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Biomedical subjects

Paul Wentworth

Publications and source records attributed to Paul Wentworth.

At least 19 recordsLinked to original sources
PubMed2006

Proatherogenic effects of the cholesterol ozonolysis products, atheronal-A and atheronal-B.

The proatherogenic properties of the cholesterol 5,6-secosterols (atheronal-A and atheronal-B), recently discovered in atherosclerotic arteries, have been investigated in terms of their effects on monocyte/macrophage function. A fluorescent analogue of atheronal-B (1) (50 microM), when cultured in either aqueous buffer (PBS) or in media containing fetal calf serum (10%), is rapidly taken-up into cultured macrophage (J774.1 or RAW 264.7) cells and accumulates at perinuclear sites (within 1 h). Co-incubation of macrophage cells (J774.1) with atheronal-A (25 microM) and atheronal-B (25 microM) when complexed with low-density lipoprotein (LDL) (100 microg/mL) leads to a significant upregulation of scavenger receptor class A (approximately 3-fold increase relative to LDL alone, p < 0.05) but not CD36, showing that cultured macrophages respond to LDL-complexed atheronals in a manner highly analogous to acetylated LDL rather than oxidized LDL. Both atheronal-A and atheronal-B in solution exhibit a dose-dependent (0-25 microM) induction of chemotaxis of cultured macrophages (p < 0.001). When complexed with LDL (100 microg/mL), atheronal-A (but not atheronal-B) induces a dose-dependent (0-25 microM, p < 0.05) upregulation of the cell-surface adhesion molecule endothelial (E)-selectin on vascular endothelial cells (HUVECs). LDL (100 microg/mL) complexed atheronal-B (25 microM) but not atheronal-A induces cultured human monocytes (THP-1) to differentiate into macrophage cell lineage. When these in vitro data are taken together with the already known effects of cholesterol 5,6-secosterols on foam cell formation and macrophage cytotoxicity, the atheronals possess biological effects that if translated to an in vivo setting could lead to the recruitment, entrapment, dysfunction, and ultimate destruction of macrophages, with the major leukocyte player in inflammatory artery disease. As such, the atheronal molecules may be a new association, in the already complex inter-relationship, between inflammation, cholesterol oxidation, the tissue macrophage, and atherosclerosis.

Animals↗
PubMed2006

Elevated levels of oxidized cholesterol metabolites in Lewy body disease brains accelerate alpha-synuclein fibrilization.

Oxidative stress, inflammation and alpha-synuclein overexpression confer risk for development of alpha-synucleinopathies-neurodegenerative diseases that include Parkinson disease and Lewy body dementia. Dopaminergic neurons undergo degeneration in these diseases and are particularly susceptible to oxidative stress because dopamine metabolism itself creates reactive oxygen species. Intraneuronal deposition of alpha-synuclein as amyloid fibrils or Lewy bodies is the hallmark of these diseases. Herein, we demonstrate that concentrations of oxidative cholesterol metabolites derived from reactive oxygen species are elevated in the cortices of individuals with Lewy body dementia relative to those of age-matched controls, and we show that these metabolites accelerate alpha-synuclein aggregation in vitro. The increase in the production of these cytotoxic cholesterol metabolites is also observed in a dopaminergic cell line that overexpresses alpha-synuclein. By extension, these data lead to the hypothesis that oxidative stress produces cholesterol aldehydes that enable alpha-synuclein aggregation, leading to a pathologic cycle.

Brain↗
PubMed2006

Cofactor-containing antibodies: crystal structure of the original yellow antibody.

Antibodies are generally thought to be a class of proteins that function without the use of cofactors. However, it is not widely appreciated that antibodies are believed to be the major carrier protein in human circulation for the important riboflavin cofactor that is involved in a host of biological phenomena. A further link between riboflavin and antibodies was discovered 30 years ago when a bright-yellow antibody, IgG(GAR), was purified from a patient with multiple myeloma who had turned yellow during the course of her disease. It was subsequently shown that the yellow color of this antibody was due to riboflavin binding. However, it was not known how and where riboflavin was bound to this antibody. We now report the crystal structure of this historically important IgG(GAR) Fab at 3.0-A resolution. The riboflavin is located in the antigen-combining site with its isoalloxazine ring stacked between the parallel aromatic moieties of TyrH33, PheH58, and TyrH100A. Together with additional hydrogen bonds, these interactions reveal the structural basis for high-affinity riboflavin binding. The ligand specificity of IgG(GAR) is compared with another riboflavin-binding antibody, IgG(DOT), which was purified from a second patient with multiple myeloma. The crystal structure of IgG(GAR) provides a starting point for attempts to understand the physiological relevance and chemical functions of cofactor-containing antibodies.

Antibodies, Monoclonal↗
PubMed2006

Small molecule oxidation products trigger disease-associated protein misfolding.

Oxidative stress and inflammation are risk factors for both the development of alpha-synucleinopathies, such as Parkinson's disease and dementia with Lewy bodies, and Alzheimer's disease, the two most common neurodegenerative disorders. These diseases are associated with the neurotoxic deposition of misassembled alpha-synuclein and amyloid-beta (Abeta) peptides, respectively. Both occur sporadically, that is, without detectable disease-related mutations, in the vast majority of cases. Small molecule oxidation products, especially secosterols derived from cholesterol and 4-hydroxynonenal derived from lipid peroxidation, found in afflicted brains, accelerate the misassembly of both Abeta and alpha-synuclein. This Account explores the mechanism of small molecule oxidation product-mediated protein misassembly and possible intervention strategies.

Aldehydes↗
PubMed2005

Immunoglobulins can utilize riboflavin (Vitamin B2) to activate the antibody-catalyzed water oxidation pathway.

We have recently discovered a reaction that all antibodies, regardless of source or antigenic specificity can catalyze, that is the reaction between singlet dioxygen ((1)O(2)(*)) and H(2)O to generate H(2)O(2). We have named this process the antibody-catalyzed water oxidation pathway (ACWOP). As part of our ongoing investigations into the possible biological role of this pathway, we have studied whether isoalloxazine-containing cofactors, that are known to be endogenous photosensitizers via Type-II pathways to generate (1)O(2)(*), such as riboflavin (RF, Vitamin B2) can trigger the ACWOP. Herein we show that regardless of the antigenic specificity or heavy and light chain composition, all antibodies and their fragments are able to intercept the (1)O(2)(*) generated by photo-oxidation of RF in the presence of oxygen (ambient aerobic conditions) to activate the ACWOP. The initial rate of HOOH generation by a panel of murine antibodies ranges from 0.218 to 0.998 microM/min. The initial rate of antibody-catalyzed HOOH production is accelerated in D(2)O and is quenched in NaN(3), highlighting the key intermediacy of (1)O(2)(*) in the process. Critically, the ACWOP is photo-activated at physiologically relevant concentrations of RF (<50 nM) suggesting that this pathway may be relevant in an in vivo setting. Finally, when activated by RF the ACWOP generates oxidants that accelerate the hemolysis of sheep RBCs hinting at a pathophysiological effect of this RF-induced photo-oxidation pathway.

Animals↗
PubMed2005

Exploring the scope of the 29G12 antibody catalyzed 1,3-dipolar cycloaddition reaction.

[Chemical reaction: See text] 29G12 is a murine monoclonal antibody programmed to catalyze the regio- and enantioselective 1,3-dipolar cycloaddition reaction between 4-acetamidobenzonitrile N-oxide 1a and N,N-dimethylacrylamide 2a (Toker, J. D.; Wentworth, P., Jr.; Hu, Y.; Houk, K. N.; Janda, K. D. J. Am. Chem. Soc. 2000, 122, 3244). Given the unique nature of 29G12 as a protein biocatalyst for this chemical reaction, we have investigated both the substrate specificity and mechanistic parameters of the 29G12-catalyzed process. These studies have shown that while 29G12 is specific for its dipole substrate 1a, the antibody is highly promiscuous with respect to the dipolarophiles it can process. 29G12 accepts a bulky hydrophobic dipolarophile cosubstrate, with rates of product formation up to 70-fold faster than with the original substrate 2a. In all cases, the respective isoxazoline products are produced with exquisite regio- and stereochemical control (78-98% ee). Comparison between the steady-state kinetic parameters from the 29G12-catalyzed reaction of 1a with the most efficient versus the original dipolarophile cosubstrate (2m and 2a, respectively), reveals that while the effective molarities (EM)s are almost identical (EM(2m)) 26 M; EM((2a)) 23 M), the affinity of 29G12 for the larger dipolarophile 2m is more than 1 order of magnitude higher than for 2a [Km(2m) 0.44 +/- 0.04 mM; Km(2a) 5.8 +/- 0.4 mM]. Furthermore, when 2m is the cosubstrate, the affinity of 29G12 for its dipole 1a is also greatly improved [Km(1a) 0.82 +/- 0.1 mM compared to Km(1a) 3.4 +/- 0.4 mM when 2a is the cosubstrate]. An analysis of the temperature dependence of the 29G12-catalyzed reaction between 1a and 2m reveals that catalysis is achieved via a decrease in enthalpy of activation (DeltaDeltaH 4.4 kcal mol(-1)) and involves a large increase in the entropy of activation (DeltaDeltaS 10.4 eu). The improved affinity of 29G12 for the nitrile oxide 1a in the presence of 2m, coupled with the increase in DeltaDeltaS during the 29G12-catalyzed reaction between 1a and 2m supports the notion of a structural reorganization of the active site to facilitate this antibody-catalyzed reaction.

Acrylamides↗
PubMed2004

Metabolite-initiated protein misfolding may trigger Alzheimer's disease.

Anfinsen showed that a protein's fold is specified by its sequence. Although it is clear why mutant proteins form amyloid, it is harder to rationalize why a wild-type protein adopts a native conformation in most individuals, but it misfolds in a minority of others, in what should be a common extracellular environment. This discrepancy suggests that another event likely triggers misfolding in sporadic amyloid disease. One possibility is that an abnormal metabolite, generated only in some individuals, covalently modifies the protein or peptide and causes it to misfold, but evidence for this is sparse. Candidate metabolites are suggested by the recently appreciated links between Alzheimer's disease (AD) and atherosclerosis, known chronic inflammatory metabolites, and the newly discovered generation of ozone during inflammation. Here we report detection of cholesterol ozonolysis products in human brains. These products and a related, lipid-derived aldehyde covalently modify Abeta, dramatically accelerating its amyloidogenesis in vitro, providing a possible chemical link between hypercholesterolemia, inflammation, atherosclerosis, and sporadic AD.

Alzheimer Disease↗
PubMed2004

Probing the antibody-catalyzed water-oxidation pathway at atomic resolution.

Antibodies can catalyze the generation of hydrogen peroxide (H2O2) from singlet dioxygen (1O2*) and water via the postulated intermediacy of dihydrogen trioxide (H2O3) and other trioxygen species. Nine different crystal structures were determined to elucidate the chemical consequences to the antibody molecule itself of exposure to such reactive intermediates and to provide insights into the location on the antibody where these species could be generated. Herein, we report structural evidence for modifications of two specific antibody residues within the interfacial region of the variable and constant domains of different murine antibody antigen-binding fragments (Fabs) by reactive species generated during the antibody-catalyzed water oxidation process. Crystal structure analyses of murine Fabs 4C6 and 13G5 after UV-irradiation revealed complex oxidative modifications to tryptophan L163 and, in 4C6, hydroxylation of the Cgamma of glutamine H6. These discrete modifications of specific residues add further support for the "active site" of the water-oxidation pathway being located within the interfacial region of the constant and variable domains and highlight the general resistance of the antibody molecule to oxidation by reactive oxygen species generated during the water-oxidation process.

Amino Acid Sequence↗
PubMed2004

Reduction of ischemia and reperfusion-induced myocardial damage by cytochrome P450 inhibitors.

Ischemia and reperfusion both contribute to tissue damage after myocardial infarction. Although many drugs have been shown to reduce infarct size when administered before ischemia, few have been shown to be effective when administered at reperfusion. Moreover, although it is generally accepted that a burst of reactive oxygen species (ROS) occurs at the onset of reperfusion and contributes to tissue damage, the source of ROS and the mechanism of injury is unclear. We now report the finding that chloramphenicol administered at reperfusion reduced infarct size by 60% in a Langendorff isolated perfused rat heart model, and that ROS production was also substantially reduced. Chloramphenicol is an inhibitor of mitochondrial protein synthesis and is also an inhibitor of a subset of cytochrome P450 monooxygenases (CYPs). We could not detect any effect on mitochondrial encoded proteins or mitochondrial respiration in chloramphenicol-perfused hearts, and hypothesized that the effect was caused by inhibition of CYPs. We tested additional CYP inhibitors and found that cimetidine and sulfaphenazole, two CYP inhibitors that have no effect on mitochondrial protein synthesis, were also able to reduce creatine kinase release and infarct size in the Langendorff model. We also showed that chloramphenicol reduced infarct size in an open chest rabbit model of regional ischemia. Taken together, these findings implicate CYPs in myocardial ischemia/reperfusion injury.

Animals↗
PubMed2004

Probing lipase/esterase libraries for lipid A hydrolases--discovery of biocatalysts for the detoxification of bacterially-expressed recombinant protein.

In our ongoing efforts to develop new methods for lipopolysaccharide (LPS) detoxification, we have screened lipase/esterase libraries for the ability to deacylate the 2'- and 3'-fatty acid chains from lipid A: the most active esterases were successfully employed to inactivate LPSs in a crude concentrated cell supernatant of E. Coli containing a recombinant single chain antibody (scFv).

Animals↗
PubMed2004

The antibody-catalyzed water oxidation pathway--a new chemical arm to immune defense?

Antibodies are the classical adaptor molecules of the immune system, linking recognition and killing of foreign pathogens. However, the recent discovery of a new property of the antibody molecule suggests a previously unexplored effector function of the immune system. All immunoglobulins, regardless of source or antigenic specificity, can catalyze the reaction between singlet (1Deltag) oxygen and water to give hydrogen peroxide (H(2)O(2)). Both the chemical and biological aspects of this pathway are being explored and intriguing new insights into how this pathway might have a role in immune defense are emerging.

Animals↗
PubMed2003

Crystallization of a carbamatase catalytic antibody Fab fragment and its complex with a transition-state analogue.

Catalytic antibodies showing carbamatase activity have significant potential in antibody-directed prodrug therapy against tumours. The Fab fragment of an IgG1 mouse monoclonal carbamatase catalytic antibody JC1 raised against a transition-state analogue, ethyl N-(3,5-dicarboxyphenyl)-P-[N-[5'-(2",5"-dioxo-1"-pyrrolidinyl)oxy-1',5'-dioxopentyl]-4-aminophenylmethyl]phosphonamidate, was obtained by digestion of the whole antibody with papain and was purified by two-step ion-exchange chromatography. Using hanging-drop vapour-diffusion crystallization techniques, three different crystal forms of the Fab fragment were obtained in the presence and absence of the transition-state analogue. All crystals diffract X-rays to between 3.5 and 3.2 A resolution. The two crystal forms grown in the presence of the transition-state analogue contain up to four or eight copies of the Fab in the asymmetric unit and diffract to 3.5 and 3.2 A, respectively. The crystal of the Fab alone is most likely to contain only two copies of the Fab in the asymmetric unit and diffracts to beyond 3.5 A. Determination of the structure will provide insights into the active-site arrangement of this antibody and will help to increase our understanding of the molecular mechanisms by which the immune system can evolve catalytic function.

Amino Acid Sequence↗
PubMed2003

Total synthesis of the depsipeptide FR-901375.

The first total synthesis of FR-901375, a novel bicyclic depsipeptide isolated from the fermentation broth of Pseudomonas chloroaphis No. 2522, has been achieved. The synthetic approach involves 13 reaction steps and is achieved in 12% overall yield. The key points in the successful synthetic strategy are a concise asymmetric synthesis of the key building block (3R,4E)-3-hydroxy-7-mercapto-4-heptenoic acid, a mild Mitsunobu macrolactonization step, and an I(2)-mediated deprotection with concomitant disulfide-bridge formation.

Depsipeptides↗
PubMed2003

Evidence for ozone formation in human atherosclerotic arteries.

Here, we report evidence for the production of ozone in human disease. Signature products unique to cholesterol ozonolysis are present within atherosclerotic tissue at the time of carotid endarterectomy, suggesting that ozone production occurred during lesion development. Furthermore, advanced atherosclerotic plaques generate ozone when the leukocytes within the diseased arteries are activated in vitro. The steroids produced by cholesterol ozonolysis cause effects that are thought to be critical to the pathogenesis of atherosclerosis, including cytotoxicity, lipid-loading in macrophages, and deformation of the apolipoprotein B-100 secondary structure. We propose the trivial designation "atheronals" for this previously unrecognized class of steroids.

Arteriosclerosis↗
PubMed2003

Investigating antibody-catalyzed ozone generation by human neutrophils.

Recent studies have suggested that antibodies can catalyze the generation of previously unknown oxidants including dihydrogen trioxide (H(2)O(3)) and ozone (O(3)) from singlet oxygen ((1)O(2)(*)) and water. Given that neutrophils have the potential both to produce (1)O(2)(*) and to bind antibodies, we considered that these cells could be a biological source of O(3). We report here further analytical evidence that antibody-coated neutrophils, after activation, produce an oxidant with the chemical signature of O(3). This process is independent of surface antibody concentration down to 50% of the resting concentration, suggesting that surface IgG is highly efficient at intercepting the neutrophil-generated (1)O(2)(*). Vinylbenzoic acid, an orthogonal probe for ozone detection, is oxidized by activated neutrophils to 4-carboxybenzaldehyde in a manner analogous to that obtained for its oxidation by ozone in solution. This discovery of the production of such a powerful oxidant in a biological context raises questions about not only the capacity of O(3) to kill invading microorganisms but also its role in amplification of the inflammatory response by signaling and gene activation.

Animals↗
PubMed2003

Soluble polymer-supported convergent parallel library synthesis.

Soluble polymer-supported convergent synthesis has for the first time been successfully exploited for parallel library synthesis; sub-libraries of tripeptide iodoarenes and arylboronic acids reacted smoothly in a multipolymer PdII-catalyzed Suzuki coupling reaction to generate a library of bisaryl-linked hexapeptides.

Combinatorial Chemistry Techniques↗
PubMed2003

Evidence for the production of trioxygen species during antibody-catalyzed chemical modification of antigens.

Recent work in our laboratory showed that products formed by the antibody-catalyzed water-oxidation pathway can kill bacteria. Dihydrogen peroxide, the end product of this pathway, was found to be necessary, but not sufficient, for the observed efficiency of bacterial killing. The search for further bactericidal agents that might be formed along the pathway led to the recognition of an oxidant that, in its interaction with chemical probes, showed the chemical signature of ozone. Here we report that the antibody-catalyzed water-oxidation process is capable of regioselectively converting antibody-bound benzoic acid into para-hydroxy benzoic acid as well as regioselectively hydroxylating the 4-position of the phenyl ring of a single tryptophan residue located in the antibody molecule. We view the occurrence of these highly selective chemical reactions as evidence for the formation of a short-lived hydroxylating radical species within the antibody molecule. In line with our previously presented hypothesis according to which the singlet-oxygen ((1)O*(2)) induced antibody-catalyzed water-oxidation pathways proceeds via the formation of dihydrogen trioxide (H(2)O(3)), we now consider the possibility that the hydroxylating species might be the hydrotrioxy radical HO(3)*, and we point to the remarkable potential of this either H(2)O(3)- or O(3)-derivable species to act as a masked hydroxyl radical HO* in a biological environment.

Animals↗