Variability of chromosomally encoded beta-lactamases from Klebsiella oxytoca

The beta-lactamase genes of Klebsiella oxytoca were previously divided into two main groups: bla(OXY-1) and bla(OXY-2). The two beta-lactamase groups were each represented by beta-lactamases with four different pIs. In each group, one form of beta-lactamase is more frequent than the others combined. The beta-lactamase gene of each representative beta-lactamase with a different pI that was not yet sequenced (pIs 5.7, 6.8 [OXY-2], 7.1, 8.2, and 8.8 [OXY-1]) was cloned and sequenced. The susceptibility patterns as well as relative rates and kinetic parameters for beta-lactam hydrolysis revealed that OXY-2 enzymes hydrolyzed several of the beta-lactams that were examined (carbenicillin, cephalothin, cefamandole, ceftriaxone, and aztreonam) at a greater rate than the OXY-1 enzymes did. Comparison of K. oxytoca beta-lactamases with plasmid-mediated extended-spectrum beta-lactamases MEN-1 and TOHO-1 implied that the threonine at position 168 present in OXY-2 beta-lactamase instead of the alanine in OXY-1 could be responsible for its modified substrate hydrolysis. In each group, the beta-lactamase with a variant pI differs from the main form of beta-lactamase by one to five amino acid substitutions. The substrate profile and the 50% inhibitory concentrations revealed that all substitutions differing from the main form of beta-lactamase were neutral except one difference in the OXY-1 group. This substitution of an Ala to a Gly at position 237 increases the hydrolysis of some beta-lactams, particularly aztreonam; decreases the hydrolysis of benzylpenicillin, cephaloridine, and cefamandole, and decreases the susceptibility to clavulanic acid (fivefold increase in the 50% inhibitory concentration).     

Synthesis of structural analogues of leukotriene B4 and their receptor binding activity

Structural analogues of leukotriene B4 (LTB4) were designed based on the plausible conformation of LTB4 (1). Joining C-7-C-9 of the conformer A or B into an aromatic ring system led to the discovery of benzene analogues 2, 4 and 6a. Joining C-4-C-9 of the conformer C or D into an aromatic ring system led to the discovery of analogues 3, 5 and 7. The compounds examined in this study were evaluated as to their inhibition of [3H] LTB4 binding to human neutrophils, and by a secondary intact human neutrophil functional assay for agonist/antagonist activity. The first analogues prepared, compounds 2-7, demonstrated moderate potency in the LTB4 receptor binding assay. The modification of these compounds by the introduction of another substituent into the aromatic ring produced a marked increase in receptor binding (28c, IC50 = 0.020 microM; 38c, IC50 = 0.020 microM; 52a, IC50 = 0.020 microM; 52b, IC50 = 0.018 microM). Most of these structural analogues of LTB4 demonstrated agonist activity. Of the analogues prepared in this study, only compound 57 demonstrated weak LTB4 receptor antagonist activity, at 10 microM.     

Structure-function relationships among wild-type variants of Staphylococcus aureus beta-lactamase: importance of amino acids 128 and 216

beta-Lactamases inactivate penicillin and cephalosporin antibiotics by hydrolysis of the beta-lactam ring and are an important mechanism of resistance for many bacterial pathogens. Four wild-type variants of Staphylococcus aureus beta-lactamase, designated A, B, C, and D, have been identified. Although distinguishable kinetically, they differ in primary structure by only a few amino acids. Using the reported sequences of the A, C, and D enzymes along with crystallographic data about the structure of the type A enzyme to identify amino acid differences located close to the active site, we hypothesized that these differences might explain the kinetic heterogeneity of the wild-type beta-lactamases. To test this hypothesis, genes encoding the type A, C, and D beta-lactamases were modified by site-directed mutagenesis, yielding mutant enzymes with single amino acid substitutions. The substitution of asparagine for serine at residue 216 of type A beta-lactamase resulted in a kinetic profile indistinguishable from that of type C beta-lactamase, whereas the substitution of serine for asparagine at the same site in the type C enzyme produced a kinetic type A mutant. Similar bidirectional substitutions identified the threonine-to-alanine difference at residue 128 as being responsible for the kinetic differences between the type A and D enzymes. Neither residue 216 nor 128 has previously been shown to be kinetically important among serine-active-site beta-lactamases.     

Comparison of the in vitro activity of several cephalosporin antibiotics against gram-negative and gram-positive bacteria resistant to cephaloridine

The in vitro activity of each of two oral [cefatrizine (BL-S640), cephalexin] and three parenteral (cefamandole, cefazolin, cephapirin) cephalosporin antibiotics was compared with that of cephalothin against 168 clinical isolates of gram-negative and gram-positive bacteria selected as resistant to 20 mug of cephaloridine per ml on the basis of agar dilution susceptibility test data. Each of the five other cephalosporins inhibited a greater percentage of gram-negative bacillary isolates than did cephalothin or cephaloridine, with minimal inhibitory concentration values ranging 2- to 50-fold lower. Significant differences between minimal inhibitory concentrations of the compounds tested were also observed in tests against strains of Streptococcus faecalis and of methicillin-resistant Staphylococcus aureus. Potential advantages of including more than a single cephalosporin antibiotic in the panel of antibiotics used for routine susceptibility testing, suggested by these observations, are discussed.     

beta-Lactamases responsible for resistance to expanded-spectrum cephalosporins in Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis isolates recovered in South Africa

Although resistance to the expanded-spectrum cephalosporins among members of the family Enterobacteriaceae lacking inducible beta-lactamases occurs virtually worldwide, little is known about this problem among isolates recovered in South Africa. Isolates of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis resistant to expanded-spectrum cephalosporins recovered from patients in various parts of South Africa over a 3-month period were investigated for extended-spectrum beta-lactamase production. Antibiotic susceptibility was determined by standard disk diffusion and agar dilution procedures. Production of extended-spectrum beta-lactamases was evaluated by using the double-disk test, and the beta-lactamases were characterized by spectrophotometric hydrolysis assays and an isoelectric focusing overlay technique which simultaneously determined isoelectric points and general substrate or inhibitor characteristics. DNA amplification and sequencing were performed to confirm the identities of these enzymes. The P. mirabilis and E. coli isolates were found to produce TEM-26-type, SHV-2, and SHV-5 extended-spectrum beta-lactamases. An AmpC-related enzyme which had a pI of 8.0 and which conferred resistance to cefoxitin as well as the expanded-spectrum cephalosporins was found in a strain of K. pneumoniae. This is the first study which has identified organisms producing different extended-spectrum beta-lactamases from South Africa and the first report describing strains of P. mirabilis producing a TEM-26-type enzyme. The variety of extended-spectrum beta-lactamases found among members of the family Enterobacteriaceae isolated from major medical centers in South Africa is troubling and adds to the growing list of countries where these enzymes pose a serious problem for antimicrobial therapy.     

NAcSDKP analogues resistant to angiotensin-converting enzyme

Two series of analogues of the tetrapeptide NAcSDKP, an inhibitor of hematopoietic stem cell proliferation, were prepared, and their enzymatic stability toward rabbit lung angiotensin-converting enzyme (ACE) was evaluated as well as their capacity to inhibit NAcSDKP hydrolysis by this enzyme. In the first series, each of the peptide bonds has been successively replaced by an aminomethylene bond. In the second one, the C-terminus of the peptide has been modified by decarboxylation or amidation. The results reported here indicate that all of these molecules but one have good stability toward the enzyme but none of the compounds is able to inhibit NAcSDKP hydrolysis by ACE.     

Inhibitor-resistant OXY-2-derived beta-lactamase produced by Klebsiella oxytoca

Klebsiella oxytoca strains are generally moderately resistant to amoxicillin and ticarcillin due to the activities of the chromosomally encoded OXY-1 and OXY-2 class A beta-lactamase families. These enzymes have the ability to hydrolyze not only penicillins but also cephalosporins, including cefuroxime, ceftriaxone, and aztreonam, and are inhibited by clavulanic acid. A Klebsiella oxytoca strain was isolated from a culture of blood from a patient who had been treated with amoxicillin-clavulanate (3 g/day) for 10 days 1 month earlier. This strain harbored an unusual phenotype characterized by resistance to amoxicillin-clavulanate. It produced an OXY-2-type beta-lactamase (pI 6.3), as confirmed by PCR amplification with primers specific for the OXY-2-encoding gene. Gene sequencing revealed a point mutation (A-->G) corresponding to the amino acid substitution Ser-->Gly at position 130. This mutant enzyme was poorly inhibited by inhibitors, and its kinetic constants compared to those of the parent enzyme were characterized by an increased Km value for ticarcillin, with a drastically reduced activity against cephalosporins, as is observed with inhibitor-resistant TEM enzymes. The substitution Ser-->Gly-130 was previously described in the inhibitor-resistant beta-lactamase SHV-10 derived from an SHV-5 variant, but this is the first report of such a mutant in OXY enzymes from K. oxytoca.     

Characterization of a chromosomal gene encoding type B beta-lactamase in phage group II isolates of Staphylococcus aureus

In contrast to most Staphylococcus aureus isolates in which the gene for staphylococcal beta-lactamase (blaZ) is plasmid borne, isolates typeable by group II bacteriophages frequently carry blaZ on the chromosome. Furthermore, the chromosomal gene encodes the type B variant of staphylococcal beta-lactamase for which the nucleotide and deduced amino acid sequences have not yet been reported. To better understand beta-lactamase production among phage group II staphylococci and the nature of the type B beta-lactamase, we determined the type and amount of enzyme produced by 24 phage group II isolates. Of these isolates, 1 did not produce beta-lactamase, 8 produced the type B enzyme, and 15 produced the type C enzyme. In all eight type B beta-lactamase-producing isolates, blaZ was located on the chromosome. This was in contrast to the type C beta-lactamase-producing isolates, in which blaZ was located on a 21-kb plasmid. The nucleotide sequence corresponding to the leader peptide and the N-terminal 85% of the mature exoenzyme form of type B S. aureus was determined. The deduced amino acid sequence revealed 3 residues in the leader peptide and 12 residues in the exoenzyme portion of the beta-lactamase that differ from the prototypic type A beta-lactamase sequence. These include the serine-to-asparagine change at residue 216 found in the kinetically similar type C enzyme, a threonine-to-lysine change at residue 128 close to the SDN loop (residues 130 to 132), and several substitutions not found in any of the other staphylococcal beta-lactamases. In summary, modern isolates of S. aureus typeable by group II phages produce type B or type C staphylococcal beta-lactamase. The type B gene resides on the chromosome and has a sequence that, when compared to the sequences of the other staphylococcal beta-lactamases, corresponds well with its kinetic properties.     

Subcapital fractures of the four ulnar metacarpal bones

A strain of Chromobacterium violaceum isolated from a fatally infected patient was found to produce a beta-lactamase. When the organism was grown in drug-free medium, beta-lactamase activity was barely detectable, but when it was grown in the presence of penicillin G, a much larger amount of activity was produced. The beta lactamase was active primarily against cephalosporins; it was sensitive to inhibition by cloxacillin but resistant to p-chloromercuribenzoate. Thus this enzyme closely resembled the common type of beta-lactamase found in Pseudomonas aeruginosa. The organism was relatively susceptible to ticarcillin, carbenicillin, and cefoxitin, which resected hydrolysis by its beta-lactamase, but was quite resistant to 11 other beta-lactam antibiotics. Production of the beta-lactamase appeared to be mediated by chromosomal genes.     

Radial glia and cell debris removal during lesion-regeneration of the lizard medial cortex

By challenging the efficiency of some of our most useful antimicrobial weapons, bacterial antibiotic resistance is becoming an increasingly worrying clinical problem. A good antibiotic is expected to exhibit a high affinity for its target and to reach it rapidly, while escaping chemical modification by inactivating enzymes and elimination by efflux mechanisms. A study of the behaviour of a beta-lactamase-overproducing mutant of Enterobacter cloacae in the presence of several penicillins and cephalosporins showed that the minimum inhibitory concentration (MIC) values for several compounds were practically independent of the sensitivity of the target penicillin binding protein (PBP), even for poor beta-lactamase substrates. This apparent paradox was explained by analysing the equation that relates the antibiotic concentration in the periplasm to that in the external medium. Indeed, under conditions that are encountered frequently in clinical isolates, the factor characterizing the PBP sensitivity became negligible. The conclusions can be extended to all antibiotics that are sensitive to enzymatic inactivation and efflux mechanisms and must overcome permeability barriers. It would be a grave mistake to neglect these considerations in the design of future antibacterial chemotherapeutic agents.     

Potent cyano and carboxamido side-chain analogues of 1', 1'-dimethyl-delta8-tetrahydrocannabinol

The synthesis and pharmacological profile of several cyano (1a-e) and carboxamido (2a-h) side-chain-substituted analogues of 1', 1'-dimethyl-Delta8-THC are described. Commercially available cyano compound 3 was transformed to the resorcinol 6 in a three-step sequence. Condensation of 6 with p-menth-2-ene-1,8-diol formed the THC 7a which, with sodium cyanide/DMSO, gave 1b. Protection of the phenol in 7a as the MOM derivative provided the common intermediate 8 for the synthesis of 1a,c,e. Compound 1d was also synthesized from 7a via the aldehyde 9a. Base hydrolysis of 1b gave the acid 10 which, via its acid chloride and subsequent treatment with the appropriate amine, formed the target compounds 2a-h. The pharmacological profile indicated that the cyano analogues 1a-e had very high CB1 binding affinity (0.36-13 nM) and high in vivo potency as agonists. Two analogues (1a,b) had extremely high potency in the mouse tetrad tests. The dimethylcarboxamido analogue 2a showed a similar profile to 1a,b. The high potency was also retained in analogue 2c. In contrast the sulfonamide analogue 2d was unique as it had greater affinity than Delta9-THC, yet it was practically devoid of agonist effects. This study suggests that the incorporation of a cyano or an amide substituent in the side chain of Delta8-THC-DMH can enhance potency and can also lead to compounds with a unique profile which have high binding affinity and are practically devoid of agonist effects.     

Chemistry of cephalosporin antibiotics. 28. Preparation and biological activity of 3-(substituted)vinyl cephalosporins

3-(Substituted)vinylcephem nuclei have been prepared by the reaction of 3-formylcephem derivatives with stabilized phosphoranes. Appropriate synthetic steps allowed preparation of a series of 3-ethoxycarbonylvinyl- and 3-carboxyvinylcephem derivatives bearing a variety of 7-acylamino functions. The phenoxyacetyl and thiopheneacetyl derivatives of the 3-cyanovinylcephem nucleus were also prepared. Although general gram-positive activity was comparable to cephalothin in many cases, against penicillin G resistant Staphylococcus aureus, the new cephalosporins were of low effectiveness. The 3-(substituted)vinyl cephalosporins had good activity against a number of gram-negative organisms. In some cases, this activity was excellent. The N-acetyl analogs had surprisingly good activity relative to N-acetyl-7-ACA. The phenylmalonoyl side-chain derivatives were shown to have an unusual antibacterial spectrum expansion (relative to previously known cephalosporins) to include activity against Serratia marcescens and Pseudomonas aeruginosa.     

Site-directed mutagenesis of beta-lactamase TEM-1. Investigating the potential role of specific residues on the activity of Pseudomonas-specific enzymes

From sequence alignments, two groups can be defined for the carbenicillin-hydrolysing beta-lactamases (CARB enzymes). One group includes the Pseudomonas-specific enzymes PSE-1, PSE-4, CARB-3, CARB-4 and also the Proteus mirabilis GN79, for which the well-conserved residue Lys 234 in all class-A beta-lactamases is changed to an arginine residue. The second group includes the enzymes PSE-3 and AER-1 which have an arginine or a lysine residue at position 165. All these enzymes also have leucine at position 68, threonine at position 104 and glycine at position 240. We engineered these mutations into the TEM-1 beta-lactamase to study their potential role in defining the substrate profile of the CARB enzymes. The mutations K234R and E240G in TEM-1 noticeably increased the hydrolysis of carboxypenicillins relative to other penicillins by approximately sixfold and twofold, respectively. The variant E240G also demonstrated an improved rate of second-generation cephalosporin and cefotaxime hydrolysis. In contrast, the substitution of Trp165 by arginine does not extend the substrate profile to alpha-carboxypenicillins nor does it noticeably modify the kinetic behavior of the enzyme. The mutations M68L and E104T do not have a large effect on the hydrolysis rate but the mutation E104T enhances the affinity of the enzyme for third-generation cephalosporins. As the mutation K234R resulted in a severe decrease in the affinity for carboxypenicillins, the double mutant E240G/K234R was constructed in an attempt to enhance the CARB character of the enzyme. Contrary to what could be expected, the additional mutation E240G for the TEM-1 K234R enzyme increases neither the catalytic constant for the carboxypenicillins nor the affinity towards these substrates. Consequently, this study strongly suggests that the three-dimensional structures of the active site of the TEM-1 enzyme and PSE-3, PSE-4 or other related enzymes are significantly different. This probably explains the discrepancy of the substrate profile between the CARB enzymes and the TEM-1 protein variants.     

6-substituted 2,4-diamino-5-methylpyrido[2,3-d]pyrimidines as inhibitors of dihydrofolate reductases from Pneumocystis carinii and Toxoplasma gondii and as antitumor agents

The synthesis and biological activity of 15 6-substituted 2,4-diamino-5-methylpyrido[2,3-d]-pyrimidines are reported. These compounds were synthesized in improved yields by modifications of procedures previously reported by us. Specifically, dimethoxyphenyl-substituted compounds with H and CH3 at the N-10 position and trimethoxyphenyl-substituted compounds with N-10 ethyl, isopropyl, and propargyl moieties were synthesized. These compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii, Toxoplasma gondii, and rat liver, and selected analogues were evaluated as inhibitors of the growth of T. gondii and tumor cells in culture. All the compounds showed increased selectivity (vs rat liver DHFR) for T. gondii DHFR compared to trimetrexate. In general, for the trimethoxy-substituted analogues, increasing the size of the N-10 substituent from a methyl group to larger groups resulted in a decrease in selectivity and potency for both P. carinii and T. gondii DHFR. For the dimethoxy-substituted analogues, N-10 methylation in general decreased potency but increased selectivity for T. gondii DHFR. In an attempt to improve the cell penetration of these analogues, the N-10 naphthyl-substituted analogues were also synthesized. These analogues displayed excellent cell penetration and inhibition of T. gondii cells in culture. Further, these analogues were potent inhibitors of the growth of tumor cells in the preclinical in-vitro screening program of the National Cancer Institute with IC50s in the nanomolar range.     

Microbial sensor for new-generation cephalosporins based in a protein-engineered beta-lactamase

A protein-engineered beta-lactamase, constructed by site-directed mutagenesis in Escherichia coli (E104M/G238S), and having broadened specificity, was able to degrade cephalosporins of first, second, and third generations. Manipulations of culture conditions allowed an increase in beta-lactamase specific activity by up to twofold. The resultant bacteria were used to construct an immersable whole-cell biosensor for the detection of new-generation cephalosporins. Cells were immobilized on agar membranes, which in turn were attached to the surface of a flat pH electrode, thus constituting a biosensor based on the detection of pH changes. The sensor was able to detect second- and third-generation cephalosporins: cefamandole (0.4-4 mM), cefotaxime (0.4-3.5 mM), and cefoperazone (0.3-1.85 mM). Response times were between 3.5 and 11 min, depending on the kind of cephalosporin tested. The biosensor was stable for at least 7 d, time during which up to 100 tests were performed.     

The role of residue 238 of TEM-1 beta-lactamase in the hydrolysis of extended-spectrum antibiotics

beta-Lactamases inactivate beta-lactam antibiotics by catalyzing the hydrolysis of the amide bond in the beta-lactam ring. The plasmid-encoded class A TEM-1 beta-lactamase is a commonly encountered beta-lactamase. It is able to inactivate penicillins and cephalosporins but not extended-spectrum antibiotics. However, TEM-1-derived natural variants containing the G238S amino acid substitution display increased hydrolysis of extended-spectrum antibiotics. Two models have been proposed to explain the role of the G238S substitution in hydrolysis of extended-spectrum antibiotics. The first proposes a direct hydrogen bond of the Ser238 side chain to the oxime group of extended-spectrum antibiotics. The second proposes that steric conflict with surrounding residues, due to increased side chain volume, leads to a more accessible active site pocket. To assess the validity of each model, TEM-1 mutants with amino acids substitutions of Ala, Ser, Cys, Thr, Asn, and Val have been constructed. Kinetic analysis of these enzymes with penicillins and cephalosporins suggests that a hydrogen bond is necessary but not sufficient to achieve the hydrolytic activity of the G238S enzyme for the extended-spectrum antibiotics cefotaxime and ceftazidime. In addition, it appears that the new hydrogen bond interaction is to a site on the enzyme rather than directly to the extended-spectrum antibiotic. The data indicate that, for the G238S substitution, a combination of an optimal side chain volume and hydrogen bonding potential results in the most versatile and advantageous antibiotic hydrolytic spectrum for bacterial resistance to extended-spectrum antibiotics.     

Activities of thyroid hormones and related compounds in an in vitro thymocyte assay

Prior studies have demonstrated that the thyroid hormones L-triiodothyronine and L-thyroxine stimulate the rapid uptake of 1-amino-cyclopentane-1-carboxylic acid into isolated rat thymocytes. In the present study the effects of several groups of thyroid hormones and structurally related compounds were investigated to determine the structure-function relations required for stimulation of this membrane process. Particular attention was given to (a) analogues with modifications at the oxygen bridge, the phenolic hydroxyl group, and the group at the 3' position of the outer ring, and (b) the steric orientation of the thyroid hormones. The following were found to be important for maximal activity: (a) the L-isomer configuration, (b) the presence of a 4'-hydroxyl group, (c) the presence of one substituent in both the inner and outer rings (3 and 3' positions), (d) the distal orientation of the 3' substituent in the outer ring of L-triiodothyronine, and (e) the lipophilic character of the 3' substituent. Of lesser importance was the presence of halogen atoms, or an oxygen atom in the ether position. Since these structure-function relations seen in thymocytes parallel in many respects those relations seen in whole-animal studies, it is believed that thymocytes will be a useful tool for further studies of thyroid hormones and their analogues.     

Analysis of ochratoxin B alone and in the presence of ochratoxin A, using carboxypeptidase A

A method is described for ochratoxin B analysis, which is adapted to the earlier described method of ochratoxin A analysis, using carboxypeptidase A (K. Hult and S. Gatenbeck, J. Assoc. Off. Anal. Chem. 59:128-129, 1976). The fluorescence spectra of ochratoxins A and B coincide too much to allow direct discrimination of the two compounds. A method using the differences in kinetic parameters of the enzymatic hydrolysis of the two compounds is suggested for the analysis of mixtures of the ochratoxins.     

Metal-substrate interactions facilitate the catalytic activity of the bacterial phosphotriesterase

The bacterial phosphotriesterase from Pseudomonas diminuta is a zinc metalloenzyme which catalyzes the hydrolysis of a variety of organophosphorus nerve agents with high efficiency. The active site of the enzyme consists of a coupled binuclear metal center embedded within a cluster of histidine residues. Potential protein-substrate interactions at the active site were probed by a systematic variation of metal identity, leaving group potential, phosphate host, and amino acid replacement. In order to determine the roles of these metal ions in binding and catalysis, the microscopic rate constants and kinetic parameters were obtained with various divalent cations. The divalent cations that were utilized in this investigation consisted of Co2+, Ni2+, Cd2+, Zn2+, Mn2+, and the mixed-metal Zn2+/Cd2+ hybrid. The leaving group potential and phosphate host were varied by altering the pKa of the departing substituted phenol or thiophenol in either a diethyl phosphate or a diethyl thiophosphate substrate. The Br?nsted plots for the nonenzymatic hydroxide catalyzed hydrolysis of these substrates showed a linear dependence between the pseudo-first-order rate constant and the pKa of the leaving group. Enzymatic activities of the wild-type enzyme with these same substrates varied by over 7 orders of magnitude over the entire experimental pKa range (4.1-10.3), and the corresponding Br?nsted plots were nonlinear. Those substrates with leaving groups with high pKa values were limited by the rate of bond cleavage while those substrates having leaving groups with low pKa values were limited by a conformational change or binding event. Thiophosphate substrates having leaving groups with high pKa values were better substrates than the corresponding phosphate analogues. These results are consistent with the direct coordination of one or both metal ions with the phosphoryl sulfur or oxygen atom of the substrate. A large dependence of the rate on the leaving group rules out the possibility of protonation of the leaving group or electrostatic interaction of the leaving group oxygen (or sulfur) with a metal ion or cationic group at the active site. The large differences in the size of the beta lg over the range of metal ions utilized by the enzyme indicate that the metal ions polarize the phosphoryl group and alter the structure of the transition state. The values of V/K(m) for the enzyme-catalyzed hydrolysis for a series of substituted thiophenol analogues were 10(2)-10(3)-fold smaller than those obtained for the hydrolysis of the corresponding phenolic substrates, suggesting that the bulkier sulfur substituent in the leaving group may induce conformational restrictions at the active site. With the zinc-substituted H201N mutant enzyme, there was a large decrease in the rate of phosphotriester hydrolysis but essentially no change in the rate of thiophosphotriester hydrolysis relative to the values observed for the zinc-substituted wild-type enzyme. These results suggest that a direct perturbation in the ligand structure of the binuclear metal center induces alterations in the mechanism of substrate hydrolysis.     

History of sexual abuse among HIV-infected women

OBJECTIVE: To study the changes in patterns and resistances of bacteria from patients with bacteremia in 11 years. METHODS: Diffusion tests were used to measure the antibiotic sensitivity of bacteria isolated from the patients with bacteremia. RESULTS: The proportion of staphylococcus epidermidis in bacteremia increased significantly. The proportion of methicillin-resistant Staphylococcus aureus (MRSA) in bacteremia caused by staphyiococcus increased markedly. Resistance of MRSA and methlcillin-sensitive staphylococcus aureus (MSSA) to penicillin, and gentamycin increased significantly. Compared 1986-1990, with 1991-1996, the resistance of S. epidermidis to penicillin, erythromycin and methicillin increased markedly, so did resistance of pseudomonas aeruginosa to carbenicillin, and piperacillin, resistance of E. coli to gentamycin and cefoperazone, resistance of klebsiella to ampicillin and carbenicillin. The resistance of enterobacteriaceae which induced beta-lactamase to the first to the third generation of cephalosporins was relatively high. CONCLUSION: The resistance of bacteria to the commonly used antibiotics was produced in various degrees.