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EDITOR MZ Khan, SENRA Academic Publishers Burnaby, British Columbia, Canada Print ISSN Online ISSN Volume 4, Number 1 Feb 1 ASSOCIATE EDITORS Errol Hassan, University of Queensland Gatton,
EDITOR MZ Khan, SENRA Academic Publishers Burnaby, British Columbia, Canada Print ISSN Online ISSN Volume 4, Number 1 Feb 1 ASSOCIATE EDITORS Errol Hassan, University of Queensland Gatton, Australia Paul CH Li, Simon Fraser University Burnaby, British Columbia, Canada EDITORIAL STAFF Jasen Nelson Walter Leung Sara Ali Hao-Feng (howie) Lai Ben Shieh MANAGING DIRECTOR Mak, SENRA Academic Publishers Burnaby, British Columbia, Canada The Canadian Journal of Pure and Applied Sciences (CJPAS-ISSN ) is a peer reviewed multi-disciplinary specialist journal aimed at promoting research worldwide in Agricultural Sciences, Biological Sciences, Chemical Sciences, Computer and Mathematical Sciences, Engineering, Environmental Sciences, Medicine and Physics (all subjects). Every effort is made by the editors, board of editorial advisors and publishers to see that no inaccurate or misleading data, opinions, or statements appear in this journal, they wish to make clear that data and opinions appearing in the articles are the sole responsibility of the contributor concerned. The CJPAS accept no responsibility for the misleading data, opinion or statements. Editorial Office : SENRA Academic Publishers th Street Burnaby British Columbia V3N 3A3 Canada CANADIAN JOURNAL OF PURE AND APPLIED SCIENCES Board of Editorial Advisors Richard Callaghan Gordon McGregor Reid University of Calgary, AB, Canada North of England Zoological Society, UK David T Cramb Pratim K Chattaraj University of Calgary, AB, Canada Indian Institute of Technology, Kharagpur, India Matthew Cooper Andrew Alek Tuen Grand Valley State University, AWRI, Muskegon, MI, USA Institute of Biodiversity, Universiti Malaysia Sarawak, Malaysia Anatoly S Borisov Dale Wrubleski Kazan State University, Tatarstan, Russia Institute for Wetland and Waterfowl Research, Stonewall, MB, Canada Ron Coley Dietrich Schmidt-Vogt Coley Water Resource & Environment Consultants, MB, Canada Asian Institute of Technology, Thailand Chia-Chu Chiang Diganta Goswami University of Arkansas at Little Rock, Arkansas, USA Indian Institute of Technology Guwahati, Assam, India Michael J Dreslik M Iqbal Choudhary Illinois Natural History, Champaign, IL, USA HEJ Research Institute of Chemistry, Karachi, Pakistan David Feder Daniel Z Sui University of Calgary, AB, Canada Texas A&M University, TX, USA David M Gardiner SS Alam University of California, Irvine, CA, USA Indian Institute of Technology Kharagpur, India Geoffrey J Hay Biagio Ricceri University of Calgary, AB, Canada University of Catania, Italy Chen Haoan Zhang Heming Guangdong Institute for drug control, Guangzhou, China Chemistry & Environment College, Normal University, China Hiroyoshi Ariga C Visvanathan Hokkaido University, Japan Asian Institute of Technology, Thailand Gongzhu Hu Indraneil Das Central Michigan University, Mount Pleasant, MI, USA Universiti Malaysia, Sarawak, Malaysia Moshe Inbar Gopal Das University of Haifa at Qranim, Tivon, Israel Indian Institute of Technology, Guwahati, India SA Isiorho Melanie LJ Stiassny Indiana University - Purdue University, (IPFW), IN, USA American Museum of Natural History, New York, NY, USA Bor-Luh Lin Kumlesh K Dev University of Iowa, IA, USA Bio-Sciences Research Institute, University College Cork, Ireland. Jinfei Li Shakeel A Khan Guangdong Coastal Institute for Drug Control, Guangzhou, China University of Karachi, Karachi, Pakistan Collen Kelly Victoria University of Wellington, New Zealand Xiaobin Shen Hamid M.K.AL-Naimiy University of Melbourne, Australia University of Sharjah, UAE Maria V Kalevitch Eric L Peters Robert Morris University, PA, USA Chicago State University, Chicago, IL, USA Xing Jin Roustam Latypov Hong Kong University of Science & Tech. Kazan State University, Kazan, Russia Leszek Czuchajowski Frances CP Law University of Idaho, ID, USA Simon Fraser University, Burnaby, BC, Canada Basem S Attili Guangchun Lei UAE University, UAE Ramsar Convention Secretariat, Switzerland David K Chiu Atif M Memon University of Guelph, Ontario, Canada University of Maryland, MD, USA Gustavo Davico SR Nasyrov University of Idaho, ID, USA Kazan State University,Kazan, Russia Andrew V Sills Russell A Nicholson Georgia Southern University Statesboro, GA, USA Simon Fraser University, Burnaby, BC, Canada Charles S. Wong Borislava Gutarts University of Alberta, Canada California State University, CA, USA Greg Gaston Sally Power University of North Alabama, USA Imperial College London, UK CONTENTS Volume 4, Number 1 February 1 LIFE SCIENCES Mohamed A Hefnawy, Mohamed I Ali and Salah Abdul-Ghany Influence of Copper and Cobalt Stress on Membrane Fluidity of Stachybotrys chartarum Rupinder Kaur, Renu Bhardwaj and Ashwani K Thukral Growth and Heavy Metal Uptake in B. juncea L. Seedlings as affected by Binary Interactions between Nickel and other Heavy Metals Magda A El-Bendary, Hala M Rifaat and Abeer A Keera Larvicidal Activity of Extracellular Secondary Metabolites of Streptomyces microflavus against Culex Pipiens K Haritha, P Udayasri, J Madhavi, KK Pulicherla and KRS Sambasiva Rao Heterologous Production of Synthetic Cationic Antimicrobial Peptide in Novel Osmotically Inducible E. coli GJ Balbir Singh, Tilak Raj, MPS Ishar, S Sateesh Kumar, RK Jaggi and Anupam Sharma In vivo Anti-Malarial Evaluation of Ocimum sanctum Linn. and O. basilicum Linn Kolawole, OM and Adesoye, AA Evaluation of the Antimalarial Activity of Bridelia ferruginea Benth Bark Nadia H Abd El-Nasser, Samia M Helmy, Amal M Ali, Abeer A Keera and Hala M Rifaat Production, Purification and Characterization of the Antimicrobial Substances from Streptomyces viridodiastaticus (NRC1) M Zaheer Khan, Babar Hussain, Syed Ali Ghalib, Afsheen Zehra and Nazia Mahmood Distribution, Population Status and Environmental Impacts on Reptiles in Manora, Sandspit, Hawkesbay and Cape Monze areas of Karachi Coast PHYSICAL SCIENCES Ling Man Tsang Some Static Spherical Classical Solutions including the Cosmological Term AE Pillay, B Ghosh, B Senthilmurugan, S Stephen and A Abd-Elhameed Ablative Laser Depth-Profiling (ICP-MS) of Reservoir Cores to Evaluate Homogeneity of Strontium and Barium Distributions Linked to Scale Deposition Part Adli A Hanna, Marwa A Sherief, Reham MM. Aboelenin and Sahar MA Mousa Preparation and Characterizations of Barium Hydroxyapatite as Ion Exchanger Chang-Hung Hung The Relationship between Elevated Alanine Transaminase and Body Mass Index in College Students Population: A Cross-Sectional Study KB Vijaya Kumar and K Gopala Krishna Naik Effect of Confinement of Gluons on Ground State Heavy Meson Spectrum in the Relativistic Harmonic Model Pardeep Kumar and Gursharn Jit Singh Transport of Vorticity in Viscoelastic Magnetic Fluid Particle Mixtures through Porous Medium Margaret A Briggs-Kamara, Alaiyi G Warmate, Yehuwdah E Chad-Umoren And Chukwuemeka M Ibechedor Neutron Activation and Flame Atomic Absorption Elemental Analyses of Selected Hair Dyes PK Bardhan, S Patra and G Sutradhar Prediction of Machinability of Sintered Iron Component using Response Surface Method SENRA Academic Publishers, Burnaby, British Columbia Vol. 4, No. 1, pp , 1 ISSN: INFLUENCE OF COPPER AND COBALT STRESS ON MEMBRANE FLUIDITY OF STACHYBOTRYS CHARTARUM *Mohamed A Hefnawy 1, Mohamed I Ali and Salah Abdul-Ghany 1 1 Department of Botany, Faculty of Science, Menoufia University, Shebin El-kom Department of Botany, Faculty of Science, Cairo University, Giza, Egypt ABSTRACT The growth of S. chartarum markedly decreased with elevated concentrations of Cu and Co in the growth medium. Total lipids and proteins in isolated plasma membrane were increased at 4mg Cu or Col -1 and decreased above this concentration while, carbohydrates markedly increased with elevated concentrations of both metals. The total amount of detected phospholipids in the membranes was decreased at 8mg l -1 of both metal ions. However, Phosphatidyl ethanolamine and phosphatidyl glycerol showed an increase at this concentration. Moreover, most of the detected fatty acids (C16:, C16:1, C18:1 and C18:) in the plasma membrane were increased with elevated concentrations of both metals to approximately 1.5- fold higher than in the control except C16:1 at 8mg Cu l -1 highly increased to 4.9 times higher than in the control. Whereas, C16: was the only fatty acid which decreased at 8mg Col -1. The unsaturation index of fatty acids at 4mg l -1 exhibited a slight decrease while, at this concentration the fluorescence polarization value of DPH in the plasma membranes markedly increased. On the other hand, at 8mgl -1 the unsaturation index was increased while, fluorescence polarization value of DPH markedly decreased. This refers that the membrane at 4mgl -1 might be less fluid and at 8mgl -1 more fluid and cannot able to control the entry of toxic metals. Keywords: S. chartarum, lipid composition, membrane fluidity, copper and cobalt stress. INTRODUCTION Living organisms are exposed in nature to heavy metals, commonly present in their ionized species. These ions exert diverse toxic effects on microorganisms. Metal exposure selects and maintains microbial variants able to tolerate their harmful effects. Varied and efficient metal resistance mechanisms have been identified in diverse species of bacteria, fungi and protists (Cervantes et al., 6). Fungal survival and tolerance to toxic metals depend on intrinsic biochemical and structural properties, physiological and genetical adaptation (Gadd and Griffiths, 1978; Gadd, 1993). Heavy metals (cobalt, nickel, cadmium and chromium) at different concentrations were toxic and inhibited the growth of Rhizoctonia solani in both solid and liquid media. Total inhibition was observed upon treatment with 1ppm of each tested heavy metal. Nickel was the most toxic to the fungus, followed by cadmium, cobalt and chromium (Singh and Singh, ). Metal resistant fungi belonged to the genera Aspergillus, Penicillium, Alternaria, Geotrichum, Fusarium, Rhizopus, Monilia and Trichoderma were isolated from wastewatertreated soil. The minimum inhibitory concentration (MIC) for Cd, Ni, Cr, Cu and Co was determined. The MIC *Corresponding author ranged from.-.5mg l -1 for Cd, followed by Ni.1-4mg l -1, Cr.3-7mg l -1, Cu.6-9mg l -1 and Co.1-5mg l -1 (Zafar et al., 7). Much work has been done on the biosorbtion of heavy metals by fungal cell wall and also the chemical changes that have happened to cell wall in presence of toxic metals. However, there is spare information on changes in chemical composition of plasma membrane of fungi cultured in toxic concentrations of metals. The effect of some metals such as aluminum on membrane proteins and lipids of plants has been described (Haug and Caldwell, 1985). Total proteins and sugars in isolated plasma membrane of Penicillium expansum were slightly increased at mg Se l -1 and decreased above this concentration. Whereas, total lipids was increased at higher concentrations of selenium in the growth medium. The fungus responds to selenium stress by increasing the biosynthesis of phospholipids, fatty acids and unsaturation index of fatty acids in plasma membrane (Hefnawy, ). Addition of different levels of lead, zinc and barium acetates in the growth medium of Neurospora indica [Tilletia indica], revealed that lead acetate and zinc acetate inhibited the synthesis of both lipids and proteins in the membrane, while barium acetate only had a mild effect. Among phosphatides, phosphatidic acid, phosphatidyl inositol and phosphatidyl ethanolamine progressively accumulated while phosphatidyl choline declined by increasing the concentrations of all 3 metal salts (Sushma et al., 1996). 14 Canadian Journal of Pure and Applied Sciences The changes in phospholipids composition and its content may play a crucial role in fungal tolerance to toxic metals. It was observed that cells of Microsporum gypseum grown in the presence of Ca exhibited increased content of phospholipids and enhances its synthesis. The rise in the levels of phospholipids was found to be due to increased synthesis of Fatty acids. Moreover, the changes in the phospholipids composition increased the membrane fluidity (Giri et al., 1995). Aluminum (1mM Al 3+ ) stimulated mycelial growth and increased membrane fluidity of Lactarius piperatus (Zel and Gogala, 1989). While, in Amanita muscaria, aluminum (1mM Al 3+ ) inhibited mycelial growth and decreased membrane fluidity (Zel et al., 1993). Hefnawy (1999) found that copper stress increased total lipids and proteins in plasma membrane of Fusarium oxysporium and decreased its fluidity. This work aims at investigation of the changes in the plasma membrane composition as a response of toxic metals and also gives light on the role of the membrane in metal tolerance by fungal cells. MATERIALS AND METHODS Organism and culture conditions. S. chartarum was isolated from heavy metal contaminated Egyptian soil, on Dox agar medium amended with 4 mgl -1 CuSO 4.5H O and CoSO 4.7H O separately. The isolated fungus was identified according to Domsch et al. (198). The fungus was grown on Dox liquid medium supplemented separately with different concentrations of Cu + and Co + (,, 4, 6, 8, 1mgl -1 ), incubating in an orbital incubating shaker (1 rpm) at 8ºC ± ºC for 7 days. The mycelial pellets were harvested, washed several times with distilled water and blotted with tissue paper prior to biochemical analysis. While, for dry mass determination the harvested pellets after washing were dried at 85ºC until constant weight was obtained. Isolation of plasma membrane enriched fraction. The method was based on that described by Touze-Soluet et al. (199) and Umura and Yoshida (1983). A known weight of fresh mycelia was ground using a Bead- Beater homogenizer with.5 mm diameter glass beads in.5m Tris-HCl (ph 7.4) buffer containing.5 M sucrose, 1mM EDTA,.1 mm MgCl, chloramphenicol (mgl -1 ) and cycloheximide (mgl -1 ). The homogenate were subjected to differential centrifugation (1g for 1 minutes, 6g for 15minutes and 15 g for minutes), using a Beckman centrifuge (L5 5 SW 8) Ultra-Centrifuge. The homogenate was fractionated, the supernatant was retained and re-centrifuged. After the final centrifugation the pellet containing the crude microsomal fraction was washed once with a 1mM phosphate buffer (ph 7.4) containing.5m sucrose and 3mM NaCl, and resedimented by recentrifugation at 15g for minutes. The washed microsomes were subjected to phase partition for further purification by placing it on the top of polyethylene glycol (PEG 4) and dextran T5 (5.6:5.6% W/W), mixed and centrifuged at 4g for 3min. The upper layer was removed and placed on top of freshly prepared lower phase and centrifuged as before. The upper layer from the second centrifugation was plasma membrane enriched fraction. Extraction and estimation of total lipids, proteins and carbohydrates. Lipids were extracted from plasma membrane enriched fraction by chloroform methanol mixture (:1, v/v) at 4ºC with occasional manual stirring for 1h. The extract was filtered and the non-lipid materials were removed by adding 1ml of.88% (w/v) KCl (Hunter and Rose, 197), the upper phase was removed and discarded. While, the lower phase was evaporated to dryness under nitrogen and the total amount of lipids were determined, using the method of Barnes and Blackstock (1973). Protein was determined according to the methods of Lowry et al. (1951), after extraction with 1 M NaOH. Sugar determination was carried out using the anthrone technique as described by Umbriet et al. (1959). Phospholipids determination. Phospholipids were extracted from plasma membrane enriched fraction obtained from (1g fresh weight) according to the method of Bligh and Dayer (1959) as detailed by Kates (197). Polar lipids were resolved by thin layer chromatography (TLC) on silica gel plates or commercially prepared plates in two dimension chromatography according to the method described by Nichols (1964). The spots were revealed by iodine vapor and outlined with a pencil. The iodine was removed in a vacuo and the chromatogram sprayed with fine mist of cupric acetate (3% w/v) in sulphric acid (8% w/v). The Plates were sprayed until translucent and then heated to approximately 17ºC for 3min. Phospholipids spots appeared dark brown and were identified by comparing R F values with standard phospholipids. The content of each spot was measured calorimetrically according to the method of Miller (1985). Fatty acid methyl ester analysis. Lipids were extracted from plasma membrane enriched fraction obtained from 1 g fresh weight mycelium in chloroform/ methanol (:1), the extract was removed by shaking with. volumes of distilled water. The chloroform layer was separated by centrifugation or allowing the tubes to stand over night, this layer was then removed and washed once with methanol / water (1:1, v/v). Butylated hydroxytoluene (.5% w/v) was added as antioxidant. The chloroform was then evaporated under nitrogen. Lipids were converted into fatty acid methyl esters by adding 5 ml of Hefnawy et al. 15 methylation reagent (conc. H SO 4 : toluene: methanol 1: 1: by vol.) to each sample. The mixture was refluxed for 1 h at 9ºC. The resulting fatty acid methyl esters were extracted with hexane and analyzed by GLC using a (Chrompack CP 9 with a column packed with CP-Sil- 58 Support Chromsorb WHP). The degree of unsaturation was expressed as unsaturation index, defined by Kates and Hagen (1964) as : mol -1 = 1x (% monoenes)/1 + x (% dienes)/1 + 3 x (% trienes)/1 Measurement of plasma membrane fluidity. To monitor the fluidity of lipid regions in the plasma membrane1, 6-diphenyl 1, 3, 5- hexatriene (DPH) was used as a probe (Shinitzky and Inbar, 1976; Shinitzky and Barenholz, 1978). A solution of mm DPH in tetrahydrofuran was diluted 1 fold with 5mM Tris/ HCl ph (7.4) containing NaCl (.9%, w/v). Freshly prepared membranes with 3 ml of diluted DPH were incubated for 3minutes at 3ºC. Fluorescence polarization measurements were made in a Fluorescence Spectrophlorometer F- at 3ºC. DPH was excited at 34nm while the emission was measured at 44nm. The degree of fluorescence polarization (P), was calculated, according to the equation P = (I//- I ) / (I//+ I ), where I//and I are the fluorescence emission intensities measured at right angles to the excitation beam, with the analyzer polarization axis parallel to, and perpendicular to, the polarization axis of the polarizer respectively. RESULTS Growth, Total carbohydrate, protein and lipid content in plasma membrane. S. chartarum was able to tolerate elevated concentrations of Cu and Co in the growth medium up to 8mg l -1. The dry mass was markedly decreased with increasing both metal ion concentrations in the medium. At 8mg Cu and Co l -1 the growth was decreased to approximately 85%. Total carbohydrate content in isolated plasma membrane were markedly increased with elevate concentrations of both metal ions. Where, total protein and lipid were increased at 4mgl -1 and decreased at 8mg -1 comparing with the control (Table 1). Polar phospholipids composition in plasma membranes. It is clear that phospholipid composition of S. chartarum membranes markedly changed in the presence of both Cu and Co in the growth medium. Some of the detected phospholipids slightly increased in the presence of 8mg Cu l -1 except that phosphatidyl coline, cardiolipin and phosphatidic acid showed a decrease at this concentration (Table and Fig. 1). At 8mg Cu l -1, phosphatidyl ethanolamine and phosphatidyl glycerol showed an increase to approximately.4, 1.1 fold Table 1. Dry mass (mg/5ml culture medium), total carbohydrate, protein and lipid (mg g -1 fresh mycelium weight) in plasma membrane fraction obtained from 1 gm fresh weight of Stachybotrys chartarum grown in the presence of different concentrations of heavy metals. ± SE of three determinations. Metals Conc. (mgl -1 ) Dry mass Carbohydrate Protein Lipids. 167± ±.4.±.4.9±. Cu 4
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