Alterations in human lymphocyte DNA caused by sulfur mustard can be mitigated by selective inhibitors of poly(ADP-ribose) polymerase.

Changes in genomic DNA caused by exposure to the cytotoxic alkylating agent, 2,2'-dichlorodiethyl sulfide (sulfur mustard; HD), alone or in combination with selective inhibitors of poly(ADP-ribose) polymerase (PARP), were analyzed as a function of HD concentration and post-exposure time. Preparations of human peripheral blood lymphocytes were exposed to HD (1x10(-8) M-1x10(-3) M), and incubated at 37 degrees C for 0-24 h. Total genomic DNA was extracted from these cells and compared with DNA from control cells of the same donor using agarose gel electrophoresis. The effects of HD on genomic DNA depended on the HD concentration and the length of the post-exposure time interval. DNA fragmentation was detected as early as 2 h after exposure to 3x10(-4) M HD, or at 24 h after exposure to 6x10(-6) M HD. The qualitative DNA pattern, as well as the extent of DNA fragmentation, changed with post-exposure time. Exposure to HD caused a time-dependent shift in the DNA cleavage pattern from an oligonucleosome-sized 'DNA ladder' characteristic of apoptotic cell death, to a 'broad band' pattern characteristic of necrotic cell death. DNA fragmentation was not observed if cells were killed with heat or with Lewisite. Treatment of cells with selective PARP inhibitors consistently altered the DNA fragmentation caused by HD exposure. The inhibitors arrested DNA fragmentation at the DNA ladder stage. This effect only was observed if the PARP inhibitors were applied within 8 h of HD exposure. We conclude that early inhibition of PARP activity can induce a switch in the mechanism of cell death caused by HD. Such a switch may be useful therapeutically to convert a lytic, pro-inflammatory cell death that includes the disintegration of dying cells (necrosis), into a slower, programmed cell death that includes absorption of dying cells (apoptosis).

Exposure of human lymphocytes to bis-(2-chloroethyl)sulfide solubilizes truncated and intact core histones.

Bis-(2-chloroethyl)sulfide (BCES) is a radiomimetic, bifunctional alkylating agent that cross-links DNA, disrupts higher-order nuclear structure and selectively kills rapidly proliferating cell types. While chemically fractionating primary, human lymphocytes after challenge with cytotoxic doses of BCES, we detected a 12,900 M(r) polypeptide in 1.0 M NaCl extracts of exposed cells that was markedly increased compared to controls. By computer-aided image analysis of polyacrylamide gels, it was detected as early as 4 h following 1 mM BCES and increased approximately 10-fold by 24 h. Two other polypeptides of 16,320 and 16,970 M(r) also were increased measurably at 24 h following BCES exposure. Altered polypeptides were found in 28 of 28 separate lymphocyte preparations ranging in cell density from 5 x 10(6)/ml to 6 x 10(7)/ml. They were not present if cells were killed with equimolar concentrations of a different cytotoxic agent, chlorovinyl-dichloroarsine (lewisite). Appearance of the polypeptides was unaffected by sulfhydryl reducing agents or pretreatment of cells with the protein synthesis inhibitor, cycloheximide. Micro sequencing resulted in a perfect match of the 12,900 M(r) polypeptide amino terminus with residues 19-27 of histone H2B. This corresponds to the exact site of H2B cleavage obtained when intact nucleosomes are treated with chymotrypsin. Sequence data from the other two altered polypeptides identified them as intact histone H2B and histone H3. Lymphocyte genomic DNA integrity also was assessed after BCES exposure and found to undergo extensive fragmentation typical of cellular necrosis. We speculate that exposure of isolated cells to BCES disrupts nucleosome structure by mechanism(s) that involve abnormal removal and perhaps proteolysis of core histones.

Active-site gorge and buried water molecules in crystal structures of acetylcholinesterase from Torpedo californica.

Buried water molecules and the water molecules in the active-site gorge are analyzed for five crystal structures of acetylcholinesterase from Torpedo californica in the resolution range 2.2-2.5 A (native enzyme, and four inhibitor complexes). A total of 45 buried hydration sites are identified, which are populated with between 36 and 41 water molecules. About half of the buried water is located in a distinct region neighboring the active-site gorge. Most of the buried water molecules are very well conserved among the five structures, and have low displacement parameters, B, of magnitudes similar to those of the main-chain atoms of the central beta-sheet structure. The active-site gorge of the native enzyme is filled with over 20 water molecules, which have poor hydrogen-bond coordination with an average of 2.9 polar contacts per water molecule. Upon ligand binding, distinct groups of these water molecules are displaced, whereas the others remain in positions similar to those that they occupy in the native enzyme. Possible roles of the buried water molecules are discussed, including their possible action as a lubricant to allow large-amplitude fluctuations of the loop structures forming the gorge wall. Such fluctuations are required to facilitate traffic of substrate, products and water molecules to and from the active-site. Because of their poor coordination, the gorge water molecules can be considered as "activated" as compared to bulk water. This should allow their easy displacement by incoming substrate. The relatively loose packing of the gorge water molecules leaves numerous small voids, and more efficient space-filling by substrates and inhibitors may be a major driving force of ligand binding.

Synthesis of a thrombospondin-like cytoadhesion molecule by cells of the trabecular meshwork.

Thrombospondin is a large extracellular-matrix protein that is released by smooth muscle cells and platelets and is distributed widely in mammalian brain. By using western blotting with a monoclonal antibody against the calcium-binding domain of thrombospondin, the authors detected a 180-kD glycated polypeptide in the trabecular meshwork tissue of normal human and porcine eyes. The epitope-bearing polypeptide was soluble in sodium dodecylsulfate/urea (SDS/urea), and apparently it was stabilized in part by disulfide bonding to the Triton X-100 and SDS/urea-insoluble pellet of this tissue. Treatment of the insoluble matrix with beta-mercaptoethanol led to an enriched extraction of the approximately 160-kD form of thrombospondin. On immunohistochemical study, the thrombospondin antibody also reacted positively with the extracellular matrix and intracellular structures of trabecular cells in primary monolayer culture. This suggested that the presence of thrombospondin in the trabecular meshwork was probably due to local synthesis. These findings are relevant to the establishment of a model in vitro for assessment of adhesion of trabecular cells. Because thrombospondin is a cell-substrate adhesion molecule, its role in the loss of cellularity that occurs in the trabecular meshwork of the aging eye and in eyes with primary open-angle glaucoma is worthy of further investigation.

Cutaneous exposure to bis-(2-chloroethyl)sulfide results in neutrophil infiltration and increased solubility of 180,000 Mr subepidermal collagens.

Exposure to bis-(2-chloroethyl)sulfide (BCES; "sulfur mustard") causes delayed formation of slowly healing skin blisters. Although the histopathology of BCES injury is well characterized [reviewed in Smith et al., J Am Acad Dermatol 32: 767-776, 1995], little is known of the cutaneous toxicity at the molecular level. To identify biological markers of exposure, epidermal and subepidermal extracts were prepared from 48 individual hairless guinea pigs (HGP) at successive 3-hr intervals following exposure to BCES vapor, and compared using gel electrophoresis, and lectin- and antisera-binding. Inflammation was assessed by measuring edema and myeloperoxidase activity. Edema reached peak levels at 15-18 hr and remained elevated above controls at 24 hr. Recruitment of neutrophils, deduced from increased myeloperoxidase, occurred as early as 3 hr after BCES exposure with maximum infiltration at 6-12 hr. Binding of concanavalin-A lectin revealed increased amounts, relative to contralateral control sites, of two approximately 180,000 Mr polypeptides in subepidermal protein extracts from the BCES-exposed skin obtained > or = 12 hr after exposure. This alteration was not found in epidermal protein extracts prepared from the same animals. Based upon the determined amino acid compositions, both polypeptides had significant collagenous triple helical content (>75%). They could be distinguished immunologically from collagen types I, III, and IV by using polyclonal antisera. We conclude that exposure of HGP skin to BCES results in an early neutrophil infiltration that precedes epidermal-dermal separation and selective alterations of the subepidermal extracellular matrix.

Acetylcholine exocytosis in PC12 cells deficient in SNAP-25.

Stimulus-induced acetylcholine (ACh) exocytosis from presynaptic nerve terminals involves two important steps: fusion of ACh loaded vesicles at presynaptic release sites, followed by release into the synaptic cleft. We studied the role of the putative vesicle fusion protein SNAP-25 in this process. The nerve growth factor-differentiated PC12 cell line was used as an experimental model. A bee venom tetradecapeptide (INLKALAALAKKIL-NH2) phospholipase A2 (PLA2) activator, mastoparan, was used to induce ACh release. Treatment of PC12 cells with appropriate antisense oligonucleotides blocked SNAP-25 expression, as judged by Western blot protein analysis with a specific monoclonal antibody. Despite apparent elimination of SNAP-25, treatment of differentiated PC12 cells with mastoparan and high (80 mM) K+ induced ACh exocytosis. The results indicate that in PC12 cells, ACh exocytosis due to mastoparan plus K+ can occur in the absence of SNAP-25.

Tissue plasminogen activator in human aqueous humor and its possible therapeutic significance.

By using an enzyme-linked immunosorbent assay, we detected a significant amount of tissue plasminogen activator (0.8 +/- 0.17 ng/ml) in the aqueous humor of ten normal human eyes. It was also identified on Western blot analysis. The ratio of tissue plasminogen activator to total protein in aqueous humor was about 30 times greater than the ratio of tissue plasminogen activator to total protein in plasma. There was evidence that at least some of the tissue plasminogen activator in the aqueous humor was synthesized locally by the structures bordering the anterior chamber of the eye. Tissue plasminogen activator may be a useful therapeutic modality in clinical conditions of delayed dissolution of fibrin in the eye.

Protein engineering of a human enzyme that hydrolyzes V and G nerve agents: design, construction and characterization.

Because of deficiencies in the present treatments for organophosphorus anticholinesterase poisoning, we are attempting to develop a catalytic scavenger that can be administered as prophylactic protection. Currently known enzymes are inadequate for this purpose because they have weak binding and slow turnover, so we are trying to make an appropriate enzyme by protein engineering techniques. One butyrylcholinesterase mutant, G117H, has the desired type of activity but reacts much too slowly. This communication describes an attempt to determine the reason for the slow reaction so that a more efficient enzyme might be designed. The results indicate that the mutation at residue 117 has resulted in a distortion of the transition state of the reaction of organophosphorus compounds with the active site serine. This information will be used to develop other mutants that avoid transition state stabilization sites.

A preliminary comparison of structural models for catalytic intermediates of acetylcholinesterase.

Determination of the three dimensional structure of Torpedo Californica acetylcholinesterase (TcAChE) provided an experimental tool for directly visualizing interaction of AChE with cholinesterase inhibitors of fundamental, pharmacological and toxicological interest. The structure revealed that the active site is located near the bottom of a deep and narrow gorge lined with 14 conserved aromatic amino acids. The structure of a complex of TcAChE with the powerful 'transition state analog' inhibitor, TMTFA, suggested that its orientation in the experimentally determined structure was very similar to that proposed for the natural substrate, acetylcholine, by manual docking. The array of enzyme-ligand interactions visualized in the TMFTA complex also are expected to envelope the unstable TI that forms with acetylcholine during acylation, and to sequester it from solvent. In our most recent studies, the crystal structures of several 'aged' conjugates of TcAChE obtained with OP nerve agents have been solved and compared with that of the native enzyme. The methylphosphonylated-enzyme obtained by reaction with soman provides a useful structural analog for the TI that forms during deacylation after the reaction of TcAChE with acetylcholine. By comparing these structures, we conclude that the same 'oxyanion hole' residues, as well as the aromatic side chains constituting the 'acyl pocket', participate in acylation (TMTFA-AChE) and deacylation (OP-AChE), and that AChE can accommodate both TIs at the bottom of the gorge without major conformational movements.

Anticholinesterases: medical applications of neurochemical principles.

Cholinesterases form a family of serine esterases that arise in animals from at least two distinct genes. Multiple forms of these enzymes can be precisely localized and regulated by alternative mRNA splicing and by co- or posttranslational modifications. The high catalytic efficiency of the cholinesterases is quelled by certain very selective reversible and irreversible inhibitors. Owing largely to the important role of acetylcholine hydrolysis in neurotransmission, cholinesterase and its inhibitors have been studied extensively in vivo. In parallel, there has emerged an equally impressive enzyme chemistry literature. Cholinesterase inhibitors are used widely as pesticides; in this regard the compounds are beneficial with concomitant health risks. Poisoning by such compounds can result in an acute but usually manageable medical crisis and may damage the CNS and the PNS, as well as cardiac and skeletal muscle tissue. Some inhibitors have been useful for the treatment of glaucoma and myasthenia gravis, and others are in clinical trials as therapy for Alzheimer's dementia. Concurrently, the most potent inhibitors have been developed as highly toxic chemical warfare agents. We review treatments and sequelae of exposure to selected anticholinesterases, especially organophosphorus compounds and carbamates, as they relate to recent progress in enzyme chemistry.

A computer model of glycosylated human butyrylcholinesterase.

The three-dimensional structure of human serum butyrylcholinesterase (BuChE) was modeled using a computer-based amino acid replacement strategy and the known coordinates of crystallized acetylcholinesterase (AChE) from Torpedo californica. The BuChE model was then energetically minimized with dynamic iterations of an adopted basis Newton-Raphson algorithm and the program CHARMM. Hypothetical glycosylation of this structure based upon the known carbohydrate composition of the enzyme was also performed. The glycosylated, minimized model predicts that the tertiary structure of BuChE could be very similar to AChE but that the entrance of the narrow channel leading toward its active site triad probably differs. All nine of the known N-linked oligosaccharides of BuChE are predicted to occur away from the putative active site channel and most are located on one face of the monomer.

Design and expression of organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase.

Serine esterases and proteases are rapidly and irreversibly inhibited by organophosphorus (OP) nerve agents. To overcome this limitation, we selected several residues that were predicted to be within 3-10 A of both the active site Ser O gamma and the oxyanion hole of human butyrylcholinesterase for mutation to His (G115H, G117H, Q119H, and G121H). In remarkable contrast with wild-type (WT) and all other His mutants tested, G117H underwent spontaneous reactivation following OP inhibition to regain 100% of original esterase activity with maximum k3 values of approximately 6.8 x 10(-5) and 16 x 10(-5) s-1 for GB (sarin) and VX, respectively, in 0.1 M Bis-Tris, 25 degrees C. The free energy of activation for k3 was 19 kcal mol-1, and measurement of pH dependence suggested that reactivation resulted from an acidic group with pKa 6.2. To evaluate further the importance of His in achieving this result, we changed the same Gly to Lys (G117K) and compared its substrate and inhibitor kinetics with those of G117H. Both mutants retained esterase activity with Km values similar to those of WT for neutral ester hydrolysis, but G117K did not reactivate. Complete reactivation proves that G117H is not irreversibly inhibited but instead functions as a catalyst for OP hydrolysis. Dephosphonylation is the rate-limiting step, and G117H effects overall rate constant enhancements of approximately 100- and 2000-fold above the uncatalyzed hydrolysis of GB and VX, respectively, at pH 6.0, 25.0 degrees C. We conclude that an appropriately positioned imidazolium ion in the oxyanion hole catalyzes dephosphonylation and, thereby, confers a novel organophosphorus acid anhydride hydrolase activity upon butyrylcholinesterase.

Organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase: synergy results in a somanase.

Organophosphorus acid anhydride (OP) "nerve agents" are rapid, stoichiometric, and essentially irreversible inhibitors of serine hydrolases. By placing a His near the oxyanion hole of human butyrylcholinesterase (BChE), we made an esterase (G117H) that catalyzed the hydrolysis of several OP, including sarin and VX [Millard et al. (1995) Biochemistry 34, 15925-15930]. G117H was limited, however, because it was irreversibly inhibited by pinacolyl methylphosphonofluoridate (soman); soman is among the most toxic synthetic poisons known. This limitation of G117H has been overcome by a new BChE (G117H/E197Q) that combines two engineered features: spontaneous dephosphonylation and slow aging (dealkylation). G117H/E197Q was compared with the single mutants BChE G117H and E197Q. Each retained cholinesterase activity with butyrylthiocholine as substrate, although kcat/Km decreased 11-, 11- or 110-fold for purified G117H, E197Q, or G117H/E197Q, respectively, as compared with wild-type BChE. Only G117H/E197Q catalyzed soman hydrolysis; all four soman stereoisomers as well as sarin and VX were substrates. Phosphonylation and dephosphonylation reactions were stereospecific. Double mutant thermodynamic cycles suggested that the effects of the His and Gln substitutions on phosphonylation were additive for PSCR or PRCR soman, but were cooperative for the PSCS stereoisomer. Dephosphonylation limited overall OP hydrolysis with apparent rate constants of 0.006, 0.077, and 0.128 min-1 for the PR/SCR, PSCS, and PRCS soman stereoisomers, respectively, at pH 7.5, 25 degrees C. We conclude that synergistic protein design converted an archetypal "irreversible inhibitor" into a slow substrate for the target enzyme.

High-throughput assays for botulinum neurotoxin proteolytic activity: serotypes A, B, D, and F.

Botulinum neurotoxins (BoNT) are zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. Because the paralytic effect of BoNT is a consequence of its enzymatic activity, selective inhibitors may be useful as drugs or as tools for further research. To expedite inhibitor discovery, we developed high-throughput, solid-phase protease activity assays for four of the seven BoNT serotypes: A, B, D, and F. Each assay consisted of a cleavable oligopeptide, based on the natural substrate sequence, labeled with fluorescein and covalently attached to maleimide-activated multiwell plates. Solutions of holotoxin or nontoxic catalytic domain of BoNT were incubated in substrate-coated wells, with or without test compounds, followed by transfer and assay of solubilized product in a multiwell fluorometer. Routine toxin concentrations ranged from 10 to 100 ng/ml, but concentrations as low as 2 ng/ml gave reproducible signals. The fluorescence assays were selective, gave very low background readings, and were stable upon prolonged storage. Using the nontoxic catalytic domain of BoNT A, we determined the relative inhibitory potencies of a family of structurally related pseudotripeptide compounds. Unlike previous methods, our assays did not employ antibodies or reverse-phase extraction steps, only well-to-well transfers, and were easily adapted to a high-throughput automated environment.

Qualitative and quantitative analyses of sialic acid in the human trabecular meshwork.

By using colloidal iron and polycationic ferritin at low pH (1.8 and 1.9, respectively) as electron histochemical stains, we localized sialic acid moieties in the trabecular meshwork of normal human eyes. The markers were distributed continuously over the entire surface of the trabecular cells, but were localized linearly as well as randomly in the basal lamina. Within the beams, the markers were located irregularly in the collagen core, but were absent from the elastic tissue and from the 100 nm banded structures in the cortical zone. Pretreatment of the tissue with neuraminidase abolished this staining which indicates that sialated moieties are present in the stained structures. Biochemical analysis with the lectin Limax flavus agglutinin revealed that the major sialated polypeptides in individual samples of human trabecular meshwork migrated at apparent molecular weights of 56, 75, 95, 128, 140, 180 and 220 kDa under reducing conditions. The fractions at approximate molecular weights of 180 and 220 kDa include the sialoglycoproteins thrombospondin and fibronectin, respectively. The total content of sialic acid in the human trabecular meshwork (patient age range, 27-57 yr) was 3.6 +/- 0.6 mumol g-1 wet weight, as determined by a colorimetric assay. We conclude that significant quantities of sialic acid are present in the normal human trabecular meshwork as neuraminidase-sensitive alpha-ketosidically linked terminal residues of the polypeptides.

Protein composition of human aqueous humor: SDS-PAGE analysis of surgical and post-mortem samples.

We analyzed the protein composition of human aqueous humor. Samples were obtained by paracentesis from 25 human eyes (age range 64-92 years) at elective cataract surgery, and from 20 age-matched post-mortem eyes within 1.5 to 18 hr after death. Individual samples were assayed for total protein, and the polypeptides were separated by qualitative SDS-PAGE into high-, medium- and low-molecular-weight ranges and then silver-stained. The clinical samples showed a remarkable consistency in the total protein values (mean +/- SEM: 12.4 +/- 2.0 mg per 100 ml) and no detectable variations in the profiles of the silver-stained proteins. Twelve major protein fractions, with apparent molecular weights of 140, 80 (doublet), 67, 60 (doublet), 35, 27, 25, 17, 14.6 and 9 kDa, were present. A preliminary analysis showed that the 17 kDa band contained a molecule resembling basic fibroblast growth factor. Two additional samples of aqueous humor from patients whose blood/aqueous barrier was compromised during paracentesis showed a quantitative and qualitative increase in the polypeptides that were present. Compared with the samples of aqueous humor obtained at surgery, the post-mortem samples exhibited a greater variability in total protein content (56.1 +/- 11.6 mg per 100 ml) and an increased number of high- and low-molecular-weight protein fractions. In view of wide differences in the clinical parameters, including ocular and systemic medications, systemic illness, surgical premedications, anesthesia and total serum protein values, the similarity in the protein profiles of the carefully drawn surgical samples is most remarkable. Our results indicate that, in patients who underwent elective cataract surgery, the levels of major proteins in human aqueous humor are not affected by wide individual variations in the clinical parameters. We attribute this finding to the care taken in the collection of aqueous humor samples.

Age-related changes in protein profiles of the normal human trabecular meshwork.

The protein profile of the trabecular meshwork from 44 normal human eyes of various ages, ranging from 5 months to 87 years, was investigated by using sodium dodecyl sulfate-polyacrylamide-gel (SDS-PAGE) electrophoresis and a highly sensitive silver-stain technique. Samples from each eye were analysed individually to avoid anomalies that may occur with tissue pooling. More than 20 protein bands were consistently visible at all ages and varied in molecular weight from 20,000 to 290,000 MW. The shifts which occurred in band prominence with increasing age included a consistent decrease in the intensity of bands at MWs 42,000 (tentatively identified as G-actin) and 58,000; a noticeable increase in the intensity of the bands at MWs 140,000 and 160,000 that correspond to alpha I and II components of type IV collagen; and a gradual disappearance of a faint band at MW 68,000 in eyes older than 31 years. Our study shows that molecular aging does occur in the trabecular meshwork. The data presented also provides a basis for comparison of the polypeptide alterations that may occur in primary open-angle glaucoma and in other disease processes.

Epinephrine-induced toxicity of human trabecular cells in vitro.

To investigate the possible side effects of epinephrine on the aqueous outflow pathway, we studied the response of human trabecular cells in vitro exposed daily to the drug at various concentrations. Epinephrine at 10(-6) M caused abnormal cytokinesis and cell retraction, inhibited mitosis and phagocytosis, and induced a 4- to 5-fold increase in cAMP. After 7-10 days of exposure, notable degenerative changes were observed in the trabecular cells. Within certain limits, these effects were reversible when the drug was withdrawn. Exposure to epinephrine at 10(-5) M caused cell death within 96 hours. When a 10(-7) M concentration of epinephrine was administered for up to 10 days, no degenerative changes were seen, but mitotic activity was reduced somewhat. Pretreatment with timolol reduced, but did not completely eliminate, the cytotoxic effects of epinephrine. Cells with loose contact to the substrate were affected most. The morphologic changes and the observed loss of intracellular actin filaments indicate that the deleterious effects of epinephrine are probably mediated through damage to cellular contractile proteins. Although the precise cytotoxic action of epinephrine in vivo remains to be established, our results for primary cell cultures indicate a toxic action of this drug at concentrations of 10(-6) M or higher on human trabecular cells and suggest that, for prolonged use, the maximal dose of this drug at target sites should be in the region of 10(-7) M.

Detection of glycated polypeptides in human aqueous humor by lectin-binding analysis.

The proteins from 30 samples of normal human aqueous humor were separated into major fractions by sodium dodecyl-sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with lectins to characterize the glycated polypeptides. The lectins used were derived from Canavalia ensiformis (concanavalin-A), Phaseolus vulgaris (erythroagglutinin), Arachis hypogaea (peanut agglutinin), and Ulex europaeus agglutinin-I. Using this microanalytical technique, the glycated polypeptides in the aqueous humor were separated into nine major fractions with apparent molecular weights of 17, 41, 50, 55, 61, 77, 80, 120 and 151 kDa. The predominant oligosaccharides detected on these fractions were complex chains usually with sialic acid residues. The possible roles of the glycated polypeptides in the aqueous humor in health and disease are discussed. Our investigation of these molecules in human aqueous humor provides baseline data for a rapid diagnostic analysis of microsamples of this fluid obtained from diseased eyes.