Histidine-rich glycoprotein inhibits the antiproliferative effect of heparin on smooth muscle cells.

Histidine-rich glycoprotein (HRGP), an alpha-glycoprotein in human plasma that is also present in platelets and macrophages, binds heparin with high affinity and neutralizes its anticoagulant activity. We now report that HRGP specifically inhibits the antiproliferative effect of heparin on arterial smooth muscle cells while other heparinoid-binding proteins do not influence mitogenesis. The multicellular inflammatory response to endothelial injury characterized, in part, by the influx of platelets and macrophages, may be associated with HRGP release into the arterial microenvironment. This release of HRGP may allow smooth muscle cell proliferation and atherogenesis by inhibiting the action of endothelial cell-derived heparinoid substances.

COPBLAM III: infusional combination chemotherapy for diffuse large-cell lymphoma.

COPBLAM III, a polychemotherapy regimen consisting of cyclophosphamide, infusional vincristine, prednisone, infusional bleomycin, doxorubicin, and procarbazine, was administered to 51 patients with diffuse large-cell lymphoma. Ninety-six percent of patients age 60 or younger achieved a complete response (CR); none have relapsed. Overall, 88% of patients are alive and well and potentially in the survival plateau. For patients greater than 60 years, CR was obtained in 73%, with 42% potentially in the survival plateau, the difference resulting in part from four relapses, three toxic deaths, and one presumed unrelated death. These results in the elderly were paralleled by a relatively reduced ability to tolerate therapy. Toxicity was primarily pulmonary, occurring in 39% of patients, two of whom died. With an overall CR rate of 84%, of which 92% are sustained at a median follow-up of 40 months, COPBLAM III represents a highly effective treatment in a sizeable cohort of patients.

A note on the description of pain and its causes.

The adjectives used by 72 psychiatric patients to describe pain were examined. Patients with physical lesions tended to have physical precipitants and physical relieving factors. Patients with psychologically based pain often recognized both physical and emotional preciptation. Most adjectives used were sensory rather than affective or evaluative.

Application of the hypersurface iterative projection method to bicyclic pyrazolidinone antibacterial agents.

Bicyclic pyrazolidinones are a class of synthetic antibacterial agents in which the beta-lactam ring is replaced by a five-membered ring. These compounds possess electronic and shape properties required for inhibiting penicillin-binding proteins essential for bacterial cell growth. A novel approach called the hypersurface iterative projection (HIP) method, which is based on three-dimensional computer graphics, allows available structure-activity information to be extrapolated to new synthetic targets. By updating the data set as the SAR evolves, the computer graphics reveal regions of parameter space to explore for optimum activity and regions yet unexplored. A large substituent parameter database is used to propose appropriate substituents. For the bicyclic pyrazolidinones, lipophilicity and particularly electron-withdrawing properties of the 3-substituent are shown to correlate strongly with minimum inhibitory concentrations (MIC). Antibacterial potency is intimately related to the activating effect of 3-substituents on chemical reactivity. The HIP method succeeded in proposing the most potent member of the series prior to synthesis and also showed when all of parameter space was reasonably well explored to the extent the chemistry allowed.

Substituent effects in cephalosporins as assessed by molecular orbital calculations, nuclear magnetic resonance, and kinetics.

For cephalosporins with different side chains at position 3, the quantum mechanically computed charge distribution in the beta-lactam carbonyl group can be correlated with observables, such as carbon-13 chemical-shift differences at C3 and C4 of the dihydrothiazine ring and alkaline rates of hydrolysis of the beta-lactam. The relationship of these properties and the theoretical transition-state energy (TSE) corroborate the fact that chemical reactivity is one important determinant affecting inhibitory activity of cephalosporins against peptidoglycan-regulating enzymes.

Conformational analogy between beta-lactam antibiotics and tetrahedral transition states of a dipeptide.

The three-dimensional structures of various penicillins and cephalosporins are compared to the spatial characteristics of glycylglycine and the tetrahedral adducts formed when a nucleophile attaches to the amide carbonyl carbon of this dipeptide. The dipeptide is taken to model the D-alanyl-D-alanine terminus of the precursors of bacterial cell-wall peptidoglycan cross-links. Least-squares fitting shows that the spatial match between the dipeptide and the antibiotic depends on the thiazolidine or dihydrothiazine ring conformation, as well as the conformation of the dipeptide. In general, the tetrahedral adducts fit somewhat better than the parent dipeptide. A previously unobserved 3-cephem conformer is found by molecular mechanics calculations to be less stable than the usual crystallographically observed conformer.

Electronic structures of cephalosporins and penicillins. 15. Inductive effect of the 3-position side chain in cephalosporins.

Induction appears to be the primary means by which the side chain at position 3 of the cephem nucleus influences the chemical reactivity of the beta-lactam ring. In vitro antibacterial activity data suggest that when the cephalosporin is in the active site of the target bacterial enzymes, the presence of a leaving group in the side chain can promote inhibition.

Electronic structures of cephalosporins and penicillins. 9. Departure of a leaving group in cephalosporins.

Molecular orbital calculations by the CNDO/2 method are used to study the potential energy surface for the stretching and rupturing of the CH2-OAc bond in a model cephalosporin structure, 7-amino-3-(acetoxymethyl)-3-cephem. The bond is easier to stretch and break when a nucleophilic group is in the vicinity of or attached to the beta-lactam carbonyl carbon (C8). The rate of acylation by a beta-lactam antibiotic at the receptor sites in bacterial cell-wall enzymes will be enhanced by a suitable leaving group at the 3' position. An orientational specificity is predicted for the direction of departure of the leaving group. Regardless of the direction the nucleophile approaches C8, the CH2-OAc bond is easiest to break when the acetate group departs from the alpha face of the molecule.

Arylpiperazines with serotonin-3 antagonist activity: a comparative molecular field analysis.

Comparative molecular field analysis (CoMFA) is applied to antagonists of the 5-HT3 receptor. Analysis is done separately on three published sets of arylpiperazines and on a combination of the three sets. d-Tubocurarine, a conformationally restricted 5-HT3 ligand, is used as a template to assist in selecting the conformation of the antagonists for CoMFA alignment. Two forms of the arylpiperazines (neutral and protonated) and three different kinds of calculated charges (Gasteiger-Hückel, AM1, and AM1 with solvation effect included) are compared. Protonated structures give better statistical results than the neutral species. The way in which charges are calculated does not greatly affect the results. In terms of molecular fields, the behavior in each separate set of compounds cannot be extrapolated to the combined set of 47 compounds. The average value of r2cv from PLS cross-validation on the combined set is 0.70 and varies between 0.56 and 0.80 depending on the orientation of the molecules in the coordinate system. The CoMFA model is tested on four compounds not in the training set: quipazine, N-methylquipazine, 4-phenyl-N-methylquipazine, and KB-6933. Mean agreement of experimental and predicted pKi values of the antagonists is 0.7 log unit. Novel structural modifications are interpreted by the CoMFA model.

Nonpeptide angiotensin II receptor antagonists: synthetic and computational chemistry of N-[[4-[2-(2H-tetrazol-5-yl)-1-cycloalken-1- yl]phenyl]methyl]imidazole derivatives and their in vitro activity.

A series of nonpeptide angiotensin II receptor antagonists was synthesized and tested in vitro to investigate requirements for recognition by and binding to AT1 receptors. Compared to a known series of N-(biphenylylmethyl)imidazoles, including losartan (DuP 753), which has a more rigid conformation in the 2'-tetrazolylbiphenyl moiety, the new series replaces the terminal phenyl with cycloalkenyls. Compounds were made with five- to seven-membered rings and with either a hydroxymethyl (3) or carboxyl (4) group at the 5 position on the imidazole ring. The effects of the lipophilicity and steric bulk of the terminal ring system, the amount of pi-electron density in the terminal ring, and the relative spatial proximity of the tetrazolyl and the middle phenyl are explored in terms of binding affinity to AT1 receptors in rat adrenal glomerulosa and rabbit aorta. The physicochemical variables of the new compounds were quantitated by computational chemistry and compared to those of losartan and its carboxyl metabolite. Potency at the AT1 receptors is maximized when the terminal ring is six-membered; an aromatic ring binds better than a cycloalkenyl ring. The 5-carboxyimidazole compounds show higher affinity than the 5-hydroxymethyl series.

Electronic structures of cephalosporins and penicillins. 4. Modeling acylation by the beta-lactam ring.

Molecular orbital calculations by the CNDO/2 method are used to study the molecular and electronic details involved in the initial phases of the opening of the beta-lactam ring of a model cephalosporin structure, 7-amino-3-acetoxymethyl-3-cephem. The effect of a simple nucleophile, OH-, approaching the carbonyl carbon center of the beta-lactam ring is monitored by following the charge redistributions that occur in the bicyclic system and in the 3 side chain. A migration of electron density to the ester oxygen of the CH2OAc group is observed with concomitant weakening of the CH2-OAc bond. The results are discussed in relation to the mechanism of acylation of bacterial cell wall enzymes by beta-lactam antibiotics and in relation to the hydrolysis of these molecules. The results indicate that the ability of the 3' substituent of cephalosporins to stabilize electron density transferred to it, i.e., the leavability of the 3' moiety, can be an important factor in activating the beta-lactam toward nucleophilic attack.

Synthesis, hydrolysis rates, supercomputer modeling, and antibacterial activity of bicyclic tetrahydropyridazinones.

Bicyclic tetrahydropyridazinones, such as 13, where X are strongly electron-withdrawing groups, were synthesized to investigate their antibacterial activity. These delta-lactams are homologues of bicyclic pyrazolidinones 15, which were the first non-beta-lactam containing compounds reported to bind to penicillin-binding proteins (PBPs). The delta-lactam compounds exhibit poor antibacterial activity despite having reactivity comparable to the gamma-lactams. Molecular modeling based on semiempirical molecular orbital calculations on a Cray X-MP supercomputer, predicted that the reason for the inactivity is steric bulk hindering high affinity of the compounds to PBPs, as well as high conformational flexibility of the tetrahydropyridazinone ring hampering effective alignment of the molecule in the active site. Subsequent PBP binding experiments confirmed that this class of compound does not bind to PBPs.

Heteroatom-activated beta-lactam antibiotics: considerations of differences in the biological activity of [[3(S)-(acylamino)-2-oxo-1-azetidinyl]oxy]acetic acids (oxamazins) and the corresponding sulfur analogues (thiamazins).

The considerable antibacterial activity of [[3(S)-(acylamino)-2-oxo-1-azetidinyl]oxy]acetic acids (oxamazins) in contrast to the lack of activity of the corresponding sulfur analogues (thiamazins) is examined in terms of physicochemical parameters, including electronegativity, IR carbonyl stretching frequencies, base hydrolysis rates, and three-dimensional molecular geometries. An X-ray structure determination of a protected thiamazin together with molecular graphics and molecular orbital calculations on model structures reveals that thiamazins would not fit as well as oxamazins in the active site of target bacterial transpeptidases. As a result of thiamazins' long N-S and S-C bond lengths, the pharmacophoric beta-lactam ring and carboxylate functionality cannot adopt the spatial relationship they have in penicillins and cephalosporins. The beta-lactam nitrogen of the monocyclic, crystalline thiamazin is 0.18 A out of the plane of its three substituents, and this distance (h) is predicted by computational chemistry methods to be higher in oxamazins. The rates of beta-lactam ring opening of an oxamazin, thiamazin, and aztreonam are comparable, even though the pyramidal character and IR data both indicate the electronegative oxygen analogue has reduced amide resonance. MNDO, AM1, and MINDO/3 correctly give a twofold potential for rotation about the N-S bond in model sulfenamides, with barrier heights ranging up to 12 kcal/mol.

3-Quaternary ammonium 1-carba-1-dethiacephems.

A series of structurally unique 1-carba-1-dethiacephems is described. The structural stability of the 1-carba-1-dethiacephem nucleus was essential for the preparation of this series of 3-quaternary ammonium carbacephems. The known p-nitrobenzyl 7 beta-(phenoxyacetamido)- 3-[(trifluoromethyl)sulfonyl]oxy]-1-carba-1-dethia-3-cephem- 4-carboxylate served as both a quaternization substrate as well as a precursor to derivatives such as allyl 7 beta-[[2-[allyloxy)carbonyl]amino-4- thiazoly] (methoxyimino)acetyl]amino]-3-[(trifluoromethyl) sulfonyl] oxy]-1-carba-1-dethia-3-cephem-4-carboxylate. Quaternization of these enol triflates was accomplished either by dissolution in acetonitrile containing the base or by dissolution in the base, with or without warning to 50 degrees C. Bases nucleophilic enough to displace the triflate include a variety of substituted pyridines and N-methylimidazole. Deprotection then produced a very active series of 1-[7 beta-[(2-amino- 4-thiazolyl)(methoxyimino)acetyl]amino]-2-carboxy-8-oxo- 1-azabicyclo[4.2.0]oct-2-en-3-yl] quaternary ammonium hydroxide inner salts. These compounds were extremely potent antibacterials against a broad range of Gram-positive and -negative bacteria including constitutive cephalosporinase producers, such as Enterobacter cloacae. The compounds exhibit similar hydrolysis kinetics and pharmacokinetics to the analogous cephalosporin-3'-quaternary ammonium salts.

Three-dimensional reconstructions of autonomic projections to the gastrointestinal tract.

Three-dimensional reconstruction protocols in confocal microscopy are typically considered in terms of rendering separate stacks of optical sections. Single stacks, however, include volumes that are often too small to permit descriptions of entire neurons, complete axonal arbors, or complex neural networks. Furthermore, traditional tissue preparation protocols generally yield specimens too limited to permit reconstructions of complex neural systems. For 3-D analyses of extensive networks such as the autonomic nervous system projections within the viscera, it is critical to incorporate appropriate tissue techniques, including suitable tracer protocols, into the reconstruction strategy. This report summarizes complementary technologies, including whole mount procedures, tracer techniques for identifying single fibers in situ, and methods of examining stacks of optical images, which make it practical to describe the complete terminal field of an individual axon in the gastrointestinal tract. Such methods establish that vagal motor axons travel long distances within their target organs, collateralize frequently, and ramify extensively. Vagal afferents have extensive, complex, and, in some cases, polytopic arbors within target tissues.

Superimposition-based protocol as a tool for determining bioactive conformations. I. Application to ligands of the glycinergic receptor (GlyR).

The natural templates (NT) approach, which is a superimposition-based protocol that has been successfully employed in several studies, is here applied to ligands of the glycine ligand-gated ion channel receptor. Bioactive conformations for glycine and its analogs were obtained using strychnine (a natural and specific competitive antagonist) as template. Experimental evidence was used to guide the superimposition protocol. Three essential regions have been defined in strychnine's structure that serve as a pharmacophore for agonist and antagonist activities. Reasonable alignments of known ligands were found in the majority of the cases. Molecular mechanics (i.e., conformational searches for the relatively flexible ligands) and molecular dynamics (for relatively rigid ligands such as strychnine and 5,6,7,8-tetrahydro-4H-isoxazolo[3,4-d]azepin-3-ol) were used to assess the energetic accessibility of the proposed bioactive conformations.

Superimposition-based protocol as a tool for determining bioactive conformations. II. Application to the GABA(A) receptor.

The natural templates (NT) superimposition method is used to determine the pharmacophoric requirements of the A subtype of the gamma-aminobutyric acid (GABA) receptor. Bioactive conformations for antagonists and agonists are found by superimposing them on a relatively rigid alkaloid bicuculline, which itself is a competitive antagonist at this ligand-gated ion channel receptor. As has been usual in the application of this modeling method, consideration of available experimental data is the cornerstone for obtaining realistic models. The identification of two substructural fragments of bicuculline permitted classification of the ligands. Analysis of the antagonists and agonists with respect to the two substructural fragments revealed two bioactive conformations of the highly flexible GABA molecule, one of which is extended with the nonhydrogenic atoms roughly coplanar torsional angles of -37 and -179 degrees at N-C-C-C and C-C-C-C (carboxyl), respectively. The second bioactive compound is clearly non planar (torsional angles of -81 and -109 degrees at N-C-C-C and C-C-C-C (carboxyl), respectively).

Conformational flexibility of the methyltetrazolethiomethyl side chain of beta-lactam antibiotics. A computer graphics study.

Analysis of X-ray crystallographic data for cephalosporins and 1- oxacephalosporins shows that, although the 1-methyl-1H- tetrazol -5- ylthiomethyl side chain at position 3 of a bicyclic beta-lactam nucleus has certain conformational preferences, it also has considerable flexibility. Both the C4 = C3-C3'-S and C3-C3'-S-C5" torsional angles are frequently observed in the vicinity of +/- 90 degrees. The distance between the tetrazole ring carbon C5" and the 4-carboxyl carbon ranges from 4.07 to 5.65 A. Mean bond lengths and bond angles for the 3 and 4 side chains are tabulated.

Examination of model enzyme and penetration systems in relation to antibacterial activity.

Recent work has shown that the activity of cephalosporins in inhibiting exocellular DD-peptidases from Streptomyces R61 and Actinomadura R39 are, at best, only poorly related to minimum inhibitory concentrations against pathogenic isolates. Taking into account the rate at which cephalosporins diffuse through porin channels, such as exist in certain Gram-negative organisms, does not help in establishing a relationship between MIC data and the kinetic data on the model enzymes. Most published cell wall permeability studies, the porin ones being a principal exception, have not examined long enough series of structurally related compounds to establish property-activity relationships.

Correlations between CNDO/2 charge distribution and 13C NMR chemical shift in 7-acylamino side chains of cephalosporins.

Molecular orbital calculations by the CNDO/2D method yield charge distributions which correlate well with the observed 13C NMR chemical shift for the amide carbon of acylamino side chains of cephalosporins. Acyl groups that withdraw electrons from the amide C-N bond and concomitantly make the amide nitrogen more negatively charged increase the chemical shift. The trends are related to the degree of amide resonance. No direct correlation was found between the chemical shift of the amide carbon and the antibacterial activity of the cephalosporins.