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Biomedical subjects

I West

Publications and source records attributed to I West.

At least 19 recordsLinked to original sources
PubMed1994

Lectin affinity chromatography.

A protocol is described for the preparation of lectin affinity chromatography columns using purified lectins and preactivated matrices. A general method is given for the purification of glycoproteins on immobilized Con A. Methods for immobilizing Con A on CDI agarose, Affi-Gel 15, and carbonyldiimidazole-activated agarose are described.

Adsorption↗
PubMed1992

Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245.

Activation of the NADPH oxidase of phagocytes in the cell-free system requires the association of several cytosolic components with membrane-bound cytochrome b. In this study we were able to fully reconstitute NADPH oxidase activity in the cell-free system with three recombinant proteins: p67-phox, p47-phox, p21rac1, and pure cytochrome b-245. Activity was dependent upon the concentration of the proteins, with maximal activity observed with roughly equimolar ratios of the cytochrome b and p67-phox (133 and 163 mol/s/mol, respectively) and concentrations of the other two proteins approximately 1 order of magnitude greater. No activity was observed in the absence of any one of these components. In addition, activation was dependent upon p21rac1 being preloaded with GTP, the cytochrome b being reconstituted with lipid, and the presence of FAD during activation. Half-maximal activity was observed at a concentration of NADPH of approximately 50 microM. These findings confirm our recent description of the membrane-bound cytochrome b as a FAD-containing flavocytochrome b containing the NADPH binding site, and implicate the three cytosolic proteins in its activation.

Cell Membrane↗
PubMed1992

Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes.

The NADPH oxidase of phagocytic cells is important for the efficient killing and digestion of ingested microbes. A very unusual low-potential cytochrome b (b-245) is the only redox molecule to have been identified in this system. The FAD-containing flavoprotein that binds NADPH and transfers electrons to the cytochrome has eluded identification for three decades. We show here that the haem/FAD ratio in the membranes does not change significantly on activation of this oxidase, indicating that the FAD is present in the membranes from the outset and not recruited from the cytosol. The FAD content of membranes from cells of patients with X-linked chronic granulomatous disease (CGD) lacking the cytochrome b was roughly one-quarter of that in normal subjects and in autosomal recessive CGD patients lacking the cytosolic protein p47-phox. Similar low amounts of FAD were present in uninduced promyelocytic (HL60) cells, suggesting that the low amount of FAD in cells from X-CGD patients was probably unrelated to this oxidase system. Cytochrome b-245 appears to bind both the haem and FAD, in a molar ratio of 2:1. The e.p.r. signal of the purified cytochrome was weak and had an asymmetric g(z) peak at g = 3.31. The purified cytochrome could be partially reflavinated (about 20%) in the presence of lipid. Amino acid sequence homology was detected between the beta-subunit of this cytochrome b and the ferredoxin-NADP+ reductase (FNR) family of reductases in the putative NADPH- and FAD-binding sites. 32P-labelled 2-azido-NADP was used as a photoaffinity label for the NADPH-binding site. Labelling that was competed off with NADP was observed in the region of the beta-subunit of the cytochrome. No labelling was seen in this region in X-CGD in three subjects in whom this cytochrome was missing and in a third in whom it was present but bore a Pro-His transposition in the putative NADPH-binding site. These studies indicate that cytochrome b-245 is a flavocytochrome, the first described in higher eukaryotic cells, bearing the complete electron-transporting apparatus of the NADPH oxidase.

Amino Acid Sequence↗
PubMed1966

Pesticide-induced illness. Public health aspects of diagnosis and treatment.

Over the past 25 years there has been a remarkable proliferation of new chemicals into our environment, among them the 57,000 different trade-named pesticide formulations now for sale in the United States. With them have come many new problems to physicians needing up-to-the minute toxocologic information, and also sizable problems in public health. Technology must be harnessed to bring to physicians, when they need it, help in the diagnosis and treatment of poisoning and other adverse effects from modern chemicals, for at present it is impossible for the practicing physician to keep up with what is known and unknown about the toxicology of all of these chemicals.

Humans↗