Phosphate Buffer Extracts Research Articles

Isolation and Characterization of Diamine Oxidase Enzyme from Mung Bean Sprouts (Vigna radiata L)

The diamine oxidase enzyme (DAO) can be isolated from mung bean sprouts (Vigna radiata L) through the process of extraction in phosphate buffer. Concentration and fractionation were performed by the addition of ammonium sulphate, (NH4)2SO4 saturation in stages and cold centrifuged at 10000 rpm. Enzyme activity of DAO crude and DAO fractionation results were tested using substrat histamin and measured with a spectrophotometer at a wavelength of 450 nm. This study revealed that the enzyme activity of the crude enzyme was1, 226 mU/mL, the fraction of 40-60% ammonium sulphate saturated had the highest activity which accounted for 3326 mU/mL and after gel filtration, the results for both activity and specific activity were 116 mU/mL and 185 mU/mg protein.

Extraction and Purification of R-phycoerythrin from Marine Red Algae.

This chapter focuses on the recovery of an R-Phycoerythrin (R-PE)-enriched fraction from marine algae. Since R-PE is a proteinaceous pigment, we have developed a simple and rapid two-step method devoted to the extraction and purification of R-PE from marine red algae. Here we describe a phosphate buffer extraction followed by anion exchange chromatography carried on a DEAE Sepharose Fast Flow column. To ensure the quality and quantity of R-PE recovery, we also indicate different methods to monitor each fraction obtained, such as spectrophotometric indicators, gel filtration, and SDS-PAGE analysis.

Nucleoside mono- and diphosphate prodrugs of 2',3'-dideoxyuridine and 2',3'-dideoxy-2',3'-didehydrouridine.

Despite their close structural similarity to nucleoside analogues such as the anti-HIV drugs AZT and d4T, 2',3'-dideoxyuridine (ddU) and 2',3'-dideoxy-2',3'-didehydrouridine (d4U) are entirely inactive against HIV in their nucleoside form. However, it has been shown that the corresponding triphosphates of these two nucleosides can effectively block HIV reverse transcriptase. Herein we report on two types of nucleotide prodrugs (cycloSal and DiPPro nucleotides) of ddU and d4U to investigate their ability to overcome insufficient intracellular phosphorylation, which may be the reason behind their low anti-HIV activity. The release of the corresponding mono- and diphosphates from these compounds was demonstrated by hydrolysis studies in phosphate buffer (pH 7.3) and human CD4 (+) T-lymphocyte CEM cell extracts. Surprisingly, however, these compounds showed low or no anti-HIV activity in tests with human CD4 (+) T-lymphocyte CEM cells. Studies of the conversion of ddUDP and d4UDP into their triphosphate metabolites by nucleoside diphosphate kinase (NDPK) showed nearly no conversion of either diphosphate, which may be the reason for low intracellular triphosphate levels that result in low antiviral activity.

Purification and characterization of iron-cofactored superoxide dismutase from Enteromorpha linza

A superoxide dismutase was purified from Enteromorpha linza using a simple and safe procedure, which comprised phosphate buffer extraction, ammonium sulphate precipitation, ion exchange chromatography on Q-sepharose column, and gel filtration chromatography on Superdex 200 10/300GL. The E. linza superoxide dismutase (ElSOD) was purified 103.6-fold, and a yield of 19.1% and a specific activity of 1 750 U/mg protein were obtained. The SDS-PAGE exhibited ElSOD a single band near 23 kDa and the gel filtration study showed ElSOD’s molecular weight is near 46 kDa in nondenatured condition, indicating it’s a homodimeric protein. El SOD is an iron-cofactored superoxide dismutase (Fe-SOD) because it was inhibited by hydrogen peroxide, insensitive to potassium cyanide. The optimal temperature for its maximal enzyme activity was 35°C, and it still had 29.8% relative activity at 0°C, then ElSOD can be classified as a cold-adapted enzyme. ElSOD was stable when temperature was below 40°C or the pH was within the range of 5–10. The first 11 N-terminal amino acids of ElSOD were ALELKAPPYEL, comparison of its N-terminal sequence with other Fe-SOD N-terminal sequences at the same position suggests it is possibly a chloroplastic Fe-SOD.

An improved phycobilin extraction method

One unique feature in cyanoprokaryotes, rhodophytes, and cryptophytes is the presence of phycobilin pigments-these water soluble pigments can absorb red, orange, yellow, and green light enhancing the spectral range available for cellular conversion to chemical energy. The presence of phycobilin pigment complexes can be detected using fluorescence, or absorbance measures. Efficient detection of these compounds is essential for use in calibrating absorbance in remote sensing or in physiological studies. The standard procedure for phycobilin analysis involves sonication, extraction in buffer potentially coupled with additional digestion steps using enzymes, repeated freeze and thawing cycles, followed by filtration, and spectrophotometric analyses. An alternative method, using asolectin-CHAPS ((3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid – AC)) solution for extraction, and three cycles of freeze/thawing/sonication, was compared to the phosphate buffer (PB) standard procedure. Cultures of both coccoid and filamentous cyanoprokaryota had improved extraction efficiency (38–80%) using AC. After two complete extractions, no pigment was detectable in AC and near baseline fluorescence was observed in the cell pellet, whereas the PB extraction method removed <90% of the phycobilins after two extractions. Phycocyanin concentration measured by AC extraction was better correlated to lipophilic pigment concentration than using phosphate buffer extraction. AC buffered to pH 6.7 was more effective than AC 3.75. One potential source of experimental error was determined to be the use of a baseline correction for the extraction buffer, not the sample.

Simple Phosphate Buffer Extraction for the Determination of Fumonisins in Masa, Maize, and Derived Products

An accurate determination of fumonisins in masa can be troublesome because of difficulties often experienced in obtaining satisfactory recoveries. In this study, a new extraction method, effective both for masa and for maize and derived products, has been developed. The method extracts fumonisins using a 0.4 M phosphate buffer (PB) at pH 7.5, without any organic solvent; the purification of the extract is performed by an immunoaffinity column and the quantification by HPLC–MS/MS. The average recovery percentages were 95.5 ± 1.9% and 96.7 ± 2.1% for fumonisin B1 and B2, respectively. The limit of detection and of quantification for both fumonisins were 10 and 30 μg kg−1, respectively. For masa and tortillas, PB pH 7.5 gave significantly higher results when compared with AOAC and other published extraction methods; for maize flour, PB gave generally higher results, even if the difference with the other extraction methods was not always significant.

Role of steaming and Toasting on the Odor, Protein Characteristics of Chickpea (Cicer arietinum L.) Flour, and Product Quality

Proteins play an important role in imparting functional attributes like texture and shape, which determine the sensory quality of the foods. Boondi, a deep fried product from chickpea (Cicer arietinum L.) flour dispersion, is a popular snack food in India. Chickpea dhal (splits) or flour was subjected to various processing conditions like steaming and toasting, to determine their effect on the chickpea flour protein characteristics and on the product quality. Dhal and flour subjected to different heat treatments showed differences in their odor profiles. The SDS-PAGE of sodium phosphate buffer extracts of steamed dhal or flour showed that the high molecular weight (HMW) proteins of 66 to 100 kDa that were present in the untreated dhal were found to be absent in steamed dhal extracts. However, SDS buffer extracts on SDS-PAGE of these steamed samples did not show any difference between untreated and thermally treated dhal samples. Phosphate buffer extracts of the thermally treated flours were subjected to gel filtration chromatography and the results indicated that the HMW protein fraction content decreased significantly in the treated dhal or flour samples compared to control. Boondi prepared from the thermally treated dhal samples resulted in the loss of spherical shape of boondi. Thus, the results indicate that thermal treatment of chickpea dhal and flour influence changes in protein characteristics, the sensory profile and quality of boondi.

First Report of Sugarcane mosaic virus on Maize in the Centre Region of France.

In 2007, sweet corn plants (Zea mays cv. Challenger) with symptoms of mosaic resembling those caused by Maize dwarf mosaic virus (MDMV) were observed in production fields in the Centre Region of France. In 10 sweet corn fields surveyed, yield production was decreased by 10 to 20%. Potyvirus-like particles were observed by electron microscopy. Twenty samples from affected plants (cv. Challenger) showing severe mosaic symptoms were tested by ELISA with rabbit polyclonal antibodies to MDMV (Montpellier SupAgro, France) and SCMV (Sugarcane mosaic virus) (Bioreba, Reinach, Switzerland). For all leaves tested, slight and strong positive reactions were observed for MDMV and SCMV, respectively, indicating that the plants were infected by SCMV. Filtered phosphate buffer extracts of infected leaves of field plants (cv. Challenger) were used to mechanically inoculate leaves of different corn plants at the three-leaf stage. Sweet corn cultivars (Atugan, Challenger, Golda, Max, and Rana) and seed corn cultivars (DK315, DK3152, DKC3420, Marcello, and PR38H20) were tested. Two weeks after inoculation, mosaic symptoms were observed on leaves. The sweet corn cultivars (with the exception of cv. Max) were considerably more sensitive to the virus. Using two lots of 1,000 seeds each (cv. Challenger), a rate of seed transmission was estimated to be 0.5 to 0.6%. Total RNA was extracted from infected leaves of cv. Challenger 3 weeks after mechanical inoculation. To confirm the potyvirus species involved, a short conserved fragment of the coat protein gene was reverse transcribed, PCR amplified and sequenced (3). The sequence was deposited in GenBank (Accession No. HM014060). Multiple sequence alignment and phylogenetic analysis clearly grouped this isolate called SCMV-MFc (Maize France centre) in the 'Maize' cluster of SCMV, which consists exclusively of isolates from maize. Nucleotide sequence comparison showed that SCMV-MXVrP, a Mexican isolate (2), and SCMV-P3, a Polish isolate (4), have the two closest sequences (285 identical nucleotides of 286). SCMV-MFc is more distantly related with the described SCMV German isolates (1). This is in contrast to SCMV isolates from the traditional Saccharum officinarum cultivars that can be clustered by geographical origin (1). This result could be a consequence of the possible seed transmission of the maize isolates of SCMV. In the future, it will be interesting to check the occurrence of SCMV-MFc in maize fields to assess its economical importance.

English

Cultivation of Spirulina platensis under salt stress conditions (0.02 M as control), 0.04 and 0.08 M NaCl led to a remarkable alteration of algal metabolism as well as an enhancement or induction of biologically active compounds. Concerning algal growth, salt stress caused a decrease in dry weight, chlorophyll a content as well as certain xanthophylls (neoxanthin and violaxanthin) while β-carotene production was stimulated especially at higher salt concentrations. Biochemical analysis of salt stressed algal revealed that lipid content was slightly increased together with certain saturated and unsaturated fatty acids especially the polyunsaturated ones (γ-inolenic acid, omega 3 fatty acid). Electrophoretic analysis of soluble protein pointed out that certain high molecular weight protein bands were not detected comparing with the protein marker. Five new protein bands of molecular weights 190, 158, 113, 77 and 28 KDa were recorded, in addition to an increase in the intensity of 6 already existing bands. Phosphate buffer and water extracts of the algal exhibited antiviral activities against both Hepatitis-A-virus-type-MBB (HAV-MBB strain, RNA virus) and Herpes simplex-virus-type-1 (HSV-1, DNA virus). Water extracts were found to be more effective than phosphate buffer extracts in inducing antiviral activities (98%) especially against HSV-1 virus. The same water extract of the salt stressed algal demonstrated higher anticoagulating activity compared with those of heparin and the positive control measured by clotting time assay. Antioxidant activity of the algal successive extracts against 2, 2 diphenyl-1-picrylhydrazyl and 2,2'- azino-bis (ethylbenzthiazoline-6- sulfonic acid) radical methods revealed moderate antioxidant activity of the non-polar algal extracts (petroleum ether) which were doubled with increasing extract concentration. The lowest activity was recorded by the partially polar (ethyl acetate) algal extract of both concentrations at all salinity levels. While the polar extracts (ethanol and water) showed higher antioxidant activities which were doubled with increasing extract concentration. Ethanolic algal extract (100 µg/ ml at 0.08 M NaCl) exhibited the highest antioxidant activity compared with those of the synthetic antioxidant butylated hydroxy anisol as standard (85.0, 89.9 and 86.0, 91.8% respectively). Key words: Spirulina platensis, antioxidant-antivirus, biochemical studies, salt stress.

A screen-printed, amperometric biosensor array incorporated into a novel automated system for the simultaneous determination of organophosphate pesticides

Organophosphate pesticides present serious risks to human and environmental health. A rapid reliable, economical and portable analytical system will be of great benefit in the detection and prevention of contamination. A biosensor array based on six acetylcholinesterase enzymes for use in a novel automated instrument incorporating a neural network program is described. Electrochemical analysis was carried out using chronoamperometry and the measurement was taken 10s after applying a potential of 0V vs. Ag/AgCl. The total analysis time for the complete assay was less than 6min. The array was used to produce calibration data with six organophosphate pesticides (OPs) in the concentration range of 10−5 M to 10−9 M to train a neural network. The output of the neural network was subsequently evaluated using different sample matrices. There were no detrimental matrix effects observed from water, phosphate buffer, food or vegetable extracts. Furthermore, the sensor system was not detrimentally affected by the contents of water samples taken from each stage of the water treatment process. The biosensor system successfully identified and quantified all samples where an OP was present in water, food and vegetable extracts containing different OPs. There were no false positives or false negatives observed during the evaluation of the analytical system. The biosensor arrays and automated instrument were evaluated in situ in field experiments where the instrument was successfully applied to the analysis of a range of environmental samples. It is envisaged that the analytical system could provide a rapid detection system for the early warning of contamination in water and food.

Purification and characterization of superoxide dismutase from garlic

An efficient and easily scaled up method to isolate superoxide dismutase from garlic is proposed. The separation and purification procedure consists of phosphate buffer extraction, heat treatment and a two-stage ultrafiltration process. The enzyme was purified 139-fold with a specific activity of 2867 U/mg protein and a yield of 91%. The native molecular mass of superoxide dismutase estimated by fast protein liquid chromatography on a Superose 6 column was 28 kDa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis showed a single band near 14 kDa, suggesting that native enzyme was homo-dimeric. The optimal pH for enzyme activity was found to be 7.0, and at this pH the enzyme exhibited maximum activity at 50 °C in 50 mM sodium phosphate buffer. Among various metal ions examined, Cu 2+ and Zn 2+ exerted a positive effect on superoxide dismutase activity, whereas Hg 2+ was found to be a strong inhibitor. The final purified enzyme had an isoelectric point of 5.1–5.4 and a sheet content of 46%, consistent with the literature values. This shows that the purified SOD folded with a reasonable secondary structure.

Abstracts of Nippon Dojo-Hiryogaku Zasshi

Abstracts of Nippon Dojo-Hiryogaku Zasshi

Statistical optimization of culture condition for enhanced hydrogen production by Thermoanaerobacterium thermosaccharolyticum W16

The optimization of culture condition for enhanced hydrogen production by Thermoanaerobacterium thermosaccharolyticum W16 was conducted using statistical experimental design and analysis. Plackett–Burman design was first used to screen the most important variables influencing hydrogen production, and subsequently central composite design was adopted to investigate the optimum value of the selected factors for achieving maximum hydrogen yield. Experimental results showed that xylose, phosphate buffer, and yeast extract had significant influence on hydrogen production and the maximum hydrogen yield of 2.39 mol/mol xylose was predicted when the concentrations of xylose, phosphate buffer, and yeast extract were 12.24 g/L, 0.170 M, and 4.11 g/L, respectively. The results were further verified by repeated experiments under optimal conditions. The excellent correlation between predicted and measured values further confirmed the validity and practicability of this statistical optimum strategy.

Implications of antioxidant enzymes in human gastric neoplasms

The present study is the first to evaluate the expression and activity of MnSOD, Cu/ZnSOD and catalase in human gastric samples, since ROS play a significant role in the pathogenesis of different forms of malignancy inducing mutations and various diseases such as gastric cancer. Biopsies and surgical samples from 53 patients (male/female 22/31, mean age 56.5+/-15.8 years) consisted of 15 healthy, 12 autoimmune atrophic gastritis, 10 Helicobacter pylori (HP) infection, 8 HP-negative chronic gastritis (CG) and 8 adenocarcinoma cases. Enzyme activity and expression were evaluated by spectrophotometry and immunoblotting after specific extraction in phosphate buffer. We found that MnSOD activity was increased in adenocarcinoma, CG and HP tissues (p<0.05-0.001), while Cu/ZnSOD was significantly lower in adenocarcinoma and HP tissues (p<0.001) when compared to the healthy control. MnSOD and Cu/ZnSOD were expressed to a significantly higher degree in adenocarcinoma and HP tissues (p<0.05 and <0.001 respectively) and to a significantly lower degree in CG tissues with respect to the healthy patients (p<0.05 and <0.001). A significant decrease in CAT activity in adenocarcinoma and HP tissues was observed (p<0.01 and <0.05). Gastric human neoplasms showed significant changes in antioxidant enzymes, that represent the first line in antioxidant protection against radical attack. The difficulties in correlating the antioxidant enzyme with the neoplasms was related to the complexity of the biochemical pathways that regulate the cellular redox balance. Our results are important in enhancing the understanding of the role that these enzymes play in the promotion/suppression of the carcinogenesis cascade in human gastric mucosa.

Single-Laboratory Validation of a High-Performance Liquid Chromatographic-Diode Array Detector-Fluorescence Detector/Mass Spectrometric Method for Simultaneous Determination of Water-Soluble Vitamins in Multivitamin Dietary Tablets

The purpose of this study was to develop a single-laboratory validated (SLV) method using high-performance liquid chromatography with different detectors [diode array detector (DAD); fluorescence detector (FLD); and mass spectrometry (MS)] for determination of 7 B-complex vitamins (B1-thiamin, B2-riboflavin, B3-nicotinamide, B6-pyridoxine, B9-folic acid, pantothenic acid, and biotin) and vitamin C in multivitamin/multimineral dietary supplements. The method involves the use of a reversed-phase octadecylsilyl column (4 microm, 250 x 2.0 mm id) and a gradient mobile phase profile. Gradient elution was performed at a flow rate of 0.25 mL/min. After a 5 min isocratic elution at 100% A (0.1% formic acid in water), a linear gradient to 50% A and 50% B (0.1% formic acid in acetonitrile) at 15 min was employed. Detection was performed with a DAD as well as either an FLD or a triple-quadrupole MS detector in the multiple reaction monitoring mode. SLV was performed using Standard Reference Material (SRM) 3280 Multivitamin/Multimineral Tablets, being developed by the National Institute of Standards and Technology, with support by the Office of Dietary Supplements of the National Institutes of Health. Phosphate buffer (10 mM, pH 2.0) extracts of the NIST SRM 3280 were analyzed by the liquid chromatographic (LC)-DAD-FLDIMS method. Following extraction, the method does not require any sample cleanup/preconcentration steps except centrifugation and filtration.

Prediction and verification of nitrogen decomposition patterns of various manure types in paddy fields by phosphate buffer extraction

Prediction and verification of nitrogen decomposition patterns of various manure types in paddy fields by phosphate buffer extraction

Exploring Equisetum arvense L., Equisetum ramosissimum L. and Equisetum telmateia L. as sources of natural antioxidants

The antioxidant and scavenging activities of above ground parts of Equisetum arvense L., Equisetum ramosissimum L. and Equisetum telmateia L. phosphate buffer (pH 7) extracts were investigated. Activities of antioxidant enzymes (superoxide dismutase, catalase, guaiacol peroxidase and glutathione peroxidase), quantities of reduced glutathione, malonyldialdehyde, superoxide and hydroxyl radicals and flavonoid, soluble protein, chlorophyll a, b and carotenoid contents were determined. The total antioxidant capacity was determined by ferric reducing antioxidant power (FRAP) assay. The Equisetum telmateia extract demonstrated scavenging and antioxidant properties better than Equisetum ramosissimum and Equisetum arvense. The ESR signal of DMPO-OH radical adducts in the presence of Equisetum telmateia phosphate buffer (pH 7) extract was reduced by 98.9% indicating that Equisetum telmateia could be a useful source of antioxidants with huge scavenging ability.

Influence of biotic stress induction on the antimicrobial and biochemical parameters of Nigella sativa

The traditionally used indigenous medicinal plant Nigella sativa was germinated under fungal induced stress for the evaluation of its antifungal activity against Aspergillus niger, Trichoderma harzianum, Fusarium solani, Helmenthosporium myedis, Rizopus solani and its antibacterial effects on Bacillus subtilis, Pasturella multocida, Escherichia coli and Staphylococcus aureus. The biochemical parameters related to stress were also studied. Crude phosphate buffer (pH: 7.0) extracts of different days post inoculation (dpi) showed significantly high antimicrobial activity (disk diffusion method) against the selected strains at 237.5µg/mL The strongest antimicrobial activity was found against S. aureus and F. solani as compared to other bacterial and fungal strains. While comparing the activity at various dpi, stronger antibacterial activity was found at dpi 4, suggesting the induction of bioactive compounds of N. sativa was due to biotic stress. On the other hand, dpi 3 showed the highest antifungal activity against the selected strains. Biochemical parameters such as peroxidases (POD), catalases (CAT), superoxide dismutase (SOD) and proteases were recorded at different dpi. The highest specific activities were obtained at dpi 3 and 4. The highest total phenolic contents (gallic acid equivalent) were also found at dpi 3. This study demonstrates that the therapeutic activity of medicinal plants should be explored multifactoriously for the treatment of different human infections diseases.

Oxalate analysis methodology for decayed wood

Oxalate from partially decayed southern pine wood was analyzed by HPLC or colorimetric assay. Oxalate extraction efficiency, assessed by comparing analysis of whole wood cubes with ground wood, showed that both wood geometries could be extracted with comparable efficiency. To differentiate soluble oxalate from total oxalate, three extraction methods were assessed, using phosphate buffer, sulphuric acid or sodium hydroxide. Oxalate values from phosphate buffer extracts were two to three times higher than those from sulphuric acid extracts by both methods of analysis. Results for HPLC analysis were not significantly different from results for colorimetric analysis within each method of extraction. Results from sodium hydroxide extraction were inconsistent. The presence of oxalate in phosphate and sulphuric acid extracts was confirmed with oxalate decarboxylase but could not be confirmed for NaOH extracts. HPLC and the colorimetric assay remain valid methods of reporting soluble oxalic acid from decayed wood and are particularly useful for comparing relative determinations between treatments or fungal isolates.

Antioxidant and scavenger activities of Allium ursinum

The antioxidative properties of bulb, leaf and stalk of Allium ursinum were investigated. Activities of antioxidant enzymes (superoxide dismutase, catalase, peroxidase, glutathione peroxidase), quantities of malonyldialdehyde, superoxide and hydroxyl radicals and reduced glutathione and also the contents of total flavonoids, chlorophylls a and b and carotenoids were determined. The extracts from all plant organs exhibited antioxidant activity, the highest having been observed in the leaves. Furthermore, ESR signal of PBN-OH radical adducts in the presence of leaves phosphate buffer (pH 7) extract was reduced for 87.61%.