limited resources.     

Effects of bovine milk lactoperoxidase system on some bacteria

Bovine lactoperoxidase (LPO) was purified from skimmed milk using amberlite CG-50-H⁺ resin, CM sephadex C-50 ion-exchange chromatography, and sephadex G-100 gel filtration chromatography. Lactoperoxidase was purified 20.45-fold with a yield of 28.8%. Purity of enzyme checked by sodium dodecyl sulphate-polyacrylamide gel electrophoresis method and a single band was observed. K _m was 0.25 mM at 20°C, V _max value was 7.95 μmol/ml min at 20°C (pH 6.0). Antibacterial study was done by disk diffusion method of Kirby-Bauer using Mueller-Hinton agar medium with slight modification. Bovine LPO showed high antibacterial activity in 100 mM thiocyanate-100 mM H₂O₂ medium for some bacteria (Brevibacillus centrosaurus, B. choshinensis, B. lyticum, Cedecea davisae, Chryseobacterium indoltheticum, Clavibacter michiganense pv. insidiosum, Kocuria erythromyxa, K. kristinae, K. rosea, K. varians, Paenibacillus validus, Pseudomonas syringae pv. populans, Ralstonia pickettii, Rhodococcus wratislaviensis, Serratia fonticola, Streptomyces violaceusniger, Vibrio cholerae-nonO1) respectively, and compared with well known antibacterial substances (levofloxacin, netilmicin). LPO system has inhibition effects on all type bacteria and concentration is really important such as LPO-100 mM thiocyanate-100 mM H₂O₂ system was proposed as an effective agent against many factors causing several diseases.     

No Diagnostic Value of Plasma Clusterin in Alzheimer's Disease

There is an urgent need for biomarkers to enable early diagnosis of Alzheimer''s disease (AD). It has recently been shown that a variant within the clusterin gene is associated with increased risk of AD and plasma levels of clusterin have been found to be associated with the risk of AD. We, therefore, investigated the diagnostic value of clusterin by quantifying clusterin using an ELISA in plasma from 171 controls, 127 patients with AD, 82 patients with other dementias and 30 patients with depression. We observed similar plasma clusterin levels in controls, AD patients and patients with other dementias, suggesting that plasma clusterin levels have no diagnostic value for AD. There was a slight, but significant, increase in plasma clusterin in patients with depression compared to all other groups tested, which may warrant further investigation.     

Risk screening of non‐native freshwater fishes at the frontier between Asia and Europe: first application in Turkey of the fish invasiveness screening kit

The aim of the present study was to assess the invasive potential of introduced non‐native and translocated fishes in Turkey (Anatolia and Thrace) by applying the Fish Invasiveness Screening Kit (FISK), a risk identification tool for freshwater fishes. From independent evaluations by two assessors of 35 species, calibration of FISK for Turkey identified a threshold score of 23, which reliably distinguished between potentially invasive (high risk) and potentially non‐invasive (medium to low risk) fishes for Anatolia (Asia) and Thrace (Europe). No species was categorized as ‘low risk’, 18 species were categorized as ‘medium risk’ and 17 as ‘high risk’ (two being ‘moderately high risk’, nine ‘high risk’, and six ‘very high risk’). The highest scoring species was gibel carp Carassius gibelio, whereas the lowest scoring species was Caucasian dwarf goby Knipowitschia caucasica, a translocated species. Assessor certainty in their responses averaged overall between ‘mostly uncertain’ and ‘mostly certain’, with red piranha Pygocentrus nattereri and topmouth gudgeon Pseudorasbora parva achieving the lowest and highest certainty values, respectively, and with overall significant differences in certainty between assessors. The results of the present study indicate that FISK is a useful and viable tool for identifying potentially invasive non‐native fishes in Turkey, a country characterized by natural biogeographical frontiers.     

Processing methods for differential analysis of LC/MS profile data

Background  

Liquid chromatography coupled to mass spectrometry (LC/MS) has been widely used in proteomics and metabolomics research. In this context, the technology has been increasingly used for differential profiling, i.e. broad screening of biomolecular components across multiple samples in order to elucidate the observed phenotypes and discover biomarkers. One of the major challenges in this domain remains development of better solutions for processing of LC/MS data.     

Cloning,Heterologous Expression,Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H

Aspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZαA vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k _cat of 189.4 s^?1 and K _m of 28.26 mM while M12 GOx had k _cat of 352.0 s^?1 and K _m of 13.33 mM for glucose at pH 5.5. Specificity constants k _cat/K _m of wt (6.70 mM^?1 s^?1) and M12 GOx (26.7 mM^?1 s^?1) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells.     

Fasciola hepatica: parasite-secreted proteinases degrade all human IgG subclasses: determination of the specific cleavage sites and identification of the immunoglobulin fragments produced

The study was focused on the relationship of Fasciola hepatica-secreted proteinases and human IgG subclasses. Each IgG was incubated at different pH values and lengths of time with either the adult parasite excretion-secretion products or the purified cysteinyl proteinases cathepsin L1 and cathepsin L2. The Ig fragments produced were isolated and characterized by Western blot analysis, and the specific cleavage sites were determined by amino acid sequence analysis. Parasite excretion-secretion products and both cathepsins L produced similar degradation patterns and cleaved all human IgG subclasses at the hinge region, yielding at pH 7.3 and 37 degrees C Fab and Fc fragments in the case of IgG1 and IgG3 or Fab(2) and Fc in IgG2 and IgG4. While IgG1 and IgG3 were readily degraded by E/S products either in the presence or in the absence of reducing agents, IgG2 and IgG4 were resistant to proteolysis and were only digested in the presence of 0.1 M dithiothreitol. The cathepsins L needed the presence of dithiothreitol to digest IgG1, IgG2, and IgG4 whereas IgG3 was identically cleaved under both reducing and nonreducing conditions. The main cleavage sites produced by E/S products, CL1, or CL2 were located at the positions peptide bonds: His237-Thr238, Glu237-Cys239, Gly233-Asp234, and Ser241-Cys242 for gamma1, gamma2, gamma3, or gamma4, respectively. The enzymes gave additional splitting sites on the middle hinge of IgG3 to produce shorter Fc fragments and also produce Fd degradation of the IgG4. No cleavage specificity differences were found between CL1 and CL2, but they differed in the kinetics of IgG3 degradation. By lowering the pH, only the E/S products produced concomitant destruction of the Fc while preserving the Fab portion. Under all the conditions assayed the enzymes produced an Fc'-like fragment of 14-15 kDa corresponding to the whole CH3 domain of the immunoglobulin. Contrary to the extensive degradation produced by cathepsins on digested proteins, its actions on IgG subclasses were specific and restricted; thus, all the fragments produced could be potentially involved in the mechanisms used by the parasite to evade the host immune response.