Comparing complex perspectives on obesity drivers: action-driven communities and evidence-oriented experts.

INTRODUCTION: The Foresight obesity map represents an expert-developed systems map describing the complex drivers of obesity. Recently, community-led causal loop diagrams have been developed to support community-based obesity prevention interventions. This paper presents a quantitative comparison between the Foresight obesity systems map and a community-developed map of the drivers of obesity. METHODS: Variables from a community-developed map were coded against the thematic clusters defined in the Foresight map to allow comparison of their sizes and strength of adjoining causal relationships. Central variables were identified using techniques from network analysis. These properties were compared to understand the similarities and differences between the systems as defined by the two groups. RESULTS: The community map focused on environmental influences, such as built physical activity environment (18% of variables) and social psychology (38%). The Foresight map's largest cluster was physiology (23%), a minimal focus in the community map (2%). Network analysis highlighted media and available time within both maps, but variables related to school and sporting club environments were unique to the community map. CONCLUSION: Community stakeholders focus on modifiable social and environmental drivers of obesity. Capturing local perspectives is critical when using systems maps to guide community-based obesity prevention.

Report of video-capsule endoscopy disruption producing episodic small bowel obstruction after prolonged retention.

INTRODUCTION: Wireless video-capsule endoscopy is a procedure which provides direct visualization of the gastrointestinal tract, particularly the jejunum and ileum. Capsule retention is the main risk associated with capsule endoscopy, occurring at a significantly elevated incidence in patients with known or suspected Crohn's disease. PRESENTATION OF CASE: A case of a prolonged retained capsule with subsequent fragmentation producing a multicentric complete small bowel obstruction in a 39 year old male patient who had undergone wireless video capsule-endoscopy approximately three years prior. Management required surgical resection of the strictured jejunum and removal of retained capsule fragments under fluoroscopic guidance. DISCUSSION: Although capsule endoscopy is capable of diagnosis, evaluation, and monitoring inflammatory bowel disease, understanding the elevated risk for capsule retention is important in this population. Specifically, prolonged capsule retention appears to increase the risk of capsule disruption, and likely the potential for intestinal perforation. CONCLUSION: Patients should therefore be carefully selected for monitoring based upon treatment compliance and offered early endoscopic or surgical intervention in the setting of questionable compliance due to the risk for capsule disruption and subsequent intestinal perforation.

Glyoxalase I inhibitors in cancer chemotherapy.

Several recent developments suggest that the GSH-dependent glyoxalase enzyme system deserves renewed interest as a potential target for antitumour drug development. This summary focuses on the design and development of new classes of tumoricidal agents that specifically target this elementary detoxification pathway in order to induce elevated concentrations of cytotoxic methylglyoxal in tumour cells. Special emphasis is placed on structure- and mechanism-based inhibitors of GlxI (glyoxalase I), the first enzyme in the pathway. A new class of bivalent transition-state analogues is described that simultaneously bind the active site on each subunit of the homodimeric human GlxI, resulting in K (i) values as low as 1 nM. Also described is a new family of bromoacyl esters of GSH that function as active-site-directed irreversible inhibitors of GlxI. Newer prodrugs for delivering the GSH-based inhibitors into tumour cells include reactive sulphoxide esters that undergo acyl exchange with endogenous GSH to give the inhibitors, and polymethacrylamide esters of the inhibitors that are potentially tumour-selective on the basis of the "enhanced permeability and retention effect". Finally, a preliminary evaluation of the efficacy of selected GlxI inhibitors in tumour-bearing mice is given.

Brief history of glyoxalase I and what we have learned about metal ion-dependent, enzyme-catalyzed isomerizations.

Glyoxalase I, a member of the metalloglutathione (GSH) transferase superfamily, plays a critical detoxification role in cells by catalyzing the conversion of cytotoxic methylglyoxal (as the diastereomeric GSH-thiohemiacetals) to S-D-lactoylglutathione via a 1,2-hydrogen transfer. The mechanism-of-action of this Zn2+-metalloenzyme has been the subject of considerable controversy over the past 50 years. Key issues relate to the role of the active-site metal ion in catalysis and how the enzyme is able to use directly both diastereomeric thiohemiacetals as substrates. The results of recent X-ray crystallographic measurements on the enzyme in complex with a transition state analogue and site-directed mutagenesis studies now strongly support a base-mediated, proton-transfer mechanism in which the bound diastereomeric substrates undergo catalytic interconversion before the 1S-diastereomer goes to product via a Zn2+-coordinated, cis-enediolate intermediate. Comparisons with chemical model systems suggest that Zn2+-coordination of thiohemiacetal substrate will dramatically increase the thermodynamic and kinetic acidity of the C1-H bond of substrate. In the human enzyme, the carboxyl group of Glu (172) is well positioned to catalyze a suprafacial proton transfer between the adjacent carbons of substrate. The Zn2+-coordinated carboxyl group of Glu(99) is a reasonable candidate to catalyze proton transfer between the Zn2+-coordinated oxygen atoms of the enediolate intermediate. Other Zn2+ metalloenzymes appear to use similar reaction mechanisms to facilitate proton transfers.

Weight-bearing exercise and markers of bone turnover in female athletes.

Weight-bearing activity provides an osteogenic stimulus, while effects of swimming on bone are unclear. We evaluated bone mineral density (BMD) and markers of bone turnover in female athletes (n = 41, age 20.7 yr) comparing three impact groups, high impact (High, basketball and volleyball, n = 14), medium impact (Med, soccer and track, n = 13), and nonimpact (Non, swimming, n = 7), with sedentary age-matched controls (Con, n = 7). BMD was assessed by dual-energy X-ray absorptiometry at the lumbar spine, femoral neck (FN), Ward's triangle, and trochanter (TR); bone resorption estimated from urinary cross-linked N-telopeptides (NTx); and bone formation determined from serum osteocalcin. Adjusted BMD (g/cm; covariates: body mass index, weight, and calcium and calorie intake) was greater at the FN and TR in the High group (1.27 +/- 0.03 and 1.05 +/- 0.03) than in the Non (1.05 +/- 0.04 and 0.86 +/- 0.04) and Con (1.03 +/- 0.05 and 0.85 +/- 0.05) groups and greater at the TR in the Med group (1.01 +/- 0.03) than in the Non (0.86 +/- 0.04) and Con (0.85 +/- 0.05) groups. Total body BMD was higher in the High group (4.9 +/- 0.12) than in the Med (4.5 +/- 0.12), Non (4.2 +/- 0.14), and Con (4.1 +/- 0.17) groups and greater in the Med group than in the Non and Con groups. Bone formation was lower in the Non group (19.8 +/- 2.6) than in the High (30.6 +/- 3.0) and Med (32.9 +/- 1.9, P < or = 0.05) groups. No differences in a marker of bone resorption (NTx) were noted. This indicates that women who participate in impact sports such as volleyball and basketball had higher BMDs and bone formation values than female swimmers.

Effect of 10%, 30%, and 60% body weight traction on the straight leg raise test of symptomatic patients with low back pain.

STUDY DESIGN: Single group test-retest repeated measures. OBJECTIVES: To determine the effects of lumbar traction with 3 different amounts of force (10%, 30% and 60% body weight) on pain-free mobility of the lower extremity as measured by the straight leg raise (SLR) test. BACKGROUND: There are several recommendations on how lumbar traction should be performed, but the duration, frequency, force, and type of technique to be applied differ among the sources. METHODS AND MEASURES: Ten subjects with subjective complaints of low back pain or radicular symptoms with a positive unilateral SLR test below 45 degrees participated in this study. The pain-free mobility of the lower extremity in the SLR test position was measured prior to and immediately following 5 minutes of static traction in the supine position. Random assignment in the order of the amount of applied traction was implemented. RESULTS: The straight leg raise measurements were found to be significantly greater immediately following 30% and 60% of body weight traction as compared to pretraction and 10% of body weight traction. The mean (SD) SLR measurements were pretraction (24.1 degrees +/- 13.0), 10% of body weight traction (27.4 degrees +/- 14.5), 30% of body weight traction (34.0 degrees +/- 14.3), 60% of body weight traction (36.5 degrees +/- 15.8). CONCLUSIONS: The results of this study indicate that traction in this group of patients improved the mobility of the lower extremity during the SLR test. Both 30% and 60% of body weight tractions were shown to be effective for increasing motion beyond pretraction levels.

Role of hydrophobic interactions in binding S-(N-aryl/alkyl-N-hydroxycarbamoyl)glutathiones to the active site of the antitumor target enzyme glyoxalase I.

Hydrophobic interactions play an important role in binding S-(N-aryl/alkyl-N-hydroxycarbamoyl)glutathiones to the active sites of human, yeast, and Pseudomonas putida glyoxalase I, as the log K(i) values for these mechanism-based competitive inhibitors decrease linearly with increasing values of the hydrophobicity constants (pi) of the N-aryl/alkyl substituents. Hydrophobic interactions also help to optimize polar interactions between the enzyme and the glutathione derivatives, given that the K(i) value for S-(N-hydroxycarbamoyl)glutathione (pi = 0) with the human enzyme is 35-fold larger than the interpolated value for this compound obtained from the log K(i) versus pi plot. Computational studies, in combination with published X-ray crystallographic measurements, indicate that human glyoxalase I binds the syn-conformer of S-(N-aryl-N-hydroxycarbamoyl)glutathiones in which the N-aryl substituents are in their lowest-energy conformations. These studies provide both an experimental and a conceptual framework for developing better inhibitors of this antitumor target enzyme.

Reaction of COTC with glutathione: structure of the putative glyoxalase I inhibitor.

The structure of the active glyoxalase I inhibitor derived from the Streptomyces griseosporeus metabolite COTC 1 has been conclusively identified by means of total synthesis as 2c. Human glyoxalase I is competitively inhibited by 2c (K(i)() = 183 +/- 6 microM) but is not inhibited by 1 itself.

Pharmacokinetics and antitumor properties in tumor-bearing mice of an enediol analogue inhibitor of glyoxalase I.

PURPOSE: The enediol analogue S-(N-p-chlorophenyl-N-hydroxycarbamoyl)glutathione (CHG) is a powerful, mechanism-based, competitive inhibitor of the methylglyoxal-detoxifying enzyme glyoxalase I. The [glycyl,glutamyl]diethyl ester prodrug form of this compound (CHG(Et)2) inhibits the growth of different tumor cell lines in vitro, apparently by inducing elevated levels of intracellular methylglyoxal. The purpose of this study was to evaluate the pharmacokinetic properties of CHG(Et)2 in plasma esterase-deficient C57BL/6 (Es-1e) mice after intravenous (i.v.) or intraperitoneal (i.p.) administration of bolus doses of CHG(Et)2. In addition, the in vivo antitumor properties of CHG(Et)2 were evaluated against murine B16 melanoma in these mice, and against androgen-independent human prostate PC3 tumor and human colon HT-29 adenocarcinoma in plasma esterase-deficient nude mice. METHODS: Pharmacokinetics were evaluated after either i.v. or i.p. administration of CHG(Et)2 at the maximally tolerated dose of 120 mg/kg to both tumor-free male and female mice and male and female mice bearing subcutaneous B16 tumors. Tissue concentrations of CHG(Et)2, CHG and the [glycyl]monoethyl ester CHG(Et) were measured as a function of time by reverse-phase C18 high-performance liquid chromatography of deproteinized tissue samples. The efficacy of CHG(Et)2 in tumor-bearing mice was evaluated after i.v. bolus administration of CHG(Et)2 at 80 or 120 mg/kg for 5 days each week for 2 weeks, or after 14 days continuous infusion of CHG(Et)2 using Alzet mini-osmotic pumps. Hydroxypropyl-beta-cyclodextrin was used as a vehicle in the efficacy studies. RESULTS: Intravenous administration of CHG(Et)2 resulted in the rapid appearance of CHG(Et)2 in the plasma of tumor-bearing mice with a peak value of 40-60 microM, followed by a first-order decrease with a half-life of about 10 min. There was a corresponding increase in the concentration of inhibitory CHG in the B16 tumors, with a maximum concentration in the range 30-60 microM occurring at 15 min, followed by a decrease to a plateau value of about 6 microM after 120 min. Neither CHG(Et)2 nor its hydrolysis products were detectable in plasma, after i.p. administration of CHG(Et)2 to tumor-free female mice. From the efficacy studies, dosing schedules were identified that resulted in antitumor effects comparable to those observed with the standard antitumor agents Adriamycin (with B16 tumors), cisplatin (with PC3 tumors), and vincristine (with HT-29 tumors). CONCLUSION: This is the first demonstration that a mechanism-based competitive inhibitor of glyoxalase I effectively inhibits the growth of solid tumors in mice when delivered as the diethyl ester prodrug.

Maillard reactions in lens proteins: methylglyoxal-mediated modifications in the rat lens.

The nonenzymatic Maillard reaction is thought to contribute to aging and cataract formation in the lens. As levels of methylglyoxal (MG) and glutathione (GSH) affect the reaction, we examined the relationship of these factors and determined the effect of a glyoxalase I inhibitor on the Maillard reaction. Rat lens cultures were maintained for up to 3 days in TC-199 medium with or without 20 m m glyceraldehyde (GLD) and 250 microm S-[N-hydroxy-N-(4-chlorophenyl) carbamoyl] glutathione diethyl ester (HCCG diester). We measured GSH, MG, D-lactate, glyoxalase I activity, immunoreactive MG-derived advanced glycation endproducts (MG-AGEs) and imidazolysine in organ cultured rat lenses. In vitro experiments with isolated rat lens proteins revealed that HCCG alone inhibited glyoxalase I activity in a dose-dependent manner. In organ cultured rat lens protein, GLD increased MG levels 24-fold, and the addition of HCCG diester further increased it by about two-fold. GSH levels fell sharply in the presence of GLD and this was prevented to some extent by the presence of HCCG diester. D-lactate production in the lens was suppressed by HCCG diester treatment. Dialysed lens proteins retained glyoxalase I activity, indicating that the enzyme was unaltered during incubation. MG-AGEs and imidazolysine levels were significantly higher (P<0.05) in GLD-treated lenses, but a combination of HCCG diester and GLD lowered immunoreactive MG-AGEs and imidazolysine levels compared to GLD alone. HCCG had no significant effect on MG-AGE formation in lens proteins incubated with GLD or MG. We conclude that exogenous GLD enhances MG and MG-AGE levels in the rat lens and that this increase is accompanied by a loss in GSH. In addition, inhibition of glyoxalase I promotes MG accumulation.

Parents' ratings of everyday cognitive abilities in very low birth weight children.

Parents' ratings of everyday cognitive functioning in very low birth weight (VLBW) children free of sensorineural impairments and normal birth weight (NBW) children were compared with the children's actual performance on psychometric measures of cognitive and motor skills. Subjects included 19 VLBW children identified at age 3 years as "suspect" for developmental problems, 19 VLBW children identified at age 3 years as "developing normally," and 30 NBW full-term peers. Results indicated that parents of the suspect VLBW group rated their children as having significant impairments in memory, language, cognitive, and motor skills, findings which were consistent with the results of concurrent psychometric assessments. When compared with psychometric test results, parents identified more children as displaying difficulties in memory, language, and cognitive skills, but fewer children with coordination difficulties. These findings suggest that the parents' ratings and the psychometric measures may be assessing somewhat different aspects of the children's functioning.

Reaction mechanism of glyoxalase I explored by an X-ray crystallographic analysis of the human enzyme in complex with a transition state analogue.

The structures of human glyoxalase I in complexes with S-(N-hydroxy-N-p-iodophenylcarbamoyl)glutathione (HIPC-GSH) and S-p-nitrobenzyloxycarbonylglutathione (NBC-GSH) have been determined at 2.0 and 1.72 A resolution, respectively. HIPC-GSH is a transition state analogue mimicking the enediolate intermediate that forms along the reaction pathway of glyoxalase I. In the structure, the hydroxycarbamoyl function is directly coordinated to the active site zinc ion. In contrast, the equivalent group in the NBC-GSH complex is approximately 6 A from the metal in a conformation that may resemble the product complex with S-D-lactoylglutathione. In this complex, two water molecules occupy the liganding positions at the zinc ion occupied by the hydroxycarbamoyl function in the enediolate analogue complex. Coordination of the transition state analogue to the metal enables a loop to close down over the active site, relative to its position in the product-like structure, allowing the glycine residue of the glutathione moiety to hydrogen bond with the protein. The structure of the complex with the enediolate analogue supports an "inner sphere mechanism" in which the GSH-methylglyoxal thiohemiacetal substrate is converted to product via a cis-enediolate intermediate. The zinc ion is envisioned to play an electrophilic role in catalysis by directly coordinating this intermediate. In addition, the carboxyl of Glu 172 is proposed to be displaced from the inner coordination sphere of the metal ion during substrate binding, thus allowing this group to facilitate proton transfer between the adjacent carbon atoms of the substrate. This proposal is supported by the observation that in the complex with the enediolate analogue the carboxyl group of Glu 172 is 3.3 A from the metal and is in an ideal position for reprotonation of the transition state intermediate. In contrast, Glu 172 is directly coordinated to the zinc ion in the complexes with S-benzylglutathione and with NBC-GSH.

Lentivirus-derived antimicrobial peptides: increased potency by sequence engineering and dimerization.

We have previously described a family of cationic amphipathic peptides derived from lentivirus envelope proteins that have properties similar to those of naturally occurring antimicrobial peptides. Here, we explored the effects of amino acid truncations and substitutions on the antimicrobial potency and selectivity of the prototype peptide, LLP1. Removal of seven residues from the C-terminus of LLP1 had little effect on potency, but abrogated haemolytic activity. Replacement of the two glutamic acid residues of LLP1 with arginine resulted in a peptide with greater bactericidal activity. We discovered that the cysteine-containing peptides spontaneously formed disulphide-linked dimers, which were 16-fold more bactericidal to Staphylococcus aureus. Monomeric and dimeric LLP1 possessed similar alpha helical contents, indicating that disulphide formation did not alter the peptide's secondary structure. The dimerization strategy was applied to magainin 2, enhancing its bactericidal activity eight-fold. By optimizing all three properties of LLP1, a highly potent and selective peptide, named TL-1, was produced. This peptide is significantly more potent than LLP1 against gram-positive bacteria while maintaining high activity against gram-negative organisms and low activity against eukaryotic cells. In addition to new antimicrobial peptides, these studies contribute useful information on which further peptide engineering efforts can be based.

A new method for rapidly generating inhibitors of glyoxalase I inside tumor cells using S-(N-aryl-N-hydroxycarbamoyl)ethylsulfoxides.

The enediol analogue S-(N-p-chlorophenyl-N-hydroxycarbamoyl)glutathione is a powerful mechanism-based competitive inhibitor of the anticancer target enzyme glyoxalase I. Nevertheless, this compound exhibits limited toxicity toward tumor cells in vitro because it does not readily diffuse across cell membranes. We describe an efficient method for indirectly delivering the enzyme inhibitor into murine leukemia L1210 cells via acyl interchange between intracellular glutathione and the cell-permeable prodrug S-(N-p-chlorophenyl-N-hydroxycarbamoyl)ethylsulfoxide. The second-order rate constant for the acyl-interchange reaction in a cell-free system is 1.84 mM-1 min-1 (100 mM potassium phosphate buffer, 5% ethanol, pH 7.5, 25 degrees C). Incubation of L1210 cells with the sulfoxide in vitro results in a rapid increase in the intracellular concentration of the glyoxalase I inhibitor (kapp = 1. 41 +/- 0.03 min-1 (37 degrees C)) and inhibition of cell growth (GI50 = 0.5 +/- 0.1 microM). This represents an improvement in both efficiency and potency over the dialkyl ester prodrug strategy in which the inhibitor is indirectly delivered into tumor cells as the [glycyl,glutamyl] diethyl or dicyclopentyl esters. The fact that pi-glutathione transferase catalyzes the acyl-interchange reaction between GSH and the sulfoxide suggests that the sulfoxide, or related compounds, might exhibit greater selective toxicity toward tumor cells that overexpress the transferase.

Mechanism-based competitive inhibitors of glyoxalase I: intracellular delivery, in vitro antitumor activities, and stabilities in human serum and mouse serum.

S-(N-Aryl-N-hydroxycarbamoyl)glutathione derivatives (GSC(O)N(OH)C6H4X, where GS = glutathionyl and X = H (1), Cl (2), Br (3)) have been proposed as possible anticancer agents, because of their ability to strongly inhibit the methylglyoxal-detoxifying enzyme glyoxalase I. In order to test this hypothesis, the in vitro antitumor activities of these compounds and their [glycyl,glutamyl] diethyl ester prodrug forms (1(Et)2-3(Et)2) have been examined. All three diethyl esters inhibit the growth of L1210 murine leukemia and B16 melanotic melanoma in culture, with GI50 values in the micromolar concentration range. Cell permeability studies with L1210 cells indicate that growth inhibition is associated with rapid diffusion of the diethyl esters into the cells, followed by enzymatic hydrolysis of the ethyl ester functions to give the inhibitory diacids. In contrast, the corresponding diacids neither readily diffuse into nor significantly inhibit the growth of these cells. Consistent with the hypothesis that cell growth inhibition is due to competitive inhibition of glyoxalase I, preincubation of L1210 cells with 2(Et)2 increases the sensitivity of these cells to the inhibitory effects of exogenous methylglyoxal. Compound 2(Et)2 is much less toxic to nonproliferating murine splenic lymphocytes, possibly reflecting reduced sensitivity to methylglyoxal and/or reduced chemical stability of the diacid inside these cells. Finally, a plasma esterase-deficient murine model has been identified that should allow in vivo testing of the diethyl esters.

Long-term neuropsychological outcomes in very low birth weight children free of sensorineural impairments.

This study investigated the neuropsychological outcomes at school age of children with very low birth weight (VLBW) free of sensorineural impairments. Subjects included 19 children with VLBW identified at age 3 as 'suspect' for developmental problems, 19 children with VLBW identified at age 3 as developing normally, and 30 children of normal birth weight (NBW). Results indicated that children in the VLBW 'suspect' group performed significantly more poorly on all of the neuropsychological measures compared to children of NBW. These findings suggest that VLBW children identified as 'suspect' for developmental problems because of impairments in cognitive skills at age 3 continued to show deficits at school age on intellectual and neuropsychological measures.

Active monomeric and dimeric forms of Pseudomonas putida glyoxalase I: evidence for 3D domain swapping.

3D domain swapping of proteins involves the interconversion of a monomer containing a single domain-domain interface and a 2-fold symmetrical dimer containing two equivalent intermolecular interfaces. Human glyoxalase I has the structure of a domain-swapped dimer [Cameron, A. D., Olin, B., Ridderström, M., Mannervik, B., and Jones, T. A. (1997) EMBO J. 16, 3386-3395] but Pseudomonas putida glyoxalase I has been reported to be monomeric [Rhee, H.-I., Murata, K., and Kimura, A. (1986) Biochem. Biophys. Res. Commun. 141, 993-999]. We show here that recombinant P. putida glyoxalase I is an active dimer (kcat approximately 500 +/- 100 s-1; KM approximately 0.4 +/- 0.2 mM) with two zinc ions per dimer. The zinc is required for structure and function. However, treatment of the dimer with glutathione yields an active monomer (kcat approximately 115 +/- 40 s-1; KM approximately 1.4 +/- 0.4 mM) containing a single zinc ion. The monomer is metastable and slowly reverts to the active dimer in the absence of glutathione. Thus, glyoxalase I appears to be a novel example of a single protein able to exist in two alternative domain-swapped forms. It is unique among domain-swapped proteins in that the active site and an essential metal binding site are apparently disassembled and reassembled by the process of domain swapping. Furthermore, it is the only example to date in which 3D domain swapping can be regulated by a small organic ligand.

Novel antimicrobial peptides derived from human immunodeficiency virus type 1 and other lentivirus transmembrane proteins.

We have previously described a conserved set of peptides derived from lentiviral envelope transmembrane proteins that are similar to the natural antimicrobial peptides cecropins and magainins in overall structure but bear no sequence homology to them or other members of their class. We describe here an evaluation of the antimicrobial properties of these virally derived peptides, designated lentivirus lytic peptides (LLPs). The results of this study demonstrate that they are potent and selective antibacterial peptides: the prototype sequence, LLP1, is bactericidal to both gram-positive and gram-negative organisms at micromolar concentrations in 10 mM phosphate buffer. Furthermore, LLP1 kills bacteria quite rapidly, causing a 1,000-fold reduction in viable organisms within 50 s. Peptides corresponding to sequences from three lentivirus envelope proteins were synthesized and characterized. Several of these peptides are selective, killing bacteria at concentrations 50- to 100-fold lower than those required to lyse erythrocytes. Development of antimicrobial agents based on these peptides may lead to improved therapeutics for the management of a variety of infectious diseases.

Endothelial cell adaptation to chronic thrombosis.

BACKGROUND: The autogenous vein graft has proven to be the most durable conduit in lower extremity vascular bypass grafts. Failures due to thrombosis, intimal hyperplasia, and progression of atherosclerotic disease commonly plague the vascular surgeon. Part of the ability of vein grafts to provide a nonthrombogenic surface relies on the capability of the endothelial cell to produce prostacyclin, a potent vasodilator and inhibitor of platelet aggregation. Once a graft fails and thromboses, little is known as to the effects of the thrombus on the function and morphology of endothelial cells. Earlier studies by this laboratory demonstrated the ability of arterialized canine vein grafts to recover function after 5 days of exposure to thrombus. This investigation sought to explore the limits of endothelial cell viability and recovery to extended periods of thrombosis. METHODS: Using a canine model of arterialized vein grafts, prostacyclin production (measured as 6-keto-PGF1a) was assessed in an ex vivo perfusion system from grafts exposed to thrombus for 10 days (group I) and 20 days (group II). Both groups underwent thrombectomy and a recovery period of 30 days. The grafts were perfused with Hanks' balanced salt solution and samples were obtained at 5 and 30 minutes to determine prostacyclin levels. Arachidonic acid was then added to a new perfusate of Hanks' solution and samples were again obtained at 5 and 30 minutes. Results were expressed as PGF/graft area (cm2/min). Representative samples of each graft underwent scanning electron microscopy. RESULTS: Without arachidonic acid, prostacyclin production of group II (20 day) grafts was greater than group I (10 day) grafts at 5 minutes of perfusion (4.31 versus 2.42, P = 0.08) and at 30 minutes (1.86 versus 0.95, P = 0.02). In response to the addition of arachidonic acid both groups increased prostacyclin production (group I, P = 0.004; group II, P = 0.12). A comparison was made between prostacyclin production at baseline and after addition of arachidonic acid. Group I grafts demonstrated a greater percent increase in prostacyclin production compared to group II (385% versus 229%, P = 0.01). Scanning electron microscopy showed no differences in endothelial coverage between the study groups. CONCLUSIONS: These results demonstrate that although endothelial cells are able to recover a basal level of prostacyclin production, the response to substrate stimulation diminishes with increased exposure time to thrombus. This diminished response may be important in understanding the ability of vein grafts to survive after a period of thrombosis.

Diffusion-dependent kinetic properties of glyoxalase I and estimates of the steady-state concentrations of glyoxalase-pathway intermediates in glycolyzing erythrocytes.

The diffusion-dependent kinetic properties of the yeast glyoxalase I reaction have been measured by means of viscosometric methods. For the glyoxalase-I-catalyzed isomerization of glutathione (GSH)-methylglyoxal thiohemiacetal to S-D-lactoylglutathione, the k(cat)/Km (3.5 x 10(6) M(-1) s(-1), pH 7, 25 degrees C) undergoes a progressive decrease in magnitude with increasing solution viscosity, using sucrose as a viscogenic agent. The viscosity effect is unlikely to be due to a sucrose-induced change in the intrinsic kinetic properties of the enzyme, as the magnitude of k(cat)/Km for the slow substrate GSH-t-butylglyoxal thiohemiacetal (3.5 x 10(3) M(-1) s(-1), pH 7, 25 degrees C) is independent of solution viscosity. Quantitative treatment of the data by means of the Stokes-Einstein diffusion law suggests that catalysis will be about 50% diffusion limited under conditions where [substrate] << Km; the encounter complex between enzyme and substrate partitions nearly equally between product formation and dissociation to form free enzyme and substrate. In a related study, the steady-state concentrations of glyoxalase-pathway intermediates in glycolyzing human erythrocytes are estimated to be in the nanomolar concentration range, on the basis of published values for the activities of glyoxalase I and glyoxalase II in lysed erythrocytes and the steady-state rate of formation of D-lactate in intact erythrocytes. This is consistent with a model of the glyoxalase pathway in which the enzyme-catalyzed steps are significantly diffusion limited under physiological conditions.