ABSTRACT Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a simple and sensitive gel X-ray film contact print technique. About 17 AlTIs were detected in the seed extracts of A. lebbeck. Two...
moreABSTRACT Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a simple and sensitive gel X-ray film contact print technique. About 17 AlTIs were detected in the seed extracts of A. lebbeck. Two groups of AlTIs-1 major (10 AlTIs; slow migration on the gel) and 1 minor (7 AlTIs; fast migration on the gel) were identified. The former was specific only toward trypsin. However, the latter was specific toward both trypsin and Helicoverpa armigera gut proteinases (HaGPs). The most potent AlTI (AlTI13) was purified to assess its in vivo bioefficacy toward HaGPs. Purification was achieved using (NH4)(2)SO4 fractionation, Sephadex G-100 column chromatography, and preparative native-polyacrylamide gel electrophoresis (PAGE). The dose dependent bioefficacies of AlTIs in the (NH4)(2)SO4 F-3 fractions (0.1%, 0.5%, and 1%) were approximately 79%, 83%, and 90%, respectively, resulting in reductions in the average larval weight of H. armigera. Artificial diet containing a single dose of AlTI13 (5 mu g/g diet) reduced the larval weight by about 76%, with 60% mortality. The half-maximal inhibitory concentrations (IC50) of AlTI13 for trypsin and HaGPs were 0.14 and 0.17 mu mol/ml, respectively. The optimum conditions for AlTI13 were pH 8 and temperatures ranging from 35 to 40 C. Reducing sodium dodecyl sulfate-PAGE analysis indicated that similar to 28 kDa Kunitz-like trypsin inhibitor was present. Thus, we showed that particularly, AlTI13 of A. lebbeck could be used as a transgene macromolecule to markedly increase insect resistance in genetically engineered plants.
This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa armigera. A bifunctional α-amylase/trypsin inhibitor was purified from the seeds of pigeonpea by native liquid phase isoelectric focusing...
moreThis paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa armigera. A bifunctional α-amylase/trypsin inhibitor was purified from the seeds of pigeonpea by native liquid phase isoelectric focusing (N-LP-IEF), affinity chromatography and preparative electrophoresis. Its in-vivo and in-vitro interaction with midgut amylases of H. armigera was studied along with growth inhibitory activity. One and two dimensional (2D) zymographic analyses revealed that the purified inhibitor is dimeric glycoprotein (60.2kDa and 56kDa) exist in a multi-isomeric form with five pI variants (pI 5.5 to 6.3). It was found to be heat labile with complete inactivation up to 80°C and stable over a wide range of pH (4-11). The slow binding and competitive type of α-amylase inhibition was observed with 0.08μM of dissociation constant (Ki) for the enzyme-inhibitor complex (EI). The internal protein sequence of two subunits obtained by mass spectrometry matched with cereal-type α-AI, a conserved domain from AAI_LTSS superfamily and sialyltransferase-like protein respectively. In-vivo studies indicated up-regulation of total midgut α-amylase activity with negative effect on growth rate of H. armigera suggesting its suitability for pest control.
Trypsin protease inhibitor have been identified and purified from extracts of Mucuna Pruriens. Mucuna Pruriens seed sample were purified using ammonium sulphate precipitation, Saphadex G-75 gel filtration chromatography technique...
moreTrypsin protease inhibitor have been identified and purified from extracts of Mucuna Pruriens. Mucuna Pruriens seed sample were purified using ammonium sulphate precipitation, Saphadex G-75 gel filtration chromatography technique including analysis of trypsin inhibitors by dot-blot method, and electrophoretic analysis and visualization of protease inhibitor bands by gel X-ray film contact print method. Saphadex G-75 gel filtration chromatography identified a small molecular weight trypsin inhibitory fraction. In the present paper we described, a simple and inexpensive procedure to detect protease inhibitors of seed by the gel-x-ray film contact print technique.
The sample from seed of Peganum harmala plant tree was extracted in hexane, chloroform, acetone and methanol and separated by thin layer chromatography on silica gel plate by using chloroform : Methanol (85:15) solvent mixture and...
moreThe sample from seed of Peganum harmala plant tree was extracted in hexane, chloroform, acetone and methanol and separated by thin layer chromatography on silica gel plate by using chloroform : Methanol (85:15) solvent mixture and detected under the ultraviolet light. Maximum bands were detected on TLC of methanol extract. Those bands were scraped and the purification of the single band of interest molecule was done by preparative silica gel column chromatography. Antimicrobial activity of single band isolated was tested against standard ATCC (American Type Culture Collection) strain of E.coli ( 25922), Klebseilla , Staphylococcus aureus (25923), Pseudomonas aeruginosa (27853), S. pneumonia (6305) using Disc diffusion method and Punch Plate technique. The Purified fraction of alkaloid show zone of growth inhibition with 10mm on ATCC Staphylococcus aureus. The sensitivity test of water extract was showing effect mostly against Staphylococcus aureus and E.coli
In this report protein content and catalase (CAT) activity were used to study the effect of hydrogen peroxide (H2O2) on leaf senescence in detached Pigeon pea leaves. A decrease in protein content measured as an indicator of leaf...
moreIn this report protein content and catalase (CAT) activity were used to study the effect of hydrogen peroxide (H2O2) on leaf senescence in detached Pigeon pea leaves. A decrease in protein content measured as an indicator of leaf senescence, and a drop in CAT activity was observed following treatment with H2O2 in Pigeon pea detached leaves compared with control leaves. However after longer incubations CAT activity significantly increased in comparison with day 1 treatment. Protein content and CAT activity were also studied in the leaves treated by 0.025, 0.05, 0.075 and 0.1 mM H2O2. The optimal concentration of H2O2 in reducing protein content seems to be 0.075 mM, whereas concentration of 0.025, 0.05, 0.075 mM increase the CAT activity while lower and higher concentrations shown the opposite effect. The observed changes revealed that H2O2 induces oxidative stress and oxidative damage thereby leaf senescence in the detached leaves of Pigeon pea.
The present study tries to examine the influence of natural and oxidative stress [hydrogen peroxide (H2O2) treatment]-induced senescence on peroxidase (POD) activity in neem (Azadirachta indica A. juss) leaves. Data indicated that...
moreThe present study tries to examine the influence of natural and oxidative stress [hydrogen peroxide (H2O2) treatment]-induced senescence on peroxidase (POD) activity in neem (Azadirachta indica A. juss) leaves. Data indicated that incubation of detached leaves in presence of H2O2 has induced POD(s), that enzyme activity is also enhanced in natural senescing leaves. Changes in POD activity and protein loss during H2O2-promoted senescence has shown similarity with natural senescence suggests that the underlying regulatory mechanisms might be the same in both stresses, at least in neem. Initial induction of POD activity under H2O2 stress suggests that PODs play a very important role during the early phases of leaf senescence. Reduction in POD activity along with the increase in protein loss at latter stages suggests that there was no correlation between PODs and senescence, at least in neem.
In this report peroxidase (POD) and polyphenol oxidase (PPO) activities were used to study the effect of hydrogen peroxide (H2O2) on leaf senescence in detached pigeonpea (Cajanus cajan [L.] Millsp.) leaves. The activities of POD and PPO...
moreIn this report peroxidase (POD) and polyphenol oxidase (PPO) activities were used to study the effect of hydrogen peroxide (H2O2) on leaf senescence in detached pigeonpea (Cajanus cajan [L.] Millsp.) leaves. The activities of POD and PPO were observed to be greater in H2O2-stressed pigeonpea leaves than in water treated control leaves. However, after longer incubations activities of these enzymes were markedly reduced. The observed changes revealed that exogenous H2O2 may induce oxidative stress tolerance by enhancing the activities of POD and PPOs. On the other hand, reduction found in H2O2-induced POD and PPO activities at later stages may be due to destruction of these proteins along with other proteins. This study will help to improve the tolerability of plants to environmental stresses by enhancing the expression of POD and PPOs.
This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa armigera. A bifunctional α-amylase/trypsin inhibitor was purified from the seeds of pigeonpea by native liquid phase isoelectric focusing...
moreThis paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa armigera. A bifunctional α-amylase/trypsin inhibitor was purified from the seeds of pigeonpea by native liquid phase isoelectric focusing (N-LP-IEF), affinity chromatography and preparative electrophoresis. Its in-vivo and in-vitro interaction with midgut amylases of H. armigera was studied along with growth inhibitory activity. One and two dimensional (2D) zymographic analyses revealed that the purified inhibitor is dimeric glycoprotein (60.2kDa and 56kDa) exist in a multi-isomeric form with five pI variants (pI 5.5 to 6.3). It was found to be heat labile with complete inactivation up to 80°C and stable over a wide range of pH (4-11). The slow binding and competitive type of α-amylase inhibition was observed with 0.08μM of dissociation constant (Ki) for the enzyme-inhibitor complex (EI). The internal protein sequence of two subunits obtained by mass spectrometry matched with cereal-type α-AI, a conserved domain from AAI_LTSS superfamily and sialyltransferase-like protein respectively. In-vivo studies indicated up-regulation of total midgut α-amylase activity with negative effect on growth rate of H. armigera suggesting its suitability for pest control.
More than 200 insect pests are found growing on pigeonpea. Insects lay eggs, attack and feed on leaves, flowers and developing pods. Plants have developed elaborate defenses against these insect pests. The present work evaluates protease...
moreMore than 200 insect pests are found growing on pigeonpea. Insects lay eggs, attack and feed on leaves, flowers and developing pods. Plants have developed elaborate defenses against these insect pests. The present work evaluates protease inhibitor (PI) based defense of pigeonpea in leaves and flowers. PIs in the extracts of these tender tissues were detected by using gel X-ray film contact print method. Up to three PIs (PI-3, PI-4 and PI-5) were detected in these tissues as against nine (PI-1-PI-9) in mature seeds. PI-3 is the major component of these tissues. Mechanical wounding, insect chewing, fungal pathogenesis and application of salicylic acid induced PIs in pigeonpea in these tissues. Induction was found to be local as well as systemic but local response was stronger than systemic response. During both local and systemic induction, PI-3 appeared first. In spite of the presence and induction of PIs in these tender tissues and seeds farmers continue to suffer yield loses. This is due to the weak expression of PIs. However the ability of the plant to respond to external stimuli by producing defense proteins does not seem to be compromised. This study therefore indicates that PIs are components of both constitutive and inducible defense and provide a ground for designing stronger inducible defense (PIs or other insect toxin based) in pigeonpea.
Simultaneous administration of caffeine (100 mg/kg, i.p., 3 days) and phenobarbital (80 mg/kg, i.p., 3 days) to adult male rats resulted in a significant decrease in hepatic cytochrome P-450 and acetanilide hydroxylase activity, compared...
moreSimultaneous administration of caffeine (100 mg/kg, i.p., 3 days) and phenobarbital (80 mg/kg, i.p., 3 days) to adult male rats resulted in a significant decrease in hepatic cytochrome P-450 and acetanilide hydroxylase activity, compared to phenobarbital administration alone. While simultaneous administration of caffeine and benzo[a]pyrene (20 mg/kg, i.p., 2 days) increased acetanilide hydroxylase, compared to benzo[a]pyrene administration, no change was seen in the cytochrome P-450 concentration. In vitro addition of 2.5 mM caffeine to microsomal incubations from untreated, phenobarbital- and benzo[a]pyrene-treated rats inhibited aminopyrine N-demethylase activity. No significant difference was seen in the extent of aminopyrine N-demethylase inhibition due to the in vitro addition of caffeine to microsomes from untreated or phenobarbital-treated rats, whereas inhibition in microsomes from benzo[a]pyrene-treated rats was greater.
Insect midgut proteases are known to be regulated by plant protease inhibitors. In the present study, the antinutritional effects of a variety of seed extracts against Periplaneta americana (Linnaeus) (Dictyoptera: Blattidae) midgut...
moreInsect midgut proteases are known to be regulated by plant protease inhibitors. In the present study, the antinutritional effects of a variety of seed extracts against Periplaneta americana (Linnaeus) (Dictyoptera: Blattidae) midgut proteases are assessed in vitro and in vivo. ...
Starchy seeds are an important food and a source of dietary ingredients in many countries. However, they suffer from extensive predation by bruchids (weevils) and other pests. α-Amylase inhibitors are attractive candidates for the control...
moreStarchy seeds are an important food and a source of dietary ingredients in many countries. However, they suffer from extensive predation by bruchids (weevils) and other pests. α-Amylase inhibitors are attractive candidates for the control of seed weevils, as these insects are highly dependent on starch as an energy source. A proteinaceous α-amylase inhibitor from the seeds of Achyranthes aspera was identified, purified and characterised. In electrophoretic analysis, two prominent amylase inhibitor activity bands (AI1 and AI2) were detected. The inhibitor was purified 9.99-fold with 1206.95 total amylase inhibitor units mg⁻¹ protein. The molecular weight of the purified inhibitor was around 6 kDa. The isolated α-amylase inhibitor was found to be resistant to heat and proteolysis. Feeding analysis of Callosobruchus maculatus larvae on a diet containing seed powder of A. aspera revealed that survival of the larvae was severely affected, with the highest mortality rate occurring on the fifth day of feeding. The isolated inhibitor inhibited the majority of amylase isoforms of C. maculatus, Tribolium confusum and Helicoverpa armigera in electrophoretic analysis and solution assays. The information obtained in the present investigation could be useful for a genetic engineering approach that would make seeds resistant to storage pest infestations.
Pigeon-pea seed extracts have been analyzed for the protease inhibitors using a new, sensitive and simple method for visualization of electrophoretically separated protease inhibitors. The visualization involves equilibrating the gel...
morePigeon-pea seed extracts have been analyzed for the protease inhibitors using a new, sensitive and simple method for visualization of electrophoretically separated protease inhibitors. The visualization involves equilibrating the gel successively in the protease assay buffer, protease solution, rinsing the gel in protease assay buffer, and exposing it to an exposed, undeveloped X-ray film. Gelatin on the film in places corresponding to the inhibitor bands remains unhydrolyzed. By this method the pigeon-pea seed extract was found to contain nine trypsin and at least seven chymotrypsin inhibitors but no papain or bromelain inhibitors.
Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a simple and sensitive gel X-ray film contact print technique. About 17 AlTIs were detected in the seed extracts of A. lebbeck. Two groups of...
moreHere, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a simple and sensitive gel X-ray film contact print technique. About 17 AlTIs were detected in the seed extracts of A. lebbeck. Two groups of AlTIs—1 major (10 AlTIs; slow migration on the gel) and 1 minor (7 AlTIs; fast migration on the gel) were identified. The former was specific only toward trypsin. However, the latter was specific toward both trypsin and Helicoverpa armigera gut proteinases (HaGPs). The most potent AlTI (AlTI13) was purified to assess its in vivo bioefficacy toward HaGPs. Purification was achieved using (NH4)2SO4 fractionation, Sephadex G-100 column chromatography, and preparative native-polyacrylamide gel electrophoresis (PAGE). The dose dependent bioefficacies of AlTIs in the (NH4)2SO4 F3 fractions (0.1%, 0.5%, and 1%) were approximately 79%, 83%, and 90%, respectively, resulting in reductions in the average larval weight of H. armigera. Artificial diet containing a single dose of AlTI13 (5 μg/g diet) reduced the larval weight by about 76%, with 60% mortality. The half-maximal inhibitory concentrations (IC50) of AlTI13 for trypsin and HaGPs were 0.14 and 0.17 μmol/ml, respectively. The optimum conditions for AlTI13 were pH 8 and temperatures ranging from 35 to 40 °C. Reducing sodium dodecyl sulfate-PAGE analysis indicated that ~ 28 kDa Kunitz-like trypsin inhibitor was present. Thus, we showed that AlTIs, particularly, AlTI13 of A. lebbeck could be used as a transgene macromolecule to markedly increase insect resistance in genetically engineered plants.
ABSTRACT Protease inhibitors play an important role in host plant defence against herbivores. However, insects have the ability to elevate the production of proteinases or resort to production of a diverse array of proteinases to offset...
moreABSTRACT Protease inhibitors play an important role in host plant defence against herbivores. However, insects have the ability to elevate the production of proteinases or resort to production of a diverse array of proteinases to offset the effect of proteinase inhibitors. Therefore, we studied the inhibition of pro‐proteinase(s) activation in the midgut of the polyphagous pest Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in response to protease inhibitors to develop appropriate strategies for the control of this pest. Gelatin coating present on X‐ray film was used as a substrate to detect electrophoretically separated pro‐proteinases and proteinases of H. armigera gut extract on native‐ and sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. Six activated pro‐proteinase bands were detected in H. armigera gut lumen, which were partially purified and characterized using substrate assays. Activated H. armigera midgut pro‐proteinase(s) showed activity maxima at pH 8 and 10, and exhibited optimal activity at 40 °C. The activation of H. armigera gut pro‐proteinase isoforms was observed in the fraction eluted on benzamidine‐sepharose 4B column. Purification and substrate assay studies revealed that 23–70 kDa polypeptides were likely the trypsin/chymotrypsin‐like pro‐proteinases. Larvae of H. armigera fed on a cocktail of synthetic inhibitors (antipain, aprotinin, leupeptin, and pefabloc) showed maximum activation of pro‐proteinases compared with the larvae fed on individual inhibitors. The implications of these results for developing plants expressing proteinase inhibitors for conferring resistance to H. armigera are discussed.
A phyto-tunnel was developed using a drilled PVC pipe. It was planted with Portulaca grandiflora and used for the treatment of a textile effluent and a dye mixture. COD, BOD, TOC, conductivity, turbidity, total suspended solids and total...
moreA phyto-tunnel was developed using a drilled PVC pipe. It was planted with Portulaca grandiflora and used for the treatment of a textile effluent and a dye mixture. COD, BOD, TOC, conductivity, turbidity, total suspended solids and total dissolved solids of the textile effluent, and dye mixture were decreased by 57, 45, 43, 52, 76, 77 and 24 % within 96 h, and 49, 62, 41, 63, 58, 71 and 33 %, within 60 h, respectively, after treatment. The effluent and dye mixture were decolorized up to 87 and 90 % within 96 and 60 h, respectively. Significant induction in activities of lignin peroxidase, tyrosinase and DCIP reductase was observed in root tissues of the plants. FTIR, HPLC and HPTLC of untreated and treated samples showed the formation of new metabolites and preferential dye removal. Phytotoxicity studies revealed the non-toxic nature of the metabolites.
Cytochrome P-450 substrate interactions were studied with cytochrome P-450 partially purified from livers of untreated, phenobarbital-treated, benzo[a]pyrene-treated and caffeine-treated rats. Partial inhibition of aminopyrine...
moreCytochrome P-450 substrate interactions were studied with cytochrome P-450 partially purified from livers of untreated, phenobarbital-treated, benzo[a]pyrene-treated and caffeine-treated rats. Partial inhibition of aminopyrine N-demethylase in presence of in vitro caffeine observed with intact microsomes was further investigated in a reconstituted system composed of partially purified cytochrome P-450 and cytochrome c reductase. Caffeine addition (in vitro) to partially purified cytochrome P-450 altered the hexobarbital, aniline and ethylisocyanide induced spectral change, and decreased NADPH oxidation in presence of substrates aminopyrine and acetanilide. NADPH oxidation was found to be increased in presence of aminopyrine and unaltered in presence of acetanilide in reconstituted system having partially purified cytochrome P-450 from caffeine-treated rats. Our studies suggest that caffeine acts as a true modifier of cytochrome P-450 and is possibly responsible for the formation of abortive complexes with aminopyrine.
ABSTRACT Seeds of pigeonpea are known to accumulate protease inhibitors (PIs), belonging to the Bowman-Burk inhibitor family. PIs are important for natural defense against phytophagous insect pests. Most insects attack crops at the early...
moreABSTRACT Seeds of pigeonpea are known to accumulate protease inhibitors (PIs), belonging to the Bowman-Burk inhibitor family. PIs are important for natural defense against phytophagous insect pests. Most insects attack crops at the early stages of seed development. Accumulation patterns of individual PIs and their relationship with each other were studied in developing seeds of 76 pigeonpea lines derived from BDN2 cultivar by ethyl methane sulfonate induced chemical mutagenesis. PIs extracted from these lines, collected between 10 and 40 days after flowering (DAF) and from mature seeds of BDN2 cultivar, were detected by using gel X-ray film contact print method. A total of 9 trypsin–chymotrypsin inhibitors were detected in mature seeds. All the nine PIs were capable of inhibiting proteases. Appearance of detectable levels of individual PIs started around 10 DAF. The PI-3 appeared early and was the most stable. It was accumulated in all the studied lines and was also detected in 84 % of the samples collected 10 DAF. In particular, 14 DAF, the individual PIs were accumulated and accumulation sequence was observed (PI-3, PI-2, PI-5, PI-7, PI-6, PI-4, PI-8, PI-9 and PI-1). Accumulation continued up to 40 DAF when seeds started hardening. Chemical mutagenesis could not produce any variation in the profile of individual PIs in the 76 studied lines in mature seeds. The process of accumulation of inhibitors is sturdy and mutagenesis fails to alter it. The robust mechanism is responsible for early appearance of PI-3. Early accumulated PIs in this study need further exploration for strengthening natural defense of pigeonpea against pests.
Eleven proteinase activity bands were detected in American cockroach (Periplaneta americana) gut. These were partially purified and characterized using a gel X-ray film contact print method. Cockroach gut proteinases (CGPs) show activity...
moreEleven proteinase activity bands were detected in American cockroach (Periplaneta americana) gut. These were partially purified and characterized using a gel X-ray film contact print method. Cockroach gut proteinases (CGPs) show activity over a broad range of pH with maximum activity between pH 6 and 10, and optimal activity at 50-70 degrees C. CGPs were partially purified by preparative gel electrophoresis and analyzed using synthetic substrates and inhibitors. Four of the proteases exhibited chymotrypsin-like (C1 to C4) activity and seven trypsin-like (T1 to T7) activity. Accuracy of the gel X-ray film contact print method is confirmed by including bovine chymotrypsin in CGP analysis. Inhibition of CGPs with different plant proteinaceous proteinase inhibitors allowed identification of potential CGP inhibitors. Our results imply that presence of several CGP activity bands, and their stability and activity over a broad pH and temperature range might contribute to adaptation of P. americana to extreme environmental conditions and the polyphagous nature of the species.
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