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In the present work, the conformation analysis, electrostatic potential calculations, and proton affinity evaluation are carried out for Cinchona alkaloids using theoretical molecular mechanics and quantum mechanical methods. The most probable conformation of the active erythro isomers at the receptor site seems to be that which enables the molecule to form intermolecular hydrogen bonds. In epiquinidine, the mutual orientation of O(12) and N(1) atoms favors intra- rather than intermolecular bonding, and this might be responsible for its inactivity. Comparison of the shape and size of the negative electrostatic potential areas provides a tentative explanation for the interaction of different erythro diastereoisomers with the same putative receptor, as well as for lack of such interaction in epiquinidine. The protonation energies calculated for cinchonidine and cinchonine confirm the higher basicity of the aliphatic N(1) as compared with that of the aromatic N(13) atom.

Nine simple and structurally flexible PAF antagonists were synthesized and their inhibitory effects on PAF induced platelet aggregation were measured. Compounds with PAF antagonistic activity exhibited a negative electrostatic potential generated by two trimethoxyphenyl groups (isocontour at -10 Kcal/mole) at various distances between the negative clouds. The optimal distance between the atoms generating the "cache-oreilles" system for exhibiting potent PAF antagonistic activity is estimated to be 11-13 A. In the flexible molecules studied, the dispersion of the electronic distribution is not necessarily favorable for anti-PAF activity. The data support the simple bipolarized model for the PAF receptor that has been proposed by the authors.

Different hypotheses of the structure of platelet-activating factor (PAF) receptor based on structure-activity relationships of agonists and antagonists are reviewed. For an agonistic effect, strong hydrophobic interactions and an ether function are required in position-1 of the glycerol backbone; chain length limitations and steric hindrance demand a small group in position-2. The unusual structural properties of non-PAF-like antagonists required 3-D electrostatic potential calculations. This method applied to seven potent antagonists suggests a strong "Cache-orielles" (ear-muff) effect, i.e., two strong electronegative wells (isocontour at -10 Kcal/mole) are located at 180 degrees to each other and at a relatively constant distance. Initial consideration of the "Cache-oreilles" effect implied the structure of a bipolarized cylinder of 10-12 A diameter for the receptor. However, very recent results on studies with agonists and antagonists structurally similar to PAF suggest that the receptor may in fact be a multi-polarized cylinder.

The sequences of class A beta-lactamases were compared. Four main groups of enzymes were distinguished: those from the gram-negative organisms and bacilli and two distinct groups of Streptomyces spp. The Staphylococcus aureus PC1 enzyme, although somewhat closer to the enzyme from the Bacillus group, did not belong to any of the groups of beta-lactamases. The similarities between the secondary structure elements of these enzymes and those of the class C beta-lactamases and of the Streptomyces sp. strain R61 DD-peptidase were also analyzed and tentatively extended to the class D beta-lactamases. A unified nomenclature of secondary structure elements is proposed for all the penicillin-recognizing enzymes.

Residue Arg220 was found to be important for the acylation of the Streptomyces albus G beta-lactamase by classical penicillins and cephalosporins bearing a carboxylate on C3 or C4. The R220L mutant exhibited strongly decreased kcat/Km values for those compounds. Conversely the acylation rates by benzylpenicillin methylester and deacetylcephalosporin C lactone were little affected, indicating a direct or indirect role of that positively charged residue in the interaction of the enzyme cavity with the negative charge of the substrate. Surprisingly that residue is not conserved in all class A beta-lactamases but when it is not present it can be seen in the known tertiary structures that the guanidinium group of another arginine side chain (Arg244) is similarly positioned. The mutation affected the behaviour of the enzyme towards cephaloridine much less than towards cephalothin. This might represent an example of substrate-assisted catalysis where the disappearance of a positive charge on the enzyme is partly compensated by the presence of a similarly charged group on one of the substrate side chains. All the experimental results are nicely explained by computer-modelling of the enzyme-substrate interactions.

Optimization by energy minimization of stable complexes occurring along the pathway of hydrolysis of benzylpenicillin and cephalosporin C by the Streptomyces albus G beta-lactamase has highlighted a proton shuttle that may explain the catalytic mechanism of the beta-lactamases of class A. Five residues, S70, S130, N132, T235 and A237, are involved in ligand binding. The gamma-OH group of T235 and, in the case of benzylpenicillin, the gamma-OH group of S130 interact with the carboxylate group, on one side of the ligand molecule. The side-chain NH2 group of N132 and the carbonyl backbone of A237 interact with the exocyclic CONH amide bond, on the other side of the ligand. The backbone NH groups of S70 and A237 polarize the carbonyl group of the scissile beta-lactam amide bond. Four residues, S70, K73, S130 and E166, and two water molecules, W1 and W2, perform hydrolysis of the bound beta-lactam compound. E166, via W1, abstracts the proton from the gamma-OH group of S70. While losing its proton, the O-gamma atom of S70 attacks the carbonyl carbon atom of the beta-lactam ring and, concomitantly, the proton is delivered back to the adjacent nitrogen atom via W2, K73 and S130, thus achieving formation of the acyl-enzyme. Subsequently, E166 abstracts a proton from W1. While losing its proton, W1 attacks the carbonyl carbon atom of the S70 ester-linked acyl-enzyme and, concomitantly, re-entry of a water molecule W'1 replacing W1 allows E166 to deliver the proton back to the same carbonyl carbon atom, thus achieving hydrolysis of the beta-lactam compound and enzyme recovery. The model well explains the differences found in the kcat. values for hydrolysis of benzylpenicillin and cephalosporin C by the Streptomyces albus G beta-lactamase. It also explains the effects caused by site-directed mutagenesis of the Bacillus cereus beta-lactamase I [Gibson, Christensen & Waley (1990) Biochem J. 272, 613-619].

In order to compare electronic and conformational properties of PAF-agonists and PAF-antagonists, 14 analogues structurally related to PAF were studied. A common conformation of the glycerol backbone was present in all agonists and all constrained or flexible antagonists. The distinction between agonists and antagonists appears to be casted on position-2 where the folded conformation of the substituent for agonists should be the most probable. In position-3 the gauche conformation can be adopted by all the analysed compounds. The electrostatic potential well at -30 kcal/mol stretches to the carbonyl group in position-2 in the folded conformation of the agonists. On the contrary, in constrained antagonists, a second negative zone appears around the carbamate group. Given the modelling results, the triethylammonium PAF analogue considered in literature as a weak agonist, was resynthesized and proved to be more potent than previously reported. These experimental results confirm our hypothesis in terms of a common conformation of agonist and antagonist PAF-like molecules.

The electronic properties of the active-sites of the structurally unrelated serine peptidases, alpha-chymotrypsin and subtilisin, have been expressed in the form of three-dimensional electrostatic potential maps derived from integrals calculated at the quantum chemistry level. As a consequence of the asymmetrical distribution of the secondary structures that occur within a 7 A sphere around the serine of the catalytic triad, the active sites are highly polarized entities and exhibit large dipole moments. One part of the active sites generates a nucleophilic suction-pump. Its isocontour at -10 kcal mol-1 defines an impressive, negatively-charged volume which bears a narrow channel in the immediate vicinity of the active-site serine 195 in alpha-chymotrypsin or 221 in subtilisin. In native alpha-chymotrypsin, there is a perfect complementation between this nucleophilic suction-pump and the positively-charged electrophilic hole that is generated by the backbone NH of Ser 195 and Gly 193. In subtilisin, generation of the complementing electrophilic hole requires binding of a carbonyl donor ligand and may be achieved by rotation of the side-chain amide of Asn 155 towards the backbone NH of Ser 221. Small variations in the atomic co-ordinates of alpha-chymotrypsin used for the calculations, the presence of water molecules in its active site and the occurrence of point mutations in the amino acid sequence of subtilisin have little effects on the shape and characteristics of the electrostatic potential.

Three-dimensional electrostatic maps were calculated for six potent antagonists of platelet-activating factor (PAF), the antagonists being selected for their apparent structural heterogeneity. The molecules examined were the compact Ginkgolides BN 52020, BN 52021 and BN 52022 (1, 2 and 3), the semi-rigid kadsurenone (4), a flexible synthetic dinor type C furanoid lignan L-652,731 (5a) and the triazolothienobenzodiazepine WEB 2086 (7). Calculation of the electrostatic potential generated around all the above molecules showed the existence of two wells of negative potential or 'cache-oreilles' (ear-muffs), i.e., the isocontours drawn at -10 kcal/mol, located at 180 degrees from each other and separated by a maximum distance of 22-27 A. Except for the synthetic dinor type C furanoid lignan (5a), the molecules also presented a moderate hydrophobic fragment, which constitutes a third point of interaction with the high-affinity binding site in rabbit and human platelets. The findings of the present study allow speculation that this high-affinity acceptor site may be a 'polarized cylinder' with a diameter of 10-12 A.

Measurement of tear turnover rate using an automated scanning fluorophotometer (FluorotronR) was compared to Schirmer test results without anesthesia in young adults with no known ocular disorder. After the instillation of fluorescein, tear film fluorescence, expressed as fluorescein concentration equivalent, decreased exponentially in a linear fashion with time. The mean tear turnover rate was 30% per minute. Contact lens wearers had higher mean tear turnover rates when compared with non-contact lens wearers, but differences were not statistically significant. Half of tested eyes demonstrated total wetting of Schirmer Tear Test StripsR. Higher mean tear turnover rates were associated with complete wetting of the Schirmer test strips. There was no significant correlation between Schirmer paper wetting of less than 30 mm and fluorescein turnover rate.

Several types of active-site-directed inactivators (inhibitors) of the Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase were tested. (i) Among the heavy-atom-containing compounds examined, K2Pt(C2O4)2 inactivates the enzyme with a second-order rate constant of about 6 X 10(-2)M-1 X S-1 and has only one binding site located close to the Zn2+ cofactor within the enzyme active site. (ii) Several compounds possessing both a C-terminal carboxylate function and, at the other end of the molecule, a thiol, hydroxamate or carboxylate function were also examined. 3-Mercaptopropionate (racemic) and 3-mercaptoisobutyrate (L-isomer) inhibit the enzyme competitively with a Ki value of 5 X 10 X 10(-9)M. (iii) Classical beta-lactam compounds have a very weak inhibitory potency. Depending on the structure of the compounds, enzyme inhibition may be competitive (and binding occurs to the active site) or non-competitive (and binding causes disruption of the protein crystal lattice). (iv) 6-beta-Iodopenicillanate inactivates the enzyme in a complex way. At high beta-lactam concentrations, the pseudo-first-order rate constant of enzyme inactivation has a limit value of 7 X 10(-4)S-1 X 6-beta-Iodopenicillanate binds to the active site just in front of the Zn2+ cofactor and superimposes histidine-190, suggesting that permanent enzyme inactivation is by reaction with this latter residue.

Fourteen patients with dry eye signs and symptoms were treated with a cellulosic ophthalmic insert and conventional artificial tear drops in a cross-over study. Each subject rated symptoms before the study and after each treatment period. At the conclusion of the study, subjects chose which of the two treatments they preferred. Thirteen people preferred the artificial tear drops, and more symptoms were alleviated by them. Tear breakup time was not significantly changed by either treatment. The insert was not well received due to subjective blurring of vision and ocular irritation. Other studies have shown patient preference for the insert, but this may be due to patient selection.

The isolated or predominant geodes of the femoral head without signs of coxarthritis or coxitis, seem to form in response to: osteochondritis dissecans in 9 cases where the sequester was only visible in profile films or oblique images for 5 cases, and only at time of operation in 2 cases; incipient necrosis in 3 adult cases, not evidenced in frontal films and only seen in profile films with the patient under axial traction; femoral head dysmorphia in 6 cases among which were 4 polyepiphyseal dysplasias and 2 coxa plana without radiographically apparent sequesters; induced increased pressure caused by congenital subluxation or a major disorder of posture and locomotion of lower limbs in 3 cases. In 2 cases no classification could be assigned to isolated or predominant geodes of the femoral head in spite of anatomic examination. The diagnosis of these isolated or predominant geodes of the head of the femur necessitates excellent films made from different angles of view (profile, "false" profile, usual profile, sometimes oblique shots, tomograms) and often enough a biopsy which must be obtained directly through the joint, not transcervically through the neck if a reliable specimen is desired. The surgical treatment indicated is voiding and packing to a maximum the geodes possibly correcting the former increased pressure discordance. But, in lots of cases the intensity of pain and discomfort remains moderate for years, so indication for surgery arises late after onset.