Effects Of Possible Inhibitors Research Articles

Protein Amyloidogenesis Investigated by Small Angle Scattering.

In the last decades, the study of the mechanisms inducing amyloid fibril formation has involved several experimental and theoretical biophysical approaches. Many efforts have been made by scientist at the borderline between biology, chemistry, biochemistry and physics in order to understand why and in which way a protein starts its amyloidogenic pattern. This fundamental research issue is evolving in parallel to the development of drugs and inhibitors able to modify protein self assembly towards amyloid fibrils. Small angle xray and neutron scattering experiments represent suitable methods to investigate protein amyloidogenesis and the possible effects of inhibitors: they are in-solution techniques, require low amount of sample and their time-resolution makes it possible to follow aggregation pattern. In this paper we review small angle x-ray and neutron scattering studies dedicated to investigate amyloid β peptide and α-synuclein, related to Alzheimer`s and Parkinson`s diseases, respectively, together with some other studies that introduced innovative models to describe with small angle scattering techniques amyloid fibrillation processes.

Abstract B039: In silico models for B cell receptor signaling in chronic lymphocytic leukemia

Abstract Recent discoveries of ZAP-70 (ζ chain–associated protein kinase of 70 kD) and Syk (spleen tyrosine kinase) inhibitors (gefitinib1 and fostamatinib2 respectively) provide promising therapeutic options for patients with chronic lymphocytic leukemia (CLL). However, the precise action of these inhibitors in the B cell receptor (BCR) signaling pathway is not well understood. This study designs an integrated mechanistic description of the BCR signaling pathway in CLL cells, and investigates the effects of possible inhibitors on this pathway. In this work, we examined several computational models of ZAP-70 and Syk regulation in the BCR signaling pathway to explain the observed differences in the clinical behaviors of ZAP+ / ZAP- phenotypes of B-CLL patients. Specifically, we characterized the effects of different ZAP-70 and Syk expression and phosphorylation levels on the BCR activation threshold. The correlations between the observed and calculated trends are reproduced quantitatively. Our calculations show that depending on the amount of Syk that is expressed in cells, increased ZAP-70 expression is correlated with decreased levels of phosphorylated ZAP-70 and Syk, and vice versa. ZAP-70 and Syk phosphorylation occur independently, but is dependent on the amount of ZAP-70 and Syk that is expressed in cells. These results support reported experimental observations.3 We also find that ZAP-70 is able to compensate for a missing Syk functionality with an increased BCR activation threshold in Syk-deficient B-CLL cells, similar to Syk-deficient B cell study.4 In addition, we find that selective inhibition of either Syk or ZAP-70 will result in a disease relapse. This suggests the used of two inhibitors rather than one for the treatment of ZAP-70+ patients, based on our model. Lastly, we find that enhanced or reduced BCR signaling is observed at low amounts of Syk expression in CLL cells. However, this computational prediction needs to be verified experimentally in order to identify a viable physiological range of the amount of Syk that is expressed in B-CLL cells for ZAP-70+ and ZAP-70- phenotypes. Our results uncover molecular mechanisms of ZAP-70 and Syk regulation in the BCR signaling pathway for its future therapeutic use in B-CLL. Our in silico network analysis will help in the development of immunotherapies targeting ZAP-70 and Syk functions to regulate B-CLL responses. Furthermore, our study provides information about responses to certain cancer therapies, and tumor progression.

Differences in the Angiogenic Response and Subsequent Growth of Plasma Cells From Myeloma and MGUS Patients Xenografted Into Zebrafish Embryos.

AbstractAbstract 2912A monoclonal gammopathy of uncertain significance (MGUS) is an obligate precursor to each case of multiple myeloma (MM). MGUS and MM samples share many of the same gene mutations and chromosomal translocations. Despite increasing genetic and genomic information gathered from patient samples, we still do not understand which mutations are responsible for the progression from MGUS to MM. In order to learn more about this progression and the possible involvement of an angiogenic switch as seen in solid tumors, we have developed a zebrafish model to study the angiogenic potential and subsequent growth of CD138+ plasma cells derived from normal human bone marrow, MGUS and myeloma patients. Immunopurified CD138+ cells were labeled with the red fluorescent dye CM DiI, suspended in Matrigelâ and injected into the perivitelline space of Tg(fli1:GFP) zebrafish embryos that had fluorescent vasculature, 48 hpf (hour-post-fertilization). Cell growth and angiogenesis were assessed by conventional and confocal fluorescent microscopy. Inhibitors of myeloma cell growth or tumor-induced angiogenesis were added to embryo water. The injection of up to 2000 plasma cells from normal human bone marrow into zebrafish embryos did not elicit an angiogenic response and the cells did not grow after injection. In contrast, the injection of as few as 100 plasma cells purified from the marrow of patients with multiple myeloma induced a brisk angiogenic response with extension of new vessels ventrally from the sub-intestinal venous system into the site of cell injection. Angiogenesis was accompanied by the growth of the CD138+ cells and, in some cases, movement of cells out of the Matrigelâ plug to distant sites in the developing embryo. MGUS cells induced minimal or no angiogenesis and grew very slowly after injection. The growth of cells from MGUS patients and de novo myeloma patients was inhibited by the addition of the VEGFR2 inhibitor SU5416 to embryo water 24 hpi (hours-post-tumor cell-injection). If the addition of SU5416 was delayed until 48 hpi, when the angiogenic response had been established, there was no effect on cell growth. We also observed that dexamethasone, bortezimib and lenalidomide, singly and in combination, inhibited the growth of MGUS and myeloma cells. These studies document clear biologic differences between normal, MGUS and myeloma plasma cells. In addition, we provide evidence that the zebrafish embryo can be used to study tumor-induced angiogenesis, tumor cell growth and the effects of possible inhibitors. We also provide some new insights into the mechanism underlying the disappointing response of angiogenic inhibitors when given to patients with multiple myeloma. Disclosures:Munshi:Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

Enhanced Stability and Decolorization of Coomassie Brilliant Blue R-250 by Dextran Aldehyde-modified Horseradish Peroxidase

Horseradish peroxidase (EC 1.11.1.7) was chemically modified by periodate-activated dextran. The activities of free and modified enzyme against organic-aqueous interface and some chemicals were determined. Modified HRP remained fully active in the presence of organic solvent for 4 h. However, the unmodified enzyme lost 50% of its activity within the first 2 h. The effects of possible inhibitors on enzyme activity were investigated. In addition, Coomassie Brilliant Blue R-250 was efficiently decolorized using the free and modified HRP. After 5 minutes of treatment, the color removal of dye was 80-90%. Modified HRP showed effective performance compared to free HRP.

Purification of a new manganese peroxidase of the white-rot fungus Schizophyllum sp. F17, and decolorization of azo dyes by the enzyme

The manganese peroxidase (MnP) was isolated and purified from Schizophyllum sp. F17 on pinewood solid-state cultures. Fractionation of MnP was performed by Sephadex G-75 gel filtration chromatography followed by DEAE-cellulose anion exchange chromatography. This purification attained 3.8% activity yield with a purification factor of 9.2. According to data on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the enzyme was 48.7 kDa. The enzyme was a glycoprotein with 18% of its weight in carbohydrates. The optimum pH and temperature of purified MnP for oxidation of DMP were 6.8 and 35 °C, respectively. This enzyme was stable in the pH range 4.0–7.0, at 25 °C for 1 h incubation period. The effects of possible inhibitors and activators of enzyme activity were investigated. The K m values of MnP for Mn 2+, H 2O 2, guaiacol, ABTS and DMP were 35.2, 6.7, 13.1, 47.8 and 122.9 μM at pH 4.5, respectively. The azo dyes such as Congo Red, Orange G and Orange IV were efficiently decolorized using the MnP purified.

Novel method for studying myelination in vivo reveals that EDTA is a potent inhibitor of myelin protein and mRNA expression during development of the rat sciatic nerve.

To probe the effects of possible inhibitors or enhancers of in vivo myelination, we have modified a technique widely used in studies of the developing neuromuscular system that involves incorporation of test compounds into a silicon rubber solution, which solidifies on contact with air. U-shaped rubber implants are inserted around the sciatic nerve of 1-day-old rats and left in place for 24-48 h. Sections from the region of the nerve lying within the implant, with or without the test compound, are then immunolabeled, examined with in situ hybridization or electron microscopy. Application of EDTA (440 microg/implant) in this way strongly suppressed the levels of the myelin-associated molecules protein P0, myelin basic protein (MBP), and galactocerebroside (Galc). mRNA levels for P0 and the myelin-related transcription factor Krox-20 were also reduced, further supporting association of the EDTA-induced effect with the myelinating Schwann cells. In contrast, no obvious differences were observed in either neurofilament (NF) protein or glial fibrillary acidic protein (GFAP) expression, suggesting absence of influence on axons or nonmyelinating Schwann cells. Despite the severely altered molecular composition of myelin in the presence of EDTA, examination in the electron microscope did not reveal any apparent ultrastructural changes in the myelin sheaths or nerve development. This work introduces a novel method for studying nerve development and shows that EDTA, which chelates divalent cations such as Ca(2+) and Mg(2+), strongly and selectively reduces levels of molecules, which, on postnatal days 1-4, are expressed in myelinating cells at much higher levels than in cells not engaged in myelination.

Purification and partial characterization of manganese peroxidase from immobilized Phanerochaete chrysosporium

Solid-state culture of the white-rot fungus Phanerochaete chrysosporium BKMF-1767 (ATCC 24725) has been carried out, using an inert support, polystyrene foam. Suitable medium and culture conditions have been chosen to favor the secretion of manganese peroxidase (MnP). The enzyme was isolated and purified from immobilized P. chrysosporium and partially characterized. Partial protein precipitation in crude enzyme was affected using ammonium sulphate, polyethylene glycol, methanol, and ethanol methods. Fractionation of MnP was performed by DEAE-Sepharose ion exchange chromatography followed by Ultragel AcA 54 gel filtration chromatography. This purification attained 23.08% activity yield with a purification factor of 5.8. According to data on gel filtration chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the enzyme was 45 000±1000 Da. The optimum pH and temperature of purified MnP were 4.5 and 30 °C, respectively. This enzyme was stable in the pH range 4.5–6.0, at 25 °C and also up to 35 °C at pH 4.5 for 1 h incubation period. MnP activity was inhibited by 2 mM NaN 3, ascorbic acid, β-mercaptoethanol and dithreitol. The K m values of MnP for hydrogen peroxide and 2.6-dimetoxyphenol were 71.4 and 28.57 μM at pH 4.5, respectively. The effects of possible inhibitors and activators of enzyme activity were investigated.

98/01877 A test system to analyse the complex physiological states of coal solubilizing fungi : Holker, U. et al. Fuel Process. Technol., 1997, 52, (1–3), 65–71

Solid-state culture of the white-rot fungus Phanerochaete chrysosporium BKMF-1767 (ATCC 24725) has been carried out, using an inert support, polystyrene foam. Suitable medium and culture conditions have been chosen to favor the secretion of manganese peroxidase (MnP). The enzyme was isolated and purified from immobilized P. chrysosporium and partially characterized. Partial protein precipitation in crude enzyme was affected using ammonium sulphate, polyethylene glycol, methanol, and ethanol methods. Fractionation of MnP was performed by DEAE-Sepharose ion exchange chromatography followed by Ultragel AcA 54 gel filtration chromatography. This purification attained 23.08% activity yield with a purification factor of 5.8. According to data on gel filtration chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the enzyme was 45000±1000 Da. The optimum pH and temperature of purified MnP were 4.5 and 30 °C, respectively. This enzyme was stable in the pH range 4.5–6.0, at 25 °C and also up to 35 °C at pH 4.5 for 1 h incubation period. MnP activity was inhibited by 2 mM NaN3, ascorbic acid, β-mercaptoethanol and dithreitol. The Km values of MnP for hydrogen peroxide and 2.6-dimetoxyphenol were 71.4 and 28.57 μM at pH 4.5, respectively. The effects of possible inhibitors and activators of enzyme activity were investigated.

P-512 - Effects of possible inhibitors of acetylcholine release on synaptosomal uptake of choline.

P-512 - Effects of possible inhibitors of acetylcholine release on synaptosomal uptake of choline.

Rapid solution assays for retroviral integration reactions and their use in kinetic analyses of wild-type and mutant Rous sarcoma virus integrases.

A rapid method for quantitating products of the oligodeoxynucleotide processing reaction in vitro has been developed to facilitate enzymatic studies of the retroviral integrases. Unlike earlier procedures, this assay does not depend on polyacrylamide gel electrphoresis but separates products by batch adsorption to PEI-cellulose. A joining assay has also been modified, to facilitate measurement of the two distinct steps in the integration reaction under parallel conditions. Since these methods allow quantitation of numerous samples in a short period of time, they are especially useful for investigation of kinetic parameters and to measure the effects of possible inhibitors of integrase. These assay systems were used to examine the enzymatic activity of wild-type Rous sarcoma virus integrase and selected mutant proteins with substitutions of single conserved amino acids. In contrast to previous studies, reactions were performed under conditions of substrate excess, and rates, rather than yields of product generated after a given period of incubation, were determined. The results showed that substitutions of several highly conserved residues in what is most likely an evolutionarily conserved catalytic domain of the integrases resulted in a 4- to 10-fold decrease in the apparent rate of processing relative to wild type, under optimized standard conditions. Changing an invariant acidic residue reduced the rate by approximately 60-fold. When joining activity was determined, the relative effects of the substitutions tested generally paralleled the results with processing. However, with both wild-type and mutant integrase proteins, the linear phase of the joining reaction was preceded by what appears to be an exponential "burst" phase.

Transport of drugs through human erythrocyte membranes: pH dependence of drug transport through labeled human erythrocytes in the presence of band 3 protein inhibitor

AbstractTo reveal the role of the band 3 anion transport protein of the erythrocyte membrane in drug transport through the membrane, the possible effects of inhibitors of anion transport on the permeability of some anionic drugs were examined. The amounts of these drugs that permeated varied markedly with the pH of the outer medium around human erythrocytes that contained the band 3 protein inhibitor 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonate (DIDS). In the pH range 7.0–8.0, the membrane permeability of drugs through DIDS‐treated erythrocytes was affected by a slight pH change (0.2), whereas that through the intact erythrocytes was not pH dependent. These results suggest that the band 3 protein acts not only as a channel for the transport of anions or some anionic drugs but also as protection for the transport system from changes in the pH of the outer medium.

Modification of vesicular dopamine and norepinephrine by mono amine oxidase inhibitors

The possible effects of inhibitors of the two forms of monoamine oxidase (types A and B) on dopamine (DA) and norepinephrine (NE) accumulation and metabolism in the cytoplasmic and microsomal (vesicular) fractions of the rat brain have been examined. It was found that, while l-DOPA treatment raised only cytoplasmic DA without affecting vesicular DA and NE, clorgyline and pargyline treatments caused significant increases in DA and NE concentrations in both cytoplasmic and vesicular fractions. The DA increase in the synaptic vesicles (200–600%) was much more pronounced than that (150%) in the cytoplasm. In contrast, deprenyl treatment increased vesicular DA only slightly without any effect on either vesicular or cytoplasmic NE. l-DOPA administration to rats pretreated with clorgyline and pargyline, but not with deprenyl, further increased cytoplasmic and vesicular DA and NE concentrations. However, excessive increases in vesicular DA lowered vesicular NE. Reserpine drastically reduced vesicular and cytoplasmic DA and NE, and l-DOPA administration to the reserpine-treated rats caused a DA increase only in the cytoplasmic fraction without affecting vesicular DA or NE. The effect of reserpine was abolished by pargyline treatment, which suggests that pargyline may interact with the reserpine-sensitive vesicular uptake. There was a significant correlation between vesicular DA and NE increase.

Incorporation of amino acids into proteins catalyzed by prostate nuclei

Purified rat ventral prostate nuclei, lacking the outer layer of their nuclear envelopes, incorporated radioactive amino acids into hot trichloroacetic acid-insoluble materials. Some properties of this reaction examined include: kinetics, pH and temperature dependence, effects of possible inhibitors, and of changes in concentration of nuclei or radioisotope. Association of radioactivity with incubated nuclei was demonstrated by light microscope autoradiography. Labelled proteins extracted from prostate nuclei and microsomes and examined by polyacrylamide gel electrophoresis yielded dissimilar patterns. This limited incorporation, catalyzed by prostate nuclei, resembles that described with rat thymus, liver and HeLa cell nuclei. While its biochemical role as reflected by assay in vitro is obscure, administration of androgen restored the reduced activity that followed castration; the process is partially androgen-dependent.