Browsing by Subject "Enzyme inhibitors"
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Item Insulin-Mediated Inhibition of Tyrosinase Activity and Protein Synthesis in Melanoma Cell Cultures(Texas Tech University, 1984-05) Ehlers, Susan ElizabethInsulin lowers basal levels of tyrosinase activity and inhibits the MSH-stimulated rise in tyrosinase in Cloudman S-91 mouse melanoma cell cultures. These cultures are very sensitive to insulin. A concentration of insulin as low as 5 x 10 M insulin produces optimum inhibition. At maximum inhibition, tyrosinase activity is reduced to approximately 50% of control levels. Insulin inhibits cellular proliferation in melanoma cells; however, inhibition of tyrosinase activity precedes this effect. Insulin also inhibits the (Bu)2 cAMP and theophylline stimulated rise in enzyme activity. This finding suggests that insulin exerts its effects at a site distal to cAMP production. Insulin, in fact, does not lower cAMP levels in melanoma cells, nor does it alter the MSH-stimulated rise of cAMP. The inhibitory effect of insulin on tyrosinase activity could not be mimicked by either (Bu)2 cGMP or 8-bromo-cGMP, suggesting that insulin does not exert its effects by altering cellular levels of this nucleotide. Insulin decreases the incorporation of [3H]-leucine into trichloracetic acid insoluble material by 50%, an inhibition which corresponds well with the observed level of reduction of tyrosinase activity. This finding suggests that the inhibition of tyrosinase activity may be caused by a general reduction in protein synthesis due to insulin treatment.Item Oral delivery and formulation of salmon calcitonin: Protection from serine proteases with ovomucoids(Texas Tech University, 2004-05) Shah, Rakhi BharatOral bioavailability of peptides and proteins is extremely low due to extensive degradation in the gastrointestinal tract and low epithelial permeability. Further, the structure and conformation of proteins are prone to alteration when exposed to formulation and process conditions. The conformation changes might lead to a loss of biological activity. Oral delivery of proteins may be enhanced by the use of enzyme inhibitors and absorption enhancers. In the present study, ovomucoids has been investigated as enzyme inhibitor in the oral delivery of salmon calcitonin. Glycyrrhetinic acid was employed as the absorption enhancer. Enzymatic degradation studies revealed that salmon calcitonin is degraded extensively by intestinal serine proteases such as trypsin, a-chymotrypsin, and elastase. Various ovomucoid species such as chicken, duck and turkey ovomucoids were investigated for their inhibitory action against the serine proteases. Duck and turkey ovomucoids stabilized salmon calcitonin against degradation in the presence of the serine proteases for an hour. In contrast, chicken ovomucoid was ineffective against a-chymotrypsin mediated degradation. The cytotoxicity studies on various enzyme inhibitors and absorption enhancers revealed that ovomucoids were safer as compared to aprotinin and protease inhibitor cocktail for long-term use up to 25 pM concentrations. Among the absorption enhancers, octylglucoside and glycyrrhetinic acid did not show any cytotoxicity on Caco-2 cell monolayer for a period of 24 hrs. Permeability studies of salmon calcitonin in Caco-2 cell monolayer suggested that salmon calcitonin is transported via passive diffusion mechanism. The permeability was enhanced in the presence of glycyrrhetinic acid. Regional permeability in rat gastrointestinal tract segments revealed that salmon calcitonin is permeated more from ileum followed by colon, jejunum, duodenum, and stomach. Therefore, the formulation of salmon calcitonin was made to bypass duodenum region and it was targeted to jejunum region. The preclinical in vivo absorption studies in rats revealed that tOVM and glycyrrhetinic acid increased the oral absorption of sCT as compared to the control without these additives. An osmotically controlled bilayered tablet coated with enteric polymers was successfully applied to prepare salmon calcitonin formulation with turkey ovomucoid and glycyrrhetinic acid. Dissolution studies were performed for a period of 4 hrs which showed dual controlled release of the drug and the inhibitor. Characterization of salmon calcitonin in the formulation using DSC, FT-IR, powder X-ray diffraction and gel electrophoresis studies revealed that the structure of salmon calcitonin was conserved after subjecting to formulation and process conditions. A seven-factor, two-level Plackett-Burman screening design and a three-factor, three-level Box-Behnken optimization design was used to evaluate the effect of critical process variables including the orifice size, coating level, amounts of sodium chloride, Polyox® NIO, Poly ox N80, Carbopol® 934P, Carbopol® 974P. Mathematical relationships, contour plots and response surface methods were employed with constrained optimization to predict levels of factors that provided optimum response. The novel formulation incorporates sCT, tOVM, and glycyrrhetinic acid with a dual-controlled release characteristics for the drug and the enzyme inhibitor.Item Oral delivery of proteins: preparation and characterization of an oral dual-controlled release formulation of insulin and ovomucoid(Texas Tech University, 2001-05) Agarwal, VikasOral bioavailability of proteins is extremely low due to extensive degradation in the gastrointestinal tract and low epithelial permeability. Further, the structure and conformation of proteins are easily altered when exposed to formulation and process conditions leading to a loss of biological activity. Oral delivery of proteins may be enhanced by the use of absorption modifiers such as enzyme inhibitors and permeation enhancers. In the present study, the role of ovomucoids has been investigated as possible absorption modifiers in the oral delivery of a model protein, insulin. Ovomucoids are enzyme inhibitors isolated from egg white of avian species. They have protease inhibitory activity and also bind to lectins on the cell surfaces through their carbohydrate moeity. However, their role in the oral delivery of proteins has not been investigated. The purpose of this study was to develop a dual controlled release formulation of insulin and ovomucoid. Enzymatic degradation studies revealed that insulin is degraded extensively in the presence of trypsin and a-chymotrypsin. Duck ovomucoid (DkOVM) stabilized insulin against degradation in the presence of trypsin and a-chymotrypsin for an hour. In contrast, chicken ovomucoid (CkOVM) was only effective against trypsin mediated degradation of insulin. Permeability studies of insulin from rat intestinal segments revealed that insulin is absorbed more from the jejunum and ileum than from the duodenum. In the presence of CkOVM and DkOVM, the permeability of insulin decreased, which may be explained in part, by the action of insulin by adipocytes. A coprecipitation technique was successfully applied to prepare microcapsules of insulin with high encapsulation efficiency. Dissolution stability studies of insulin microcapsules in the presence of enzymes revealed considerable improvement in the availability of insulin with ovomucoids even at the end of 6 hours. Characterization of insulin in the microcapsules using DSC, FT-IR, powder x-ray diffraction and size exclusion chromatography revealed that the structure of insulin was conserved after subjecting it to formulation and process conditions. A three-factor, three-level optimization design was used to evaluate the effect of critical process variables including the rate of addition of polymeric solution, compression pressure and volume of water with respect to polymeric solution. Mathematical relationships, contour plots and response surface methods were employed with constrained optimization to predict levels of factors that provide optimum response. The predicted and observed values were in close agreement. The release of DkOVM was delayed from the formulation. The novel formulation incorporates controlled release characteristics of both protein and inhibitor that may enhance the stability and availability of proteins with less potential for inhibitor related toxicity.Item The effects of chemical modification on spinach nitrite reductase(Texas Tech University, 1996-08) Dose, Michelle MarleneIt was demonstrated that chemical modification of spinach nitrite reductase with phenylglyoxal, pyridoxal-5'-phosphate, and N-bromosuccinimide eliminated the ferredoxin linked activity of the enzyme. It is believed that the former two reagents modify specific arginine and lysine residues, respectively, and thereby interfere with the electrostatic interaction between nitrite reductase and ferredoxin. The latter reagent, Nbromosuccinimide, is believed to modify a specific tryptophan which is needed to assist in the transfer of electrons fi"om the [2Fe-2S] cluster of ferredoxin to the [4Fe-4S] cluster of nitrite reductase. The purpose of this dissertation was to determine the arginine, lysine, and tryptophan residues necessary for nitrite reductase activity. Chemical modification with [C] phenylglyoxal resulted in the identification of two arginine residues, R375 and R556, that appear to be located at the ferredoxinbinding site. Chemical modification with [^H] pyridoxal-5'-phosphate resulted in the indentification of one lysine residue at postion 437 that also appeared to be located at the ferredoxin-binding site of spinach nitrite reductase. These residues are likely to be involved in the electrostatic interaction between ferredoxin and the enzyme. Modification of the enzyme with N-bromosuccinimide led to the identification of a tryptophan residue, W92, that appeared to be at the ferredoxin-binding site and suggested that it assists in the transfer of electrons from ferredoxin to nitrite reductase.Item The effects of site-directed mutation on spinach nitrite reductase(Texas Tech University, 2004-05) Tripathy, Jatindra NathFerredoxin-nitrite oxidoreductase (NiR) catalyzes the six electron reduction of nitrite to ammonia, with reduced ferredoxin (Fd) serving as the electron donor. Chemical modification studies of spinach chloroplast nitrite reductase suggested that tryptophan at 92 (W92) position in NiR is located near the Fd-binding site of the enzyme and plays an important role in the NiR-catalyzed reaction. W92 is absolutely conserved in ferredoxin-dependent NiR's from higher plants, algae, and photosynthetic bacteria, consistent with an essential role for this tryptophan. The ferredoxin dependent nitrite reductase from spinach was expressed in Escherichia coli using the pET30b expression system. Coexpression of bacterial cysG gene with pET30b-NiR or inclusion of 6-aminolevulinic acid, a heme precursor, in the growth medium, resulted in increased amount of pure protein per liter of culture and greater specific activity. W92 was mutated to alanine (A), valine (V), and tyrosine (Y) in order to further test the role of W92 in the NiR-catalyzed reaction. The wild-type NiR and its W92 variants, all were purified to homogeneity, and all exhibited essentially identical visible-region spectra. The Fd-linked specific activity of the W92A variant was only 20% of that observed with the wild-type NiR. The W92V variant exhibited a specific activity 30% of the wild-type level and the W92Y variant exhibited a specific activity 37% of the wild-type level. A very similar pattern was observed for the methyl viologen-linked specific activity of these NiR variants (methyl viologen is a non-physiological electron donor). The binding constant (Kd) for nitrite was 15 (±5) µM for the wild-type NiR and all of its W92 variants. The Kd for Fd was 1 (±0.2) µM for wild-type NiR and ranged from 2 µM to 7 µM for the three W92 variants. Km values for nitrite and ferredoxin were measured for wild-type NiR and its W92 variants, allowing calculation of the catalytic efficiencies. The W92A variant displayed 80% lower catalytic efficiency with respect to nitrite, and 40% lower catalytic efficiency with respect to ferredoxin, compared to the wild-type enzyme. The catalytic efficiencies, with respect to nitrite, for W92V and W92Y variants decreased by approximately 25% compared to the wild-type enzyme. However, the catalytic efficiencies, with respect to ferredoxin, for W92V and W92Y variants were greater than that of wild-type NiR.Item The nature of N-5 reactivity in flavins and D-amino acid oxidase-inhibitor complexes(Texas Tech University, 1978-12) Choi, Jung-DoNot availableItem Thermal dependence of pyrithiobac efficacy in Amaranthus palmeri(Texas Tech University, 1999-12) Light, Ginger G.Variability in weed control following pyrithiobac applications has been observed under field conditions. The influence of temperature on this variability was investigated. Results from field studies performed over two growing seasons identified plant and air temperatures at the time of herbicide treatment that correlated with differences in whole-plant efficacy (R^ = 0.90). Based on the field data, weed control with pyrithiobac was acceptable at application temperatures of 20 to 34° C. To investigate a potential source of thermal limitations on pyrithiobac efficacy, the thermal dependence of in vitro inhibition of acetolactate synthase (ALS), the site of action for pyrithiobac, was examined. A crude leaf extract of ALS was obtained from seedling Amaranthus palmeri. Relative inhibitor potency (I50) values were obtained at saturating substrate conditions for temperatures from 10 to 50° 0. Regression analysis of field activity against I50 values showed the two data sets to be highly correlated (F^ = 0.88). Historic air temperature data sets collected over eleven growing seasons were evaluated to assess the probability, duration, and frequency of the temperature range where acceptable weed control occurred. The recommended thermal range occurred during 59 to 93% of the daylight hours in a typical growing season. Conversely, up to 41% of pyrithiobac applications are potentially adversely affected by application temperature. Computer images of seasonal temperature data with a color oveday corresponding to the temperatures below, within, and above the recommended application temperature range were developed to provide a visualization of seasonal efficacy. The thermal dependence of enzyme/herbicide interactions may provide another means of understanding environmental factors limiting herbicidal efficacy and predicting herbicide inhibition at the whole-plant level.