Isolation, characterisation and expression patterns of a RAD51 ortholog from Pleurotus ostreatus

AB: Using degenerated primers for conserved regions of RecA homologs we have isolated a gene from Pleurotus ostreatus that shows characteristic features of RAD51 homologs. The encoded amino acid sequence of P. ostreatus RAD51 (PoRAD51) shows greatest sequence similarities with RAD51 from Coprinus cinereus (89% identity). Furthermore the genomic organisation of PoRAD51 is almost identical to that of RAD51 from C. cinereus. Northern analysis shows that the expression of PoRAD51 is found in vegetative mycelium, and fruit body tissue, and that it is expressed at elevated levels in lamellae/basidia and following DNA damage. A sporulation deficient mutant strain of P. ostreatus (ATTC 58937) showed expression patterns of the RAD51 gene that are similar those of the normal sporulating strain

De zuivering en de eigenschappen van de replicatieve vorm van het RNA van cowpea-mozaiekvirus

Purified infectious preparations of cowpea mosaic virus (CPMV) consist of three centrifugal components with sedimentation coefficients of 58, 95 and 115 S . These are referred to as top (T), middle (M) and bottom (B) component and contain 0, 24 and 33% RNA respectively (Van Kammen, 1967). All three components are isometric particles with a diameter of 28 mp and have serologically similar capsids. Van Kammen (1968) demonstrated that both RNA- containing components of the virus are necessary for infection.This thesis deals with the isolation and properties of the replicative form (RF) of CPMV-RNA. Some properties of the virus and its RNA are studied to provide reference values for the isolation of the RF.After the virus had been separated into its components by means of centrifugation in a zonal rotor, the buoyant densities of middle and bottom component were determined in a CsCl gradient. They proved to be 1.412 g/cm 3and 1.469 g/cm 3respectively. From both middle and bottom component the RNA was extracted with phenol. This RNA was homogeneous in the analytical ultracentrifuge and had sedimentation coefficients of 26.4 S and 33.5 S respectively. By applying Spirin's (1963) relationship between the sedimentation coefficient of an RNA and its molecular weight (M = 1550 x S 2.1) molecular weights were calculated as 1.5 x 10 6dalton and 2.5 x 10 6dalton for middle and bottom component RNA. In a Cs 2 SO 4 gradient the mixture of both RNAs showed only one band with a mean density of 1.628 g/cm 3.The RF was isolated from CPMV-infected primary leaves of Vigna unguiculata . After homogenization of the leaves the fraction sedimenting at 15,000 x g was used for extraction of the RNA. This fraction consisting mainly of chloroplasts, membranes, nuclear fragments and nucleoli was suspended in a small volume of a buffer solution composed of 0.1 M glycine + NaOH pH 9.5, 0.1 M NaCl, 0.005 M Na 3 EDTA, 1% sodiumdodecylsulphate and 3% diethylpyrocarbonate. This mixture was deproteinized by three extractions with phenol. The nucleic acid was precipitated as the quaternary ammonium salt by adding 0.33% cetyltrimethylammoniumbromide. The precipitate was collected by centrifugation and by repeated washing with 0.1 M sodiumacetate in 70% alcohol converted into the sodium salt. This treatment removed even small traces of protein from the nucleic acid preparation. DNA was broken down by DNase and the single-stranded RNA was hydrolyzed by incubation with RNase A and RNase T 1 .Subsequently the solution was passed through a Sephadex G 200 column (2.5 x 35 em) to separate the breakdown products from the remaining high molecular weight material. The sharp frontrunning peak was collected. The nucleic acid in this peak proved to have the properties expected for the RF of CPMV- RNA. It was RNase resistent in 1 x SSC. It showed a sharp helix-coil transition curve in 1 x SSC with a T m of 94° C as measured bij the temperature dependent RNase resistance. Its buoyant density proved to be 1.60 g/cm 3.The yield of RF was 10 μg per 100 g of leaf material. A modified isolation procedure was worked out for the determination of the sedimentation coefficient of the RF. To overcome breakdown due to the effect of the RNase incubation, the singlestranded RNA was precipitated in high salt. For this the RNA solution was brought to 2 M NaCl and frozen at -20° C. After slowly thawing at 4° C the precipitated singlestranded RNA was centrifuged off. The resulting supernatant, containing the doublestranded RNA, s-RNA and some contaminating single-stranded RNA, was subjected to gel filtration on a Sephadex G 200 column (2-5 x 35 cm). The peak eluting just after the void volume of the column was collected and centrifuged on a 5 to 20% linear sucrose gradient. After centrifugation the gradient was fractionated and each fraction was tested for RNase resistance. The RF sedimented with a peak at 15 S and a shoulder at 18 to 19 S. Studier's (1965) formula (S 20, w = 0.0882 x M 0.346) permits the calculation of the molecular weight of double-stranded RNA from its sedimentation coefficient. The molecular weights calculated were 2.8 x 10 6dalton and 5.0 x 10 6dalton. This suggested that there are two RFs in plants infected by CPMV: one for the middle component RNA and one for the bottom component RNA. , Electron microscopy provided independent data on the length distribution of the double-stranded RNA. Again the single-stranded RNA was precipitated from the RNA solution by treatment with high salt as described above. After gel filtration the peak eluting just after the void volume of the column was collected and subjected to equilibrium centrifugation in a Cs 2 SO 4 gradient with a starting density of 1.60 g/cm 3. After equilibrium had been reached, the material banding at a density between 1.58 g/cm 3and 1.62 g/cm 3was collected and used for electron microscopy. Electron microscopy was carried out according to the spreading method of Kleinschmidt et al. (1962). The frequency distribution of the lengths of 397 molecules showed that molecules varied in length from 0.1 μto 2.4 μ. No molecules longer than 2.4 μwere found. The relative frequency distribution of the length of the double-stranded RNA, i.e. the amount of RNA (number of molecules x length) having a certain length, indicated that a large amount of the double-stranded RNA consisted of molecules of the expected length of 1.48 μand 2.45 μrespectively, which was calculated from the base translation of 3.17 Å published by Granboulan and Franklin (1966). This again suggested that both middle component RNA and bottom component RNA each induce their own replicative structure.The double-stranded RNA has been used in molecular hybridization experiments with viral RNA and RNA of the separate components. A saturation curve was determined to gain information on the amount of single-stranded 32P-labeled RNA necessary to replace the homologous RNA completely from the double-stranded structure. Two micrograins of double-stranded RNA were heated for 20 minutes in a closed tube with increasing amounts of 32P-labeled CPMV-RNA. to 105° C to get separation of the strands of the double-stranded structure. Thereafter the tube with the hybridisation mixture was kept at 70° C for 2 hours to obtain annealing of the complementary strands. The tube was cooled on ice, opened and the mixture was incubated with 100 μg RNase A and 150 U RNase T 1 to break down the single-stranded RNA. The RNase-resistent RNA was precipitated with TCA and collected on millipore filters. The amount of annealed exogeneous RNA was determined from the radioactivity of the RNase- resistent RNA. With 2 μg double-stranded RNA, saturation was reached at 50 pg of virus-RNA.The hybridization product was subjected to equilibrium centrifugation in a Cs 2 SO 4 gradient and was shown to have a density of 1.58 g/cm 3. This proved that a true hybrid was formed during the annealing process.Hybridization was also carried out with separate middle and bottom component RNAs. The preliminary results show that with middle component RNA alone 85% of the maximum hybridization level was reached, and with bottom component RNA alone 14%. Turnip yellow mosaic virus RNA did not give any hybridization. These results suggest that M-RNA and B-RNA hybridized independently with the minus strands of the double-stranded structures, which indicates that no overlap occurs in base sequence between M-RNA and B-RNA. This means that M-RNA and B-RNA do not have any genes in common.The final conclusion is that the cowpea mosaic virus genome consists of two different pieces of RNA, both carrying a different part of the genetic information of the virus and both inducing their own replicative structure in the host cell

Antibacterial Effects of the Essential Oils of CommonlyConsumed Medicinal Herbs Using an In Vitro Model.

The chemical composition and antibacterial activity of essential oils from 10 commonly consumed herbs: Citrus aurantium, C. limon, Lavandula angustifolia, Matricaria chamomilla, Mentha piperita, M. spicata, Ocimum basilicum, Origanum vulgare, Thymus vulgaris and Salvia officinalis have been determined. The antibacterial activity of these oils and their main components; i.e. camphor, carvacrol, 1,8-cineole, linalool, linalyl acetate, limonene, menthol, a-pinene, b-pinene, and thymol were assayed against the human pathogenic bacteria Bacillus subtilis, Enterobacter cloacae, Escherichia coli O157:H7, Micrococcus flavus, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enteritidis, S. epidermidis, S. typhimurium, and Staphylococcus aureus. The highest and broadest activity was shown by O. vulgare oil. Carvacrol had the highest antibacterial activity among the tested components

Flow cytometric evaluation of the effects of 3-bromopyruvate (3BP) and dichloracetate (DCA) on THP-1 cells: a multiparameter analysis

Two human leukemia cells K562 and THP-1, the breast cancer lines MCF-7 and ZR-75-1, and the melanoma line MDA-MB-435S were compared by flowcytometry for their behaviour at increasing levels of 3BP. K562 and THP-1 responded to 3BP by membrane depolarization and increased ROS; MCF-7 and ZR-75-1 showed decreased polarization and low ROS increase; MDA-MB-435S had limited depolarization and no ROS increase. THP-1 cells exposed to a range of 3BP concentrations in combination with DCA showed increase of polarization, slight ROS increase, and weakened nuclear integrity. 3BP and DCA show no synergism, but have complementary destructive effects on THP-1 cells. The data led to the conclusion that the THP-1 cells do not carry a functional membrane monocarboxylate transporter (MCT) or that 3BP circumvents MCT binding and can enter these cells independently

Agaricus Blazei Hot Water Extract Shows Anti Quorum Sensing Activity in the Nosocomial Human PathogenPseudomonas Aeruginosa

The edible mushroom Agaricus blazei Murill is known to induce protective immunomodulatory action against a variety of infectious diseases. In the present study we report potential anti-quorum sensing properties of A. blazei hot water extract. Quorum sensing (QS) plays an important role in virulence, biofilm formation and survival of many pathogenic bacteria, including the Gram negative Pseudomonas aeruginosa, and is considered as a novel and promising target for anti-infectious agents. In this study, the effect of the sub-MICs of Agaricus blazei water extract on QS regulated virulence factors and biofilm formation was evaluated against P. aeruginosa PAO1. Sub-MIC concentrations of the extract which did not kill P. aeruginosa nor inhibited its growth, demonstrated a statistically significant reduction of virulence factors of P. aeruginosa, such as pyocyanin production, twitching and swimming motility. The biofilm forming capability of P. aeruginosa was also reduced in a concentration-dependent manner at sub-MIC values. Water extract of A. blazei is a promising source of antiquorum sensing and antibacterial compounds. Keywords: Agaricus blazei; mushroom; antiqourum sensing activity; antimicrobial activity; antibiofilm activity; Pseudomonas aeruginosa

Anti-inflammatory properties of the medicinal mushroom Cordyceps militaris might be related to its linear (1¿3)-ß-D-glucan.

The Ascomycete Cordyceps militaris, an entomopathogenic fungus, is one of the most important traditional Chinese medicines. Studies related to its pharmacological properties suggest that this mushroom can exert interesting biological activities. Aqueous (CW and HW) and alkaline (K5) extracts containing polysaccharides were prepared from this mushroom, and a ß-D-glucan was purified. This polymer was analysed by GC-MS and NMR spectrometry, showing a linear chain composed of ß-D-Glcp (1¿3)-linked. The six main signals in the 13C-NMR spectrum were assigned by comparison to reported data. The aqueous (CW, HW) extracts stimulated the expression of IL-1ß, TNF-a, and COX-2 by THP-1 macrophages, while the alkaline (K5) extract did not show any effect. However, when the extracts were added to the cells in the presence of LPS, K5 showed the highest inhibition of the pro-inflammatory genes expression. This inhibitory effect was also observed for the purified ß-(1¿3)-D-glucan, that seems to be the most potent anti-inflammatory compound present in the polysaccharide extracts of C. militaris. In vivo, ß-(1¿3)-D-glucan also inhibited significantly the inflammatory phase of formalin-induced nociceptive response, and, in addition, it reduced the migration of total leukocytes but not the neutrophils induced by LPS. In conclusion, this study clearly demonstrates the anti-inflammatory effect of ß-(1¿3)-D-glucan

Protective effect of Phellinus linteus polysaccharide extracts against thioacetamide-induced liver fibrosis in rats: a proteomics analysis

Background: The hepatoprotective potential of Phellinus linteus polysaccharide (PLP) extracts has been described. However, the molecular mechanism of PLP for the inhibition of liver fibrosis is unclear. This study aims to investigate the molecular protein signatures involved in the hepatoprotective mechanisms of PLP via a proteomics approach using a thioacetamide (TAA)-induced liver fibrosis rat model. Methods: Male Sprague-Dawley rats were divided into three groups of six as follows: Normal group; TAA group, in which rats received TAA only; and PLP group, in which rats received PLP and TAA. Liver fibrosis was induced in the rats by repeated intraperitoneal injections of TAA at a dose of 200 mg/kg body weight twice a week for 4 weeks. PLP was given orally at a dose of 50 mg/kg body weight twice a day from the beginning of the TAA treatment until the end of the experiment. The development of liver cirrhosis was verified by histological examination. Liver proteomes were established by two-dimensional gel electrophoresis. Proteins with significantly altered expression levels were identified by matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry and the differentially expressed proteins were validated by immunohistochemical staining and reverse transcription polymerase chain reaction. Results: Histological staining showed a remarkable reduction in liver fibrosis in the rats with PLP treatment. A total of 13 differentially expressed proteins including actin, tubulin alpha-1C chain, preprohaptoglobin, hemopexin, galectin-5, glutathione S-transferase alpha-4 (GSTA4), branched chain keto acid dehydrogenase hterotetrameric E1 subunit alpha (BCKDHA), glutathione S-transferase mu (GSTmu); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); thiosulfate sulfurtransferase (TFT); betaine-homocysteine S-methyltransferase 1 (BHMT1); quinoid dihydropteridine reductase (QDPR); ribonuclease UK114 were observed between the TAA and PLP groups. These proteins are involved in oxidative stress, heme and iron metabolism, cysteine metabolism, and branched-chain amino acid catabolism. Conclusion: The proteomics data indicate that P. linteus may be protective against TAA-induced liver fibrosis via regulation of oxidative stress pathways, heat shock pathways, and metabolic pathways for amino acids and nucleic acids