Aldehyde dehydrogenase expression in Metaphire posthuma as a bioindicator to monitor heavy metal pollution in soil

Background Soil contamination and associated pollution plays a detrimental role in soil flora and fauna. Soil is processed and remodeled by subterranean earthworms, accordingly are referred to as soil chemical engineers. These worms, besides processing carbon and nitrogen, serve as minors for processing metals. In heavy metal contaminated soils, they accumulate heavy metals, which in turn cause altered gene expression, including aldehyde dehydrogenase (ALDH) enzymes. This study explores the possibility of ALDH expression in earthworms as a novel biomarker for the heavy metal contamination of soil. Results Earthworms cultured in contaminated soils accumulated significantly higher levels of Pb and Cd. Similarly, significantly higher levels of ALDH enzyme activities were observed in earthworms cultured in soils contaminated with Pb and Cd. The ALDH activity was found to be highest in worms cultured in 5 ppm heavy metal contaminated soils. Although, ALDH activities decreased as the heavy metal concentration in soil increased, they were significantly higher when compared to control worms cultured in uncontaminated soils. The accumulation of heavy metal in earthworms measured after 28 days decreased as the heavy metal concentration in soil increased. Conclusions Levels of ALDH expression correlated with total Pb and Cd concentration in the earthworm tissue. This study showed that the ALDH activity in earthworms could potentially be used as a biomarker to show heavy metal pollution in soil.Central Department of Biotechnology, Tribhuvan Universit

Phytochemical Analysis of Acacia ehrenbergiana (Hayne) Grown in Qatar: Identification of Active Ingredients and Their Biological Activities

Acacia ehrenbergiana (Hayne), also known as Salam, is a highly drought resistant shrub distributed in North and East Africa, and the Arabian Peninsula. The plant is gathered for its gum and fiber, and is an important legume species for indigenous populations. In this study, the phytochemical analysis, antibacterial, and antioxidant properties of various alcoholic and aqueous extracts of Acacia ehrenbergiana grown in Qatar were investigated. The qualitative phytochemical screening of this species exhibited the presence of glycosides, tannins, flavonoids, terpenoids, saponins, phenol, and anthraquinones in various extracts. The agar diffusion method was performed to check the antibacterial activity. The acetone and ethanol extracts showed 85% antibacterial activity of the control against Gram-negative E. coli, while the acetone extract had 65% activity against the Bacillus Gram-positive species. The highest activity against Staphylococcus aureus was 65% for the butanol extract. The antioxidant capacities were evaluated by the DPPH method. Various extracts exhibited antioxidant activities similar to or higher than standard antioxidants, with the highest percent inhibition of 95% for the acetone and ethanol extracts. The acetone extracts were further purified by reverse phase combiflash chromatography followed by HPLC. Three of the pure compounds isolated were subjected to MS, FTIR, and NMR spectral analysis and were found to be stigmasterol, spinasterol, and theogallin. In conclusion, the observed antibacterial and antioxidant activities as well as the presence of secondary metabolites with potential medicinal activities makes Acacia ehrenbergiana a potent valuable endemic medicinal plant

Phytochemical Analysis of <i>Anastatica hierochuntica</i> and <i>Aerva javanica</i> Grown in Qatar: Their Biological Activities and Identification of Some Active Ingredients

Plant-derived compounds and their extracts are known to exhibit chemo preventive (antimicrobial, antioxidant and other) activities. The levels of such chemo preventive compounds vary depending on environmental factors, including the regions where they grow. Described in this study are: (i) a phytochemical analysis of the two plants grown in the desert environment of Qatar, viz., Anastatica hierochuntica and Aerva javanica; (ii) the antibacterial, antifungal and antioxidant activities of various solvent extracts of these plants; (iii) a report on the isolation of several pure compounds from these plants. The phytochemical screening indicated the presence of glycosides, tannins, flavonoids, terpenoids, saponins, phenol and anthraquinones in various extracts of each of the plants. Antibacterial and antioxidant activities were studied using agar diffusion and DPPH methods, respectively. The extracts of Anastatica hierochuntica as well as Aerva javanica inhibit the growth of both gram-positive and gram-negative bacterial species. Various extracts of the two plants also exhibited higher or similar antioxidant activities as those of the standard antioxidants, α-tocopherol and ascorbic acid. The extracts of these plants were further purified by HPLC and characterized by IR and NMR techniques. This process has led to identification of β-sitosterol, campesterol and methyl-9-(4-(3,4-dihydroxy-1′-methyl-5′-oxocyclohexyl)-2-hydroxycyclohexyl)nonanoate from Anastatica hierochuntica, and lupenone, betulinic acid, lupeol acetate and persinoside A and B from Aerva javanica. The results reported herein suggests that Anastatica hierochuntica and Aerva javanica are potent sources of phytomedicines

Relative contribution of human erythrocyte aldehyde dehydrogenase to the systemic detoxification of the oxazaphosphorines

ABSTRACT: Detoxification of cyclophosphamide is effected, in part, by hepatic class 1 aldehyde dehydrogenase (ALDH-1)-catalyzed oxidation of aldophosphamide, a pivotal aldehyde intermediate, to the nontoxic metabolite, carboxyphosphamide. This enzyme is found in erythrocytes as well. Detoxification of aldophosphamide may also be effected by enzymes, viz. certain aldo-keto reductases, that catalyze the reduction of aldophosphamide to alcophosphamide. Such enzymes are also found in erythrocytes. Not known at the onset of this investigation was whether the contribution of erythrocyte ALDH-1 and/or aldo-keto reductases to the overall systemic detoxification of circulating aldophosphamide is significant. Thus, NAD-linked oxidation and NADPH-linked reduction of aldophosphamide catalyzed by relevant erythrocyte enzymes were quantified. ALDH-1-catalyzed oxidation of aldophosphamide (160 M) to carboxyphosphamide occurred at a mean (؎ SD) rate of 5.0 ؎ 1.4 atmol/min/rbc (red blood cell). Aldo-keto reductase-catalyzed reduction of aldophosphamide (160 M) to alcophosphamide occurred at a much slower rate, viz. 0.3 ؎ 0.2 atmol/min/rbc. Thus, at a pharmacologically relevant concentration of aldophosphamide, viz. 1 M, estimated aggregate erythrocyte ALDH-1-catalyzed aldophosphamide oxidation, viz. 2.0 mol/min, was only about 3% of estimated aggregate hepatic enzyme-catalyzed aldophosphamide oxidation, viz. 72 mol/min; however, this rate is greater than the estimated flow-limited rate of aldophosphamide delivery to the liver by the blood, viz. 1.5 mol/min. These observations/considerations suggest an important in vivo role for erythrocyte ALDH-1 in systemic aldophosphamide detoxification. Erythrocyte ALDH-1-effected oxidation of other aldehydes to their corresponding acids, e.g. retinaldehyde to retinoic acid, may also be of pharmacological and/or physiological significance since a wide variety of aldehydes are known to be substrates for ALDH-1

Synthesis and characterization of vanadium (IV)-flavonoid complexes and its antioxidant ability toward superoxide and radical scavenging

In this project Vanadium complex -Vanadium (IV) - flavone was synthesized using vanadium(IV) acetylacetonate (VO(acac)2) complex and 3-hydroxy-6-methyl flavone ligand. The complex stability was checked using FTIR and UV-vis spectroscopies. Peackes around 990 cm-1 conforms the formation of (V=O) in the complex, as well as (V-O) around 790 cm-1. In UV-Vis spectrum peak around 400-450 nm was noticed, which conforms the formation of the vanadium complex that correspond to the ligand to metal charge transfer (LMCT) transition. The radical scavenging abilities of vanadium complex were investigated using DPPH. The anti-oxidant activity using (BHA) as a standard reference, the complex synthesized displayed strong DPPH antioxidant radical scavenging activity compared to VO(acac)2 and BHA, with IC50 value of (105, 95 and 96) mM respectively. The absorbance in which the reducing power occurred were found to be (0.397, 0.825 and 0.228) for the complex, VO(acac)2 and BHA

Isolation and Physicochemical Characterization of Laccase from Ganoderma lucidum-CDBT1 Isolated from Its Native Habitat in Nepal

At present, few organisms are known to and capable of naturally producing laccases and white rot fungi are one such group. In the present study, three fungal species, namely, Ganoderma lucidum-CDBT1, Ganoderma japonicum, and Lentinula edodes, isolated from their native habitat in Nepal were screened for laccase production, and G. lucidum-CDBT1 was found to express highest levels of enzyme (day 10 culture media showed 0.92 IU/mg total protein or 92 IU/mL laccase activity with ABTS as substrate). Lignin extracted from rice straw was used in Olga medium for laccase production and isolation from G. lucidum-CDBT1. Presence of lignin (5 g/L) and copper sulfate (30 μM) in the media increased the extracellular laccase content by 111% and 114%, respectively. The laccase enzyme produced by G. lucidum-CDBT1 was fractionated by ammonium sulfate and purified by DEAE Sepharose anion exchange chromatography. The purified enzyme was found to have a molecular mass of 43 kDa and exhibits optimal activity at pH 5.0 and 30°C. The isolated laccase was thermally stable for up to 70°C for 1 h and exhibited broad pH stability. The kinetic constants, Km, Vmax, and Kcat, determined using 2,2′-azinobis-(-3-ethylbenzothiazoline-6-sulfonic acid) as substrate were found to be 110 μM, 36 μmol/min/mg, and 246 min−1, respectively. The isolated thermostable laccase will be used in future experiments for delignification process

Development of Novel Reagents for Visualizing Latent Fingerprints

Chemical composition of fingerprints includes the presence of salts, ions, fatty acids, lipids, amino acids, nucleobases, nucleotides, and nucleic acids. Many methods used to detect latent fingerprints on porous surfaces such as paper exploit the reactivity of amino acids with a number of different reagents, including ninhydrin and lawsone (2-hydroxy-1,4-naphthoquinone; HNQ). HNQ, also known as hennotannic acid, is a natural yellow-orange dye present in the leaves of henna plant (Lawsonia inermis) as well as in the flower of water hyacinth (Eichhornia crassipes). It is used as natural dye to color hair and skin. HNQ has been shown to react with amino acids, accordingly used to detect fingerprints on paper. HNQ has also been shown to be a sensor for the detection of anions (changes its color from yellow to orange-red in the presence of anions such as OH-, CN-, etc.). The presence of nucleobases, nucleotides and/or nucleic acids that are also part of the chemical composition of fingerprints, has not been exploited to detect latent fingerprints. In this study we propose utilize the reactivity of HNQ, nucleobases and nucleotides to detect and enhance the detection of latent fingerprints. HNQ reacts with amines and form Schiff-bases that are expected to be strongly colored, whose colors can be further enhanced upon exposure to a base (NH3) or anions (OH- and/or CN). In this regard, we have synthesized a series of HNQ-amine derivatives using microwave green synthetic methods (reactants dissolved in methanol and the reaction carried out at 150°C for 5 min). The compounds formed in the above reactions are strongly colored as judged visually and by spectrophotometry. Under similar conditions, adenine and guanine bases or adenosine and guanosine nucleosides (with amine substituents) also react with aromatic aldehydes to form Schiff-bases. The aromatic aldehyde derivatives of these purine nucleobases or nucleosides are expected to be highly fluorescent. Again, we have synthesized a series of aromatic imine derivatives of adenine, guanine, adenosine and guanosine as above. As judged visually and by spectrophotometry, these compounds exhibit strong fluorescence. The spectroscopic characterization of the compounds described above is on-going. Further, we have used these compounds to detect and enhance detection of latent fingerprints. The latter involved (a) application of powdered HNQ, adenine, guanine, adenosine or guanosine on latent fingerprints or cyanoacrylate coated latent fingerprints, (b) powder deposition aromatic amines or aldehydes, (c) followed by microwave synthesis of Schiff-base derivatives directly on the surface of the fingerprints using a commercially available domestic microwave oven (medium power for 3–10 min). The resulting fingerprints developed as above with HNQ and aromatic amines are strongly colored. Further exposure of thus formed colored fingerprints to NH3 strongly enhanced their color. The fingerprints developed as above using purine nucleobases or nucleosides with aromatic aldehydes were strongly fluorescent. Through this study, we have successfully synthesized Schiff-base derivatives of HNQ and aromatic amines as well as purine nucleobases or nucleosides and aromatic aldehydes. Further, we also suggest a novel method for the detection of latent fingerprints and its successful application on nonporous surfaces using the newly synthesized compounds.qscienc

Secretory Laccase from Pestalotiopsis Species CDBT-F-G1 Fungal Strain Isolated from High Altitude: Optimization of Its Production and Characterization

Microorganisms producing laccases may be used for the pretreatment of lignocellulosic biomass to recover fermentable sugar. Very few fungi and other microbes growing in high altitudes have been tested for this purpose. As part of this study, we have collected soil samples from different parts of the Kathmandu Valley and the Rautah at district of Nepal (1600 to 2303 m above sea level) and successfully cultured 53 different isolates of microorganisms. Among the 53 isolates obtained 30 were Actinomycetes, 20 were Streptomycetes, and three were fungi). These isolates were tested for laccase expression using guaiacol, tannic acid, and 1-naphthol as substrates. Twelve of the 53 isolates tested positive for the expression of laccase. Among the laccase- positive isolates, a fungal species designated as CDBT-F-G1was found to produce high levels of laccase. This isolate was identified as Pestalotiopsis species based on 18S rRNA sequencing. Pestalotiopsis spp. CDBT-F-G1 isolate grows efficiently in PDB media containing 1% Kraft lignin at pH 5 and 30 °C and secretes 20 ± 2 U/mL laccase in culture medium. Further optimization of growth conditions reveled that addition of (i) metal salts, e.g., 1 mM magnesium sulfate (51 ± 25 U/mL); (ii) agitation of cultures at 200 rpm (51 ± 9U/mL); (iii) surfactants, e.g., 0.75 mM Tween 80 (54 ± 14 U/mL); (iv) 40% dissolved O2 (57 ± 2 U/mL) and inducers, e.g., 1 mM gallic acid (69 ± 11 U/mL), further promote laccase production by Pestalotiopsis spp. CDBT-F-G1 isolate. On the other hand, 0.1 mM cysteine inhibited laccase production. The secretory laccase obtained from fermentation broth of CDBT-F-G1 was partially purified by ammonium sulfate (13-fold purification with specific activity 26,200 U/mg) and acetone (14-fold purification with specific activity 31,700 U/mg) precipitation methods. The enzyme has an approximate molecular mass of 43 kDa, pH and temperature optima werepH6 and 60 °C, respectively. Vmax and Km were 100 μmol/min and 0.10 mM, respectively, with ABTS as the substrate. Given the above characteristics, we believe Pestalotiopsis spp. CDBT-F-G1 strain native to high altitudes of Nepal could be used to pretreat lignocellulosic biomass to efficiently recover fermentable sugars

Production, Purification, and Characterization of Thermostable Alkaline Xylanase From Anoxybacillus kamchatkensis NASTPD13

Anoxybacillus kamchatkensis NASTPD13 used herein as a source for thermostable alkaline xylanase were isolated from Paudwar Hot Springs, Nepal. NASTPD13 cultured at 60°C, pH 7 and in presence of inorganic (ammonium sulfate) or organic (yeast extract) nitrogen sources, produced maximum xylanase enzyme. Xylanase production in the cultures was monitored by following the ability of culture media to hydrolyze beech wood xylan producing xylooligosaccharide and xylose by thin layer chromatography (TLC). The extracellular xylanase was isolated from optimized A. kamchatkensis NASTPD13 cultures by ammonium sulfate (80%) precipitation; the enriched xylanase preparation was dialyzed and purified using Sephadex G100 column chromatography. The purified xylanaseshowed 11-fold enrichment with a specific activity of 33 U/mg and molecular weight were37 kDa based on SDS-PAGE and PAGE-Zymography. The optimum pH and temperature of purified xylanase was 9.0 and 65°C respectively retainingmore than 50% of its maximal activity over a broad range of pH (6–9) and temperature (30–65°C). With beech wood xylan, the enzyme showed Km 0.7 mg/ml and Vmax 66.64 μM/min/mg The xylanase described herein is a secretory enzyme produced in large quantities by NASTPD13 and is a novel thermostable, alkaline xylanase with potential biotechnological applications