Antimicrob Agents Chemother, 2000 Jul, 44(7), 1970 - 3 Cloning and sequence of the gene encoding a novel cefotaxime-hydrolyzing beta-lactamase (CTX-M-9) from Escherichia coli in Spain; Sabate M et al.; A new CTX-M-type beta-lactamase (CTX-M-9) has been cloned from a clinical cefotaxime-resistant Escherichia coli strain . Despite the close identity that exists between the CTX-M-9 and Toho-2 beta-lactamases (88%), the 35 amino acids located between residues Ala-185 and Ala-219 are totally different in both enzymes . Outside of this region there are only six amino acids substitutions between both proteins. J Antimicrob Chemother, 2000 Jun, 45(6), 783 - 8 Analysis of the effects of -42 and -32 ampC promoter mutations in clinical isolates of Escherichia coli hyperproducing ampC; Caroff N et al.; Escherichia coli usually produces only very small amounts of a constitutive AmpC beta-lactamase, but clinical strains overproducing this enzyme have been isolated . Three different ampC promoters of E . coli clinical strains were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene in the pKK232-8 reporter plasmid and their relative strengths were compared by two different methods . The strength of the promoters from AmpC hyperproducers was 70- to 120-fold higher than those from a low-level AmpC producer . One of the strong promoters, which differs from strain K12 at bases -88, -82, -42, -18, -1 and +58, was mutated to abolish the -42 mutation . This change resulted in a 43-fold decrease in CAT concentration . In another promoter, with eight different mutations at positions -88, -82, -32, -18, -1, +5, +24 and +58, the -32T-->A transversion, which created perfect homology with the -35 consensus sequence, was reverted; this led to a 13-fold decrease in CAT concentration . The -42 and -32 mutations play an important role in E . coli resistance to beta-lactams by increasing ampC transcription. Microbiol Res, 2000 Apr, 155(1), 1 - 6 A search for beta-lactamase in chlamydiae, mycoplasmas, planctomycetes, and cyanelles: bacteria and bacterial descendants at different phylogenetic positions and stages of cell wall development; Claus H et al.; Bacteria from different phylogenetic positions such as chlamydiae, mycoplasmas, planctomycetes and also endosymbiotic murein-containing cyanelles were investigated for the production of beta-lactamases . No beta-lactamase activity was found in bacteria lacking murein such as Chlamydia pneumoniae, Mycoplasma pneumoniae, Pirellula marina and Planctomyces maris . In the murein-containing cyanelles of Cyanophora paradoxa no beta-lactamase activity could be detected. Appl Biochem Biotechnol, 2000 Jan-Mar, 83(1-3), 163 - 9; discussion 169-71, 297-313 Catalytic mechanism of an abzyme displaying a beta-lactamase-like activity; Avalle B et al.; A catalytic IgG (Ab2) displaying a beta-lactamase-like activity was previously obtained by using the antiidiotypic pathway: the particularity of this antibody is that it is a true antiidiotype of the beta-lactamase active site . We have previously demonstrated that this IgG has retained some of the structural information displayed by the beta-lactamase active site, evident from data that polyclonal anti-Ab2 antibodies (Ab3) recognize beta-lactamase . In this article, we investigated the catalytic mechanism of the abzyme compared to that of the enzyme . The experimental data allow us to hypothesize the catalytic residues required for catalysis. Scand J Infect Dis, 2000, 32(2), 217 - 8 Moraxella catarrhalis endocarditis: case report and review of the literature; Stefanou J et al.; A case of bacterial endocarditis caused by Moraxella catarrhalis in an apparently immunocompetent Greek male is presented, which was diagnosed after a 2-month history of low-grade fever of unknown origin . The agent seems to be a rare pathogen, but due to the high mortality rate, it should always be considered in the differential diagnosis of relevant cases . Beta-lactamase production by many strains complicates the choice of antibiotic. Antimicrob Agents Chemother, 2000 Jun, 44(6), 1725 - 7 A new SHV-derived extended-spectrum beta-lactamase (SHV-24) that hydrolyzes ceftazidime through a single-amino-acid substitution (D179G) in the -loop; Kurokawa H et al.; A new SHV-derived extended-spectrum beta-lactamase (SHV-24) conferring high-level resistance to ceftazidime but not cefotaxime and cefazolin was identified in Japan . This enzyme was encoded by a transferable 150-kb plasmid from an Escherichia coli clinical isolate . The pI and K(m) for CAZ of this enzyme were 7.5 and 30 microM, respectively . SHV-24 was found to have a D179G substitution in the Omega-loop of the enzyme. Antimicrob Agents Chemother, 2000 Jun, 44(6), 1538 - 43 The Legionella (Fluoribacter) gormanii metallo-beta-lactamase: a new member of the highly divergent lineage of molecular-subclass B3 beta-lactamases; Boschi L et al.; A metallo-beta-lactamase determinant was cloned from a genomic library of Legionella (Fluoribacter) gormanii ATCC 33297(T) constructed in the plasmid vector pACYC184 and transformed into Escherichia coli DH5alpha, by screening for clones showing a reduced susceptibility to imipenem . The product of the cloned determinant, named FEZ-1, contains a 30-kDa polypeptide and exhibits an isoelectric pH of 7.6 . Sequencing revealed that FEZ-1 is a molecular-class B beta-lactamase which shares the closest structural similarity (29.7% of identical residues) with the L1 enzyme of Stenotrophomonas maltophilia, being a new member of the highly divergent subclass B3 lineage . All the residues that in L1 are known to be directly or indirectly involved in coordination of the zinc ions were found to be conserved also in FEZ-1, suggesting that the geometry of zinc coordination in the active site of the latter enzyme is identical to that of L1 . Unlike L1, however, FEZ-1 appeared to be monomeric in gel permeation chromatography experiments and exhibited a distinctive substrate specificity with a marked preference for cephalosporins and meropenem . The properties of FEZ-1 overall resembled those of a beta-lactamase previously purified from the same strain of L . gormanii (T . Fujii, K . Sato, K . Miyata, M . Inoue, and S . Mitsuhashi, Antimicrob . Agents Chemother . 29:925-926, 1986) and are as yet unique among class B enzymes, reinforcing the notion that considerable functional heterogeneity can be encountered among members of this class . A system for overexpression of the bla(FEZ-1) gene in E . coli, based on the T7 phage promoter, was also developed. Proteins, 2000 Jul 1, 40(1), 23 - 8 pK(a) calculations for class C beta-lactamases: the role of Tyr-150; Lamotte-Brasseur J et al.; The Poisson-Boltzmann method was used to compute the pK(a) values of titratable residues in a set of class C beta-lactamases . In these calculations, the pK(a) of the phenolic group of residue Tyr150 is the only one to stand out with an abnormally low value of 8.3, more than one pK(a) unit lower than the measured reference value for tyrosine in solution . Other important residues of the catalytic pocket, such as the conserved Lys67, Lys315, His314, and Glu272 (hydrogen-bonded to the ammonium group of Lys315), display normal protonation states at neutral pH . pK(a) values were also computed in catalytically impaired beta-lactamase mutants . Comparisons between the relative k(cat) values and the Tyr150 pK(a) value in these mutants revealed a striking correlation . In active enzymes, this pK(a) value is always lower than the solution reference value while it is close to normal in inactive enzymes . These results thus support the hypothesis that the phenolate form of Tyr150 is responsible for the activation of the nucleophilic serine . The possible roles of Lys67 and Lys315 during catalysis are also discussed . Protein Eng, 2000 Apr, 13(4), 267 - 74 Structure-function analysis of alpha-helix H4 using PSE-4 as a model enzyme representative of class A beta-lactamases; Savoie A et al.; We extracted maximum information for structure-function analysis of the PSE-4 class A beta-lactamase by random replacement mutagenesis of three contiguous codons in the H4 alpha-helix at amino acid positions Ala125, Thr126, Met127, Thr128 and Thr129 . These positions were predicted to interact with suicide mechanism-based inhibitors when examining the PSE-4 three-dimensional model . Structure-function studies on positions 125-129 indicated that in PSE-4 these amino acids have a role distinct from those in TEM-1, in tolerating substitutions at Ala125 and being invariant at Met127 . The importance of Met127 was suspected to be implicated in a structural role in maintaining the integrity of the H4 alpha-helix structure together, thus maintaining the important Ser130-Asp131-Asn132 motif positioned towards the active site . At the structural level, the H4 region was analyzed using energy minimization of the H4 regions of the PSE-4 YAM mutant and compared with wild-type PSE-4 . The Tyr 125 of the mutant YAM formed an edge to face pi-pi interaction with Phe 124 which also interacts with the Trp 210 with the same interactions . Antibiotic susceptibilities showed that amino acid changes in the the H4 alpha-helix region of PSE-4 are particularly sensitive to mechanism based-inhibitors . However, kinetic analysis of PSE-4 showed that the two suicide inhibitors belonging to the penicillanic acid sulfone class, sulbactam and tazobactam, were less affected by changes in the H4 alpha-helix region than clavulanic acid, an inhibitor of the oxypenam class . The analysis of H4 alpha-helix in PSE-4 suggests its importance in interactions with the three clinically useful inhibitors and in general to all class A enzymes. Farmaco, 2000 Feb, 55(2), 134 - 5 An efficient method for the synthesis of sulbactam pivoxil; Changov LS et al.; Sulbactam pivoxil, a prodrug of the beta-lactamase inhibitor sulbactam, was prepared in high yield by reacting the sodium salt of sulbactam with chloromethyl pivalate in a polar solvent, then diluting the reaction mixture with water and isolating the product by filtration . Dimethyl sulfoxide was found to be the solvent of choice among several aprotic organic solvents. Antimicrob Agents Chemother, 2000 May, 44(5), 1387 - 90 Contributions of the AmpC beta-lactamase and the AcrAB multidrug efflux system in intrinsic resistance of Escherichia coli K-12 to beta-lactams; Mazzariol A et al.; The roles of the AmpC chromosomal beta-lactamase and the AcrAB efflux system in levels of intrinsic resistance and susceptibility of Escherichia coli to beta-lactams were studied with a set of isogenic strains . MICs of ureidopenicillins, carbenicillin, oxacillin, and cloxacillin were drastically reduced by the inactivation of AcrAB, whereas those of the earlier cephalosporins were affected mostly by the loss of AmpC beta-lactamase. J Infect Dis, 2000 Apr, 181(4), 1376 - 87 Epub 2000 Apr 13. Analysis of Moraxella catarrhalis by DNA typing: evidence for a distinct subpopulation associated with virulence traits; Bootsma HJ et al.; Two DNA typing methods, probe-generated restriction fragment length polymorphism analysis and single-adapter amplified fragment length polymorphism analysis, were used to study the genetic relationships among 90 Moraxella catarrhalis strains . Both methods were found to be highly concordant, generating a dendrogram with 2 main branches . The division of the M . catarrhalis population into 2 subspecies was supported by analysis of the 16S rRNA sequences . Both beta-lactamase-positive and beta-lactamase-negative strains were found in all main branches, suggesting horizontal transfer of the beta-lactamase gene . In contrast, 2 virulence traits, complement resistance and adherence to epithelial cells, were strongly associated with 1 of the 2 subspecies . The branch depth suggested that complement-resistant adherent strains diverged from a common ancestor more recently than did complement-sensitive nonadherent strains . These findings suggest the existence of subpopulations of M . catarrhalis that differ in virulence, and they may have implications for vaccine development. Mol Microbiol, 2000 Apr, 36(1), 93 - 104 Genesis of BRO beta-lactamase-producing Moraxella catarrhalis: evidence for transformation-mediated horizontal transfer; Bootsma HJ et al.; The dramatic rise in BRO-producing M . catarrhalis strains observed in the last decades is without precedence . The aim of this study was to elucidate the events that led to the emergence of BRO-1 and BRO-2 beta-lactamases . Previously, we showed bro1 and bro2 to be >99% identical . Data presented here suggested that bro2 was acquired by a fortuitous event and inserted between M . catarrhalis genes orf1 and orf3 . Subsequently, bro1 evolved from bro2 . Promoter-up mutations increased fitness of bro2, explaining its present predominance . The highly conserved nature of bro compared with orf1 and orf3 suggested that acquisition has occurred relatively recently . The random distribution of bro among M . catarrhalis fingerprint types indicated that bro has spread by horizontal transfer . Sequence analysis revealed that 80-200 bp is generally cotransferred with bro, serving as regions of homology that target bro to the same chromosomal locus . A region of 160 bases upstream of bro1 lacked polymorphism, indicating it was derived from the original strain that acquired bro2 . We observed that bro was readily transferred by transformation between M . catarrhalis strains in vitro, suggesting a mechanism by which bro has disseminated . In conclusion, we have been able to reconstruct the steps that led to the emergence of BRO-producing M . catarrhalis. J Biol Chem, 2000 Jun 9, 275(23), 17693 - 9 The cysteine-rich protein A from Helicobacter pylori is a beta-lactamase; Mittl PR et al.; Among the large number of hypothetical proteins within the genomes of Helicobacter pylori, there is a family of unique and highly disulfide-bridged proteins, designated family 12, for which no function could originally be assigned . Sequence analysis revealed that members of this family possess a modular architecture of alpha/beta-units and a stringent pattern of cysteine residues . The H . pylori cysteine-rich protein A (HcpA), which is a member of this family, was expressed and refolded from inclusion bodies . Six pairs of cysteine residues, which are separated by exactly seven residues, form disulfide bridges . HcpA is a beta-lactamase . It slowly hydrolyzes 6-aminopenicillinic acid and 7-aminocephalosporanic acid (ACA) derivatives . The turnover for 6-aminopenicillinic acid derivatives is 2-3 times greater than for ACA derivatives . The enzyme is efficiently inhibited by cloxacillin and oxacillin but not by ACA derivatives or metal chelators . We suggest that all family 12 members possess similar activities and might be involved in the synthesis of the cell wall peptidoglycan . They might also be responsible for amoxicillin resistance of certain H . pylori strains. J Biol Chem, 2000 Jun 9, 275(23), 17428 - 33 Functionally accepted insertions of proteins within protein domains; Collinet B et al.; Experiments were designed to explore the tolerance of protein structure and folding to very large insertions of folded protein within a structural domain . Dihydrofolate reductase and beta-lactamase have been inserted in four different positions of phosphoglycerate kinase . The resultant chimeric proteins are all overexpressed, and the host as well as the inserted partners are functional . Although not explicitly designed, functional coupling between the two fused partners was observed in some of the chimeras . These results show that the tolerance of protein structures to very large structured insertions is more general than previously expected and supports the idea that the natural sequence continuity of a structural domain is not required for the folding process . These results directly suggest a new experimental approach to screen, for example, for folded protein in randomized polypeptide sequences. J Antimicrob Chemother, 2000 Apr, 45(4), 517 - 20 Combinatorial biochemistry and shuffling of TEM, SHV and Streptomyces albus omega loops in PSE-4 class A beta-lactamase; Sanschagrin F et al.; The class A PSE-4 beta-lactamase was used for studying the importance of amino acids in the omega (Omega) loop and its interactions for hydrolysis of beta-lactam antibiotics . By cassette mutagenesis, we replaced the amino acids 163-179 Omega loop in PSE-4 with TEM-1, SHV-1 and Streptomyces albus G beta-lactamase Omega loops . Phenotypic analysis of Escherichia coli recombinants expressing the Omega loop PSE-4 mutant enzymes gave MICs and kinetic data similar to those of wild-type PSE-4. Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3160 - 5 Protonation of the beta-lactam nitrogen is the trigger event in the catalytic action of class A beta-lactamases; Atanasov BP et al.; The pH dependence of the pK(a) values of all ionizable groups and of the electrostatic potential at grid points corresponding to catalytically important atoms in the active site of TEM-1 beta-lactamase has been calculated by a mean-field approach for reaction intermediates modeled on the basis of energy minimized x-ray crystallographic coordinates . By estimating electrostatic contributions to the free energy changes accompanying the conversion of the free enzyme into the acylenzyme reaction intermediate, we found that acid-catalyzed protonation of the beta-lactam nitrogen is energetically favored as the initiating event, followed by base-catalyzed nucleophilic attack on the carbonyl carbon of the beta-lactam group . N-protonation is catalyzed through a hydrogen-bonded cluster involving the 2-carboxylate group of the substrate, the side chains of S130 and K234, and a solvent molecule . Nucleophilic attack on the carbonyl carbon is carried out by the side chain of S70 with proton abstraction catalyzed by a water molecule hydrogen-bonded to the side chain of E166 . Stabilization of ion pairs in the active site through interactions with distant clusters of charged residues in the enzyme was concluded to be an important driving force of the catalytic mechanism. FEMS Microbiol Lett, 2000 Mar 15, 184(2), 303 - 6 Expression of beta-lactamase in Coxiella burnetii transformants; Suhan ML et al.; Coxiella burnetii can be transformed to ampicillin resistance by electroporation with plasmids encoding beta-lactamase . However, non-plasmid emergence of resistance to ampicillin also develops . To validate the usefulness of the bla gene marker for selection and detection, transformed C . burnetii were examined for beta-lactamase expression by use of immunoblotting after SDS-PAGE . The 29-kDa mature form of the beta-lactamase protein was detected in C . burnetii lysates . Quantitation of these immunoblot signals showed that C . burnetii surprisingly expressed low levels of beta-lactamase . The results validate the use of plasmid-encoded ampicillin resistance as a means for selecting C . burnetii transformants; they also suggest that levels of ampicillin used for selection pressure should be empirically determined and that detection of beta-lactamase by antibody blotting done to confirm transformants. Curr Microbiol, 2000 May, 40(5), 302 - 5 Development of a plasmid vector for easy selection of strong promoters; Pineiro SA et al.; A promoter vector pACPR33 for Escherichia coli based on the promotorless ampicillin-resistance gene from pBR322 has been constructed . The promoter of the ampicillin-resistance gene was deleted and replaced by a suitable multiple cloning site . Molecular cloning of promoters into the polylinker resulted in activation of the ampicillin resistance in E . coli . The plasmid contains a functional origin of DNA replication and a tetracycline resistance gene for E . coli, and a chloramphenicol resistance gene for S . aureus . The vector permitted direct detection of promoter activity, especially strong promoters, by easy iodometric determination of beta-lactamase activity in liquid or solid media. Biotechnol Bioeng, 2000 Apr 5, 68(1), 121 - 5 Rapid in vivo evolution of a beta-lactamase using phagemids; Long-McGie J et al.; RNA viruses are capable of undergoing extremely rapid evolution due to their high rates of reproduction, small genome size, and a high frequency of spontaneous mutagenesis . Here we demonstrate that a virus-like, evolutionary state can be created by propagating a phagemid population in a hypermutator strain of Escherichia coli in the presence of a helper phage . This enables one to subject individual phagemid-encoded genes to rapid in vivo evolution . We applied this approach to TEM-1 beta-lactamase which confers resistance to 0.05 mg/L of the antibiotic cefotaxime . After 3 weeks of in vivo evolution we were able to isolate a double mutant, E104K/G238S, of the enzyme which confers a 500-fold increased level of resistance to cefotaxime compared to the starting enzyme . In two independent experiments we obtained a triple mutant, E104K/G238S/T263M, which confers a 1000-fold increase in resistance compared to the wild type enzyme . The same three mutations have been previously observed in TEM-4 beta-lactamase which was discovered in a highly cefotaxime-resistant clinical isolate . The probability of randomly obtaining a beta-lactamase carrying three identical point mutations is less than 10(-10) . This indicates that phagemid evolution can rapidly reproduce evolution occurring in nature . FEMS Microbiol Lett, 2000 Mar 1, 184(1), 85 - 9 Characterisation of extended-spectrum beta-lactamases of the SHV family using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP); Chanawong A et al.; Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been developed to extend the identification of SHV beta-lactamases previously characterised by PCR-single strand conformational polymorphism (PCR-SSCP) analysis alone . Eight bacteria, each producing a different SHV beta-lactamase, were used in this study . These bacteria harbour bla(SHV-1), bla(SHV-2a), bla(SHV-3), bla(SHV-4), bla(SHV-5) (two strains), bla(SHV-11) and bla(SHV-12) . All isolates were characterised by PCR-SSCP and PCR-RFLP with DdeI and NheI digestion . By a combination of these techniques, the genes encoding these beta-lactamases could be differentiated from each other . In addition, the PCR-RFLP technique theoretically can be applied to distinguish the genes encoding SHV-7, SHV-9, SHV-10, SHV-15, SHV-17 and SHV-24 from those encoding other SHV variants . We report a simple PCR-RFLP technique that can be used in epidemiological studies to enable the rapid characterisation of known SHV beta-lactamases in a combination with the previously published PCR-SSCP analysis. Bioorg Med Chem, 1999 Dec, 7(12), 2899 - 904 Synthesis and beta-lactamase inhibitory activity of new 6beta-cysteinesulfonamidopenicillanic acids; Changov LS et al.; New 6beta-cysteinesulfonamidopenicillanic acids and their sulfoxides were synthesized by sulfonylation of 6beta-aminopenicillanic acid or its (S)-sulfoxide with (R)-N-benzyloxycarbonylcysteinesulfonyl chloride ethyl ester (2a, 1b) and (R)-N-benzyloxycarbonylcysteinesulfonyl chloride benzyl ester (2a, 2b) . The corresponding 6beta-cysteinesulfonamidopenicillanic acids sulfones 1c and 2c were prepared by oxidation of the sulfoxides 1b and 2b with potassium permanganate in aqueous medium . When combined with ampicillin some of the compounds reduced the minimal inhibitory concentrations of ampicillin against beta-lactamase producing strains. Biotechnol Bioeng, 2000 Mar 5, 67(5), 505 - 12 Enhanced protein renaturation by temperature-responsive polymers; Lin SC et al.; The application of temperature-sensitive polymer (PNIPAAm) for the renaturation of beta-lactamase from inclusion bodies was investigated . It was observed that PNIPAAm was more effective than PEG in enhancing protein renaturation . At a concentration of 0.1%, PNIPAAm improved the yield of beta-lactamase activity by 41% from 46 . 5 to 65.4 IU/mL, compared to 26% with PEG from 46.5 to 58.7 IU/mL . Kinetic study indicated that PNIPAAm did not significantly affect the initial rate of protein renaturation but did increase final activity yield . In the presence of PEG and PNIPAAm, the activity yields increased with temperature, indicating that hydrophobic interactions between denatured protein and polymer molecules contributed to the enhanced protein renaturation with polymers . The sequential addition approach, aiming at enhancing protein renaturation by reducing local protein concentration during renaturation, was also shown effective in enhancing protein renaturation, especially in the presence of polymers . With the sequential addition approach, the activity yield was increased by 60 . 5% from 46.5 to 74.6 IU/mL with PNIPAAm . Similar behavior was also observed with PEG . PNIPAAm exhibited similar behavior as PEG on the renaturation of beta-lactamase in terms of temperature effect and concentration effect, indicating that the mechanism for enhanced protein renaturation for the two polymers might be similar . PNIPAAm exhibits a lower critical solution temperature (LCST) of 32 degrees C and can be effectively separated from aqueous solution and recycled . A protein renaturation process employing PNIPAAm, which offers the advantages of enhanced renaturation efficiency, minimum loss of protein aggregates, and ease of polymers recycling, was proposed . Antimicrob Agents Chemother, 2000 Feb, 44(2), 444 - 6 Molecular characterization of the beta-lactamases from clinical isolates of Moraxella (Branhamella) catarrhalis obtained from 24 U.S . medical centers during 1994-1995 and 1997-1998; Richter SS et al.; The beta-lactamases from 403 Moraxella (Branhamella) catarrhalis clinical isolates obtained during 1994-1995 and 1997-1998 U.S . multicenter surveillance studies were characterized by isoelectric focusing . The overall prevalences of the BRO-1 and BRO-2 enzymes among beta-lactamase-positive isolates were estimated to be 97.5 and 2.5%, respectively . The minimum inhibitory concentrations (MICs) of ampicillin for all BRO-2-producing isolates were </=1 microg/ml; however, numerous beta-lactamase-positive isolates for which the ampicillin MICs were </=1 microg/ml produced the BRO-1 enzyme (88 . 1%). J Antimicrob Chemother, 2000 Jan, 45(1), 101 - 4 Detrimental effect of the combination of R164S with G238S in TEM-1 beta-lactamase on the extended-spectrum activity conferred by each single mutation; Giakkoupi P et al.; The non-naturally occurring TEM-1 beta-lactamase mutant R164S:G238S, as well as the R164S (TEM-12) and G238S (TEM-19) beta-lactamases, were constructed and expressed in Escherichia coli under isogenic conditions . Comparison of susceptibilities to beta-lactam antibiotics and substrate profiles showed that the combination of R164S with G238S drastically reduced the extended-spectrum activity of the respective single mutants. Antimicrob Agents Chemother, 1999 Dec, 43(12), 3014 - 7 Distribution of beta-lactamases in actinomycetes; Ogawara H et al.; The distribution of beta-lactamase activities in a collection of actinomycete strains was surveyed . Six of 127 strains were found to produce beta-lactamase . This low frequency was in contrast to the case with Streptomyces species . The producing strains were not related phylogenetically . MICs of benzylpenicillin did not correlate with beta-lactamase production. Enferm Infecc Microbiol Clin, 1999 Oct, 17(8), 401 - 4 {Incidence of extended spectrum beta lactamase in Escherichia coli in a university hospital from 1994-1996}; Sabate M et al.; BACKGROUND: The aim of this study was to evaluate the incidence of TEM- and SHV-type extended spectrum betalactamases (ESBLs) in Escherichia coli in a 700-bed teaching hospital between 1994 and 1996 . MATERIAL AND METHODS: Strains that presented reduced diameters to third-generation cephalosporins, as identified by disc diffusion techniques, were studied . The betalactamases involved were characterized by determination of the isoelectric point, hydrolysis profile, gene detection by polymerase chain reaction (PCR), and sequencing of the amplified products . RESULTS: 96 strains (1.4%) out of 7,054 strains of E . coli isolated between 1994 and 1996 showed decreased susceptibility to third-generation cephalosporins and only 4 strains (0.06%) produced ESBLs . Two strains produced SHV-2 and two produced TEM-12 . CONCLUSIONS: The contribution of ESBL production to resistance to third-generation cephalosporins was low: 0.06% of all the E . coli strains isolated between 1994 and 1996. J Bacteriol, 1999 Nov, 181(22), 6922 - 8 Deletion of the pyc gene blocks clavulanic acid biosynthesis except in glycerol-containing medium: evidence for two different genes in formation of the C3 unit; Perez-Redondo R et al.; The beta-lactamase inhibitor clavulanic acid is formed by condensation of a pyruvate-derived C3 unit with a molecule of arginine . A gene (pyc, for pyruvate converting) located upstream of the bls gene in the clavulanic acid gene cluster of Streptomyces clavuligerus encodes a 582-amino-acid protein with domains recognizing pyruvate and thiamine pyrophosphate that shows 29.9% identity to acetohydroxyacid synthases . Amplification of the pyc gene resulted in an earlier onset and higher production of clavulanic acid . Replacement of the pyc gene with the aph gene did not cause isoleucine-valine auxotrophy in the mutant . The pyc replacement mutant did not produce clavulanic acid in starch-asparagine (SA) or in Trypticase soy broth (TSB) complex medium, suggesting that the pyc gene product is involved in the conversion of pyruvate into the C3 unit of clavulanic acid . However, the beta-lactamase inhibitor was still formed at the same level as in the wild-type strain in defined medium containing D-glycerol, glutamic acid, and proline (GSPG medium) as confirmed by high-pressure liquid chromatography and paper chromatography . The production of clavulanic acid by the replacement mutant was dependent on addition of glycerol to the medium, and glycerol-free GSPG medium did not support clavulanic acid biosynthesis, suggesting that an alternative gene product catalyzes the incorporation of glycerol into clavulanic acid in the absence of the Pyc protein . The pyc replacement mutant overproduces cephamycin. Proc Natl Acad Sci U S A, 1999 Nov 9, 96(23), 13029 - 33 High pressure fosters protein refolding from aggregates at high concentrations; St John RJ et al.; High hydrostatic pressures (1-2 kbar), combined with low, nondenaturing concentrations of guanidine hydrochloride (GdmHCl) foster disaggregation and refolding of denatured and aggregated human growth hormone and lysozyme, and beta-lactamase inclusion bodies . One hundred percent recovery of properly folded protein can be obtained by applying pressures of 2 kbar to suspensions containing aggregates of recombinant human growth hormone (up to 8.7 mg/ml) and 0.75 M GdmHCl . Covalently crosslinked, insoluble aggregates of lysozyme could be refolded to native, functional protein at a 70% yield, independent of protein concentration up to 2 mg/ml . Inclusion bodies containing beta-lactamase could be refolded at high yields of active protein, even without added GdmHCl. J Cell Sci, 1999 Nov, 112 ( Pt 22), 3889 - 98 The sorting determinant guiding Hsp150 to the COPI-independent transport pathway in yeast; Suntio T et al.; The COPI coatomer is thought to be required in yeast directly for retrograde transport from the Golgi to the endoplasmic reticulum (ER), and directly or indirectly for ER-to-Golgi transport . Unexpectedly, the secretory glycoproteins Hsp150 and invertase have been found not to require COPI for ER exit . The features according to which cargo proteins are selected for the COPI-independent pathway are not known . The ER form of Hsp150 has three distinct domains: an N-terminal fragment of 54 amino acids (subunit I) is followed by 11 repeats of a 19 amino acid peptide plus a unique C-terminal fragment of 114 amino acids (subunit II) . By fusing heterologous proteins to different Hsp150 domains and expressing them in sec21-1 and sec21-3 mutants with temperature-sensitive mutations in the gamma-COPI subunit, we show here that the repeats of subunit II function as sorting determinants for COPI-independent ER exit . The C-terminal fragment of Hsp150 could be replaced by E . coli beta-lactamase or rat nerve growth factor receptor ectodomain (NGFRe), and subunit I could be deleted, without inhibiting COPI-independent transport . However, when the repetitive region was omitted and beta-lactamase was fused directly to the C terminus of subunit I, COPI was required for efficient ER exit . Mass spectroscopic analysis demonstrated that both subunit I and II of Hsp150 were extensively O-glycosylated, suggesting that the O-glycosylation pattern was not decisive for cargo selection. FEBS Lett, 1999 Oct 8, 459(2), 166 - 72 Circular beta-lactamase: stability enhancement by cyclizing the backbone; Iwai H et al.; We have cyclized the polypeptide backbone of beta-lactamase with a short peptide loop as a novel method for protein stabilization, using intein-mediated protein ligation . Successful cyclization was proven by mass spectrometry and subsequent re-linearization by proteolytic cleavage, as well as by resistance against carboxypeptidase . Under the conditions of the experiment, no disulfide bond is present . The circular form of beta-lactamase was found to be significantly more stable against irreversible aggregation upon heating than the linear form . The circular form could be purified from the linear one either by this heat treatment or by a his-tag which became exopeptidase-resistant by cyclization . The increased stability of the circular form is probably due to the decreased conformational entropy in the unfolded state and in the intermediate states . While the introduction of additional disulfide bonds for protein stabilization follows the same rationale, the cyclization strategy may disturb the structure less and thus constitute a general method for stabilizing those proteins with N- and C-termini in close proximity. Pharmacol Ther, 1999 Aug, 83(2), 141 - 51 Class A beta-lactamases--enzyme-inhibitor interactions and resistance; Yang Y et al.; Beta-Lactamases of Ambler's Class A are the most commonly encountered mechanism of bacterial resistance to beta-lactam antibiotics . In the face of selective pressure arising from use of either newer cephalosporins or beta-lactam/beta-lactamase inhibitor combinations, mutations arose among Class A beta-lactamase genes, leading to resistance . Clavulanic acid, a naturally occurring clavam, and the penicillanic acid sulfones sulbactam and tazobactam are the inhibitors in clinical use . This review focuses on the mechanism of inhibition by these currently marketed beta-lactamase inhibitors and on the mechanism by which specific amino acid substitutions might lead to resistance . The key amino acid positions important for inhibitor-resistance include Met69, Ser130, Arg244, Arg275, and Asn276 . Ser130 is vital to the chemical mechanism of inhibition . Arg244 appears to be coordinated to Arg275 and Asp276 by hydrogen bonds . Arg244 is involved in positioning beta-lactams, especially penicillins and beta-lactamase inhibitors, via their carboxyl groups . Site-directed mutagenesis studies confirm the role of Arg244 and its coordinating partners in beta-lactam turnover and in the reactions leading to enzyme inactivation . This mechanism is dependent on the donation of a proton via a water coordinated to Arg244 and Val216 to clavulanic acid to allow formation of a favorable leaving group . This proton donation is probably not required for formation of a favorable leaving group for the sulfone inhibitors sulbactam and tazobactam . Therefore, some amino acid substitutions have differing effects on inhibition by clavulanic acid compared with the penicillanic acid sulfones . Met69 may play a more structural role in beta-lactam positioning within the oxyanion hole. Protein Eng, 1999 Sep, 12(9), 761 - 9 Susceptibility of beta-lactamase to core amino acid substitutions; Petrosino JF et al.; To determine which amino acids in TEM-1 beta-lactamase are important for its structure and function, random libraries were previously constructed which systematically randomized the 263 codons of the mature enzyme . A comprehensive screening of these libraries identified several TEM-1 beta-lactamase core positions, including F66 and L76, which are strictly required for wild-type levels of hydrolytic activity . An examination of positions 66 and 76 in the class A beta-lactamase gene family shows that a phenylalanine at position 66 is strongly conserved while position 76 varies considerably among other beta-lactamases . It is possible that position 76 varies in the gene family because beta-lactamase mutants with non-conservative substitutions at position 76 retain partial function . In contrast, position 66 may remain unchanged in the gene family because non-conservative substitutions at this location are detrimental for enzyme structure and function . By determining the beta-lactam resistance levels of the 38 possible mutants at positions 66 and 76 in the TEM-1 enzyme, it was confirmed that position 76 is indeed more tolerant of non-conservative substitutions . An analysis of the Protein Data Bank files for three class A beta-lactamases indicates that volume constraints at position 66 are at least partly responsible for the low tolerance of substitutions at this position. J Mol Biol, 1999 Sep 10, 292(1), 19 - 37 Activities of constitutive promoters in Escherichia coli; Liang S et al.; The in vivo activities of seven constitutive promoters in Escherichia coli have been determined as functions of growth rate in wild-type relA+ spoT+ strains with normal levels of guanosine tetraphosphate (ppGpp) and in ppGpp-deficient DeltarelADeltaspoT derivatives . The promoters include (i) the spc ribosomal protein operon promotor Pspc; (ii) the beta-lactamase gene promotor Pblaof plasmid pBR322; (iii) the PLpromoter of phage lambda; (iv) and (v) the replication control promoters PRNAIand PRNAIIof plasmid pBR322; and (vi) and (vii) the P1 and P2 promoters of the rrnB ribosomal RNA operon . Each strain carried an operon fusion consisting of one of the respective promoter regions linked to lacZ and recombined into the chromosome at the mal locus of a lac deletion strain . The amount of 5'-terminal lacZ mRNA and of beta-galactosidase activity expressed from these promoters were determined by standard hybridization or enzyme activity assays, respectively . In addition, DNA, RNA and protein measurements were used to obtain information about gene dosage, rRNA synthesis and translation rates . By combining lacZ mRNA hybridization data with gene dosage and rRNA synthesis data, the absolute activity of the different promoters, in transcripts/minute per promoter, was determined . In ppGpp-proficient (relA+ spoT+) strains, the respective activities of rrnB P1 and P2 increased 40 and fivefold with increasing growth rate between 0.7 and 3.0 doublings/hour . The activities of Pspc, PL, Pbla, and PRNAIincreased two- to threefold and reached a maximum at growth rates above 2.0 doublings/hour . In contrast, PRNAIIactivity decreased threefold over this range of growth rates . In ppGpp-deficient (DeltarelA DeltaspoT) bacterial strains, the activities of rrnB P1 and P2 promoters both increased about twofold between 1.6 and 3.0 doublings/hour, whereas the activities of Pspc, PL, Pbla, and PRNAI, and PRNAIIwere about constant . To explain these observations, we suggest that the cellular concentration of free RNA polymerase increases with increasing growth rate; for saturation the P1 and P2 rRNA promoters require a high RNA polymerase concentration that is approached only at the highest growth rates, whereas the other promoters are saturated at lower polymerase concentrations achieved at intermediate growth rates . In addition, the data indicate that the respective rrnB P1 and PRNAIIpromoters were under negative and positive control by ppGpp . This caused a reduced activity of rrnB P1 and an increased activity of PRNAIIduring slow growth in wild-type (relA+ spoT+) relative to ppGpp-deficient (DeltarelA DeltaspoT) bacterial strains . J Immunoassay, 1999 Aug, 20(3), 151 - 83 A comparison of performances of four enzymes used in ELISA with special reference to beta-lactamase; Khatkhatay MI et al.; Horse radish peroxidase, alkaline phaosphatase and beta-D-galactosidase are widely used as labels in the development of enzyme immunoassays (EIAs) . Enzyme beta-lactamase, though introduced as a label in late seventies has not yet become very popular inspite of having the necessary features of an enzyme to be used in EIAs . The present article reviews assays developed with this enzyme, highlights its salient features and brings out an argument in favour of its wide spread use in EIAs. Biochim Biophys Acta, 1999 Aug 17, 1433(1-2), 153 - 8 Role of ser-237 in the substrate specificity of the carbapenem-hydrolyzing class A beta-lactamase Sme-1; Sougakoff W et al.; The role of the serine residue found at position 237 in the carbapenemase Sme-1 has been investigated by constructing a mutant in which Ser-237 was replaced by an alanine . The S237A mutant showed a catalytic behavior against penicillins and aztreonam very similar to that of Sme-1 . By contrast, S237A was characterized by a reduced catalytic efficiency against cephems, such as cephalothin and cephaloridine . In addition, the weak activity of Sme-1 against the cephamycin cefoxitin was hardly detectable with the mutant enzyme . Finally, the Ser-237-->Ala mutation resulted in a marked decrease in catalytic activity against imipenem, showing that Ser-237 contributes to the carbapenemase activity of the class A beta-lactamase Sme-1. J Mol Biol, 1999 Aug 13, 291(2), 481 - 90 Unfolding and refolding of human glyoxalase II and its single-tryptophan mutants; Dragani B et al.; Here the structure of human glyoxalase II has been investigated by studying unfolding at equilibrium and refolding . Human glyoxalase II contains two tryptophan residues situated at the N-terminal (Trp57) and C-terminal (Trp199) regions of the molecule . Trp57 is a non-conserved residue located within a "zinc binding motif" (T/SHXHX57DH) which is strictly conserved in all known glyoxalase II sequences as well as in metal-dependent beta-lactamase and arylsulfatase . Site-directed mutagenesis has been used to construct single-tryptophan mutants in order to characterize better the guanidine-induced unfolding intermediates . The denaturation at equilibrium of wild-type glyoxalase II, as followed by activity, intrinsic fluorescence and CD, is multiphasic, suggesting that different regions of varying structural stability characterize the native structure of glyoxalase II . At intermediate denaturant concentration (1.2 M guanidine) a molten globule state is attained . The reactivation of the denatured wild-type enzyme occurs only in the presence of Zn(II) ions . The results show that Zn(II) is essential for the maintenance of the native structure of glyoxalase II and that its binding to the apoenzyme occurs during an essential step of refolding . The comparison of unfolding fluorescence transitions of single-trypthophan mutants with that of wild-type enzyme indicates that the strictly conserved "zinc binding motif" is located in a flexible region of the active site in which Zn(II) participates in catalysis . J Biol Chem, 1999 Aug 13, 274(33), 23052 - 60 Effects on substrate profile by mutational substitutions at positions 164 and 179 of the class A TEM(pUC19) beta-lactamase from Escherichia coli; Vakulenko SB et al.; We investigated the effects of mutations at positions 164 and 179 of the TEM(pUC19) beta-lactamase on turnover of substrates . The direct consequence of some mutations at these sites is that clinically important expanded-spectrum beta-lactams, such as third-generation cephalosporins, which are normally exceedingly poor substrates for class A beta-lactamases, bind the active site of these mutant enzymes more favorably . We employed site-saturation mutagenesis at both positions 164 and 179 to identify mutant variants of the parental enzyme that conferred resistance to expanded-spectrum beta-lactams by their enhanced ability to turn over these antibiotic substrates . Four of these mutant variants, Arg(164) --> Asn, Arg(164) --> Ser, Asp(179) --> Asn, and Asp(179) --> Gly, were purified and the details of their catalytic properties were examined in a series of biochemical and kinetic experiments . The effects on the kinetic parameters were such that either activity with the expanded-spectrum beta-lactams remained unchanged or, in some cases, the activity was enhanced . The affinity of the enzyme for these poorer substrates (as defined by the dissociation constant, K(s)) invariably increased . Computation of the microscopic rate constants (k(2) and k(3)) for turnover of these poorer substrates indicated either that the rate-limiting step in turnover was the deacylation step (governed by k(3)) or that neither the acylation nor deacylation became the sole rate-limiting step . In a few instances, the rate constants for both the acylation (k(2)) and deacylation (k(3)) of the extended-spectrum beta-lactamase were enhanced . These results were investigated further by molecular modeling experiments, using the crystal structure of the TEM(pUC19) beta-lactamase . Our results indicated that severe steric interactions between the large 7beta functionalities of the expanded-spectrum beta-lactams and the Omega-loop secondary structural element near the active site were at the root of the low affinity by the enzyme for these substrates . These conclusions were consistent with the proposal that the aforementioned mutations would enlarge the active site, and hence improve affinity. Antimicrob Agents Chemother, 1999 Aug, 43(8), 1881 - 7 Construction and characterization of mutants of the TEM-1 beta-lactamase containing amino acid substitutions associated with both extended-spectrum resistance and resistance to beta-lactamase inhibitors; Stapleton PD et al.; Extended-spectrum TEM beta-lactamases (ESBLs) do not usually confer resistance to beta-lactamase inhibitors such as clavulanate or tazobactam . To investigate the compatibility of the two phenotypes we used site-directed mutagenesis of the bla(TEM-1) gene to introduce into the TEM-1 beta-lactamase amino acid substitutions that confer the ESBL phenotype: TEM-12 (Arg164-->Ser), TEM-26 (Arg164-->Ser plus Glu104-->Lys), TEM-19 (Gly238-->Ser), and TEM-15 (Gly238-->Ser plus Glu104-->Lys) . These were combined with three sets of substitutions that confer inhibitor resistance: TEM-31 (Arg244-->Cys), TEM-33 (Met69-->Leu), and TEM-35 (Met69-->Leu and Asn276-->Asp) . Introduction of the Arg244-->Cys substitution gave rise to inhibitor-resistant hybrid enzymes that either lost ESBL activity (TEM-12, TEM-15, and TEM-19) or had reduced activity (TEM-26) against ceftazidime . In contrast, the introduction of Met69-->Leu or Met69-->Leu plus Asn276-->Asp substitutions did not significantly affect the abilities of the enzymes to confer resistance to ceftazidime, although increased susceptibility to cefotaxime was observed with Escherichia coli strains that expressed the TEM-19 and TEM-26 beta-lactamases . With the exception of the TEM-12 beta-lactamase, introduction of the Met69-->Leu substitution did not give rise to enzymes with increased resistance to clavulanate compared to that of the TEM-1 beta-lactamase . However, introduction of the double substitution Met69-->Leu plus Asn276-->Asp in the ESBLs did give rise to low-level (TEM-19, TEM-15, and TEM-26) or moderate-level (TEM-12) clavulanate resistance . None of the hybrid enzymes were as resistant to clavulanate as the corresponding inhibitor-resistant TEM beta-lactamase mutant, suggesting that active-site configuration in the ESBLs limits the degree of clavulanate resistance conferred. Antonie Van Leeuwenhoek, 1999 Jan-Feb, 75(1-2), 125 - 33 Biosynthesis and molecular genetics of clavulanic acid; Jensen SE et al.; The biosynthesis of clavulanic acid and related clavam metabolites is only now being elucidated . Understanding of this pathway has resulted from a combination of both biochemical studies of purified biosynthetic enzymes, and molecular genetic studies of the genes encoding these enzymes . Clavulanic acid biosynthesis has been most thoroughly investigated in Streptomyces clavuligerus where the biosynthetic gene cluster resides immediately adjacent to the cluster of cephamycin biosynthetic genes . A minimum of eight structural genes have been implicated in clavulanic acid biosynthesis, although more are probably involved . While details of the early and late steps of the pathway remain unclear, synthesis proceeds from arginine and pyruvate, as the most likely primary metabolic precursors, through the monocyclic beta-lactam intermediate, proclavaminic acid, to the bicyclic intermediate, clavaminic acid, which is a branch point leading either to clavulanic acid or the other clavams . Conversion of clavaminic acid to clavulanic acid requires side chain modification as well as inversion of ring stereochemistry . This stereochemical change occurs coincident with acquisition of the beta-lactamase inhibitory activity which gives clavulanic acid its therapeutic and commercial importance . In contrast, the other clavam metabolites all arise from clavaminic acid with retention of configuration and lack beta-lactamase inhibitory activity. Chem Biol, 1999 Aug, 6(8), 541 - 51 Rapid fluorescence-based reporter-gene assays to evaluate the cytotoxicity and antitumor drug potential of platinum complexes; Sandman KE et al.; BACKGROUND: The need for new platinum antitumor drugs is underscored by the usefulness of cisplatin and carboplatin in chemotherapy and the resistance of many tumors to these compounds . Combinatorial chemistry could aid in the search for cisplatin analogs if fast, high-throughput assays were available . Our goal was to develop rapid cell-based assays suitable for high-throughput screening that accurately predict the cytotoxicity of platinum complexes . We examined the effects of platinum complexes and other agents on reporter-gene expression in cancer cells . RESULTS: HeLa Tet-On cells with inducible enhanced green fluorescent protein (EGFP) were prepared . Cisplatin and other cis-disubstituted platinum complexes inhibited EGFP expression, with a strong positive correlation between EGFP inhibition and cytotoxicity . By contrast, trans-{Pt(NH(3))(2)Cl(2)}, other trans-platinum complexes, methyl methanesulfonate or heat shock stimulated EGFP expression . Northern and nuclear run-on analyses revealed that the changes in EGFP expression were at the level of transcription . In another reporter-gene assay in Jurkat cells, cisplatin, but not trans-{Pt(NH(3))(2)Cl(2)} or K(2){PtCl(4)}, inhibited beta-lactamase expression, as measured by hydrolysis of the fluorescent substrate CCF2 . CONCLUSIONS: The EGFP results indicate that cytotoxic stress enhances transcription from the inducible promoter, whereas compounds able to form the 1,2-intrastrand platinum-DNA cross-links repress transcription . Both fluorescence-based reporter-gene assays afford promising new approaches to platinum anticancer drug discovery. Can J Microbiol, 1999 Apr, 45(4), 299 - 303 Construction of a genomic map of Moraxella (Branhamella) catarrhalis ATCC 25238 and physical mapping of virulence-associated genes; Nguyen KT et al.; A physical genome map of the Moraxella catarrhalis type strain (ATCC 25238) has been constructed using pulsed field gel electrophoresis . Macrorestriction analyses of the genome of M . catarrhalis were performed by digestion with the restriction enzymes SmaI, NotI, and RsrII, which cleave the single circular chromosome into 9, 10, and 6 fragments, respectively . The chromosomal fragments generated by pulsed field gel electrophoresis were converted to a linkage map utilizing a combination of partial digestions, and cross-hybridizations . Moraxella catarrhalis, like a number of other respiratory pathogens, has a relatively small genome estimated at 1750 kilobase pairs or about 40% of the size of the Escherichia coli genome . The locations of the four ribosomal RNA operons (rrnLS) were determined by Southern hybridization and by digestion with I-CeuI endonuclease . A number of genes involved in virulence have been placed onto the physical map by Southern hybridization including those encoding the predominant outer-membrane proteins and the chromosomal gene encoding beta-lactamase. FEBS Lett, 1999 Jun 25, 453(3), 305 - 7 Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution; Doi N et al.; Protein-engineering techniques have been adapted for the molecular design of biosensors that combine a molecular-recognition site with a signal-transduction function . The optical signal-transduction mechanism of green fluorescent protein (GFP) is most attractive, but hard to combine with a ligand-binding site . Here we describe a general method of creating entirely new molecular-recognition sites on GFPs . At the first step, a protein domain containing a desired molecular-binding site is inserted into a surface loop of GFP . Next, the insertional fusion protein is randomly mutated, and new allosteric proteins that undergo changes in fluorescence upon binding of target molecules are selected from the random library . We have tested this methodology by using TEM1 beta-lactamase and its inhibitory protein as our model protein-ligand system . 'Allosteric GFP biosensors' constructed by this method may be used in a wide range of applications including biochemistry and cell biology. Int J Antimicrob Agents, 1999 Jun, 12(1), 27 - 31 Beta-lactamases and outer membrane investigations in beta-lactam-resistant Comamonas acidovorans strains; Ravaoarinoro M et al.; Imipenem-induced beta-lactamase (level of expression, specific activity and kinetic parameters (Vmax and Km) in response to nitrocefin) and outer membrane proteins (OMPs) (hydrophobicity, permeability and electrophoretic pattern) were characterized in, one beta-lactam sensitive (PAC-9), one resistant (PAC-1) and two resistant laboratory mutants (PAC-9M, PAC-9M2) of Comamonas acidovorans strains . Beta-lactamases from both mutant strains showed different Vmax values compared to the parental strains . Beta-lactam resistance was found to be associated in PAC-1 with inducible beta-lactamase production and OMP alteration by the appearance of a 102-KDa protein . Moreover, PAC-1 was less permeable to nitrocefin than PAC-9 . These data indicate that C . acidovorans resistance to beta-lactam resulted from synergy between beta-lactamase and OMP alterations. Clin Lab Sci, 1999 Mar-Apr, 12(2), 115 - 8 Drug-induced hemolytic anemias: increasing complications to therapeutic interventions; Wright MS; There are recent reports of severe drug-induced immune hemolysis caused by several different classes of drugs . Second and third generation cephalosporins, diclofenac, fludarabine, carboplatin, and beta-lactamase inhibitors are among the drugs associated with severe or fatal hemolysis . Studies on patients who exhibit hemolysis after ingesting these drugs indicate that the four classical mechanisms of drug-induced hemolytic anemia may overlap . These studies appear to support the unified theory for induction of drug-induced immune hemolytic anemia. Biochim Biophys Acta, 1999 Jun 15, 1432(1), 125 - 36 OHIO-1 beta-lactamase mutants: the Arg244Ser mutant and resistance to beta-lactams and beta-lactamase inhibitors; Lin S et al.; Mutations at residue 244 (Ambler numbering system) in the class A TEM beta-lactamase confer resistance to inactivation by beta-lactamase inhibitors and result in diminished turnover of beta-lactam substrates . The Arg244Ser mutant of the OHIO-1 beta-lactamase, an SHV family enzyme, demonstrates variable susceptibilities to beta-lactamase inhibitors and has significantly reduced catalytic efficiency . The minimum inhibitory concentrations (MICs) for Escherichia coli DH5alpha expressing the Arg244Ser beta-lactamase were reduced when compared to the strain bearing the OHIO-1 beta-lactamase: ampicillin, 512 vs . 8192 micrograms ml-1; cephaloridine, 4 vs . 32 micrograms ml-1, respectively . The MICs for the beta-lactam beta-lactamase inhibitor combinations demonstrated resistance only to ampicillin-clavulanate, 16/8 vs . 8/4 micrograms ml-1 respectively . In contrast, there was increased susceptibility to ampicillin-sulbactam, ampicillin-tazobactam, and piperacillin-tazobactam . When compared to the OHIO-1 beta-lactamase homogenous preparations of the Arg244Ser beta-lactamase enzyme demonstrated increased Km and decreased kcat values for benzylpenicillin (Km=17 vs . 50 microM, kcat=345 vs . 234 s-1) and cephaloridine (Km=97 vs . 202 microM, kcat=1023 vs . 202 s-1) . Although the Ki and IC50 values were increased for each inhibitor when compared to OHIO-1 beta-lactamase, the turnover numbers (tn) required for inactivation were increased only for clavulanate . For the Arg244Ser mutant enzyme of OHIO-1, the increased Ki, decreased tn for the sulfones, and different partition ratio (kcat/kinact) support the notion that not all class A enzymes are inactivated in the same manner, and that certain class A beta-lactamase enzymes may react differently with identical substitutions in structurally conserved amino acids . The resistance phenotype of a specific mutations can vary depending on the enzyme. J Antimicrob Chemother, 1999 Apr, 43(4), 447 - 58 Inhibitor-resistant TEM beta-lactamases: phenotypic, genetic and biochemical characteristics; Chaibi EB et al.; Beta-lactamases represent the main mechanism of bacterial resistance to beta-lactam antibiotics . The recent emergence of bacterial strains producing inhibitor-resistant TEM (IRT) enzymes could be related to the frequent use of beta-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam in hospitals and in general practice . The IRT beta-lactamases differ from the parental enzymes TEM-1 or TEM-2 by one, two or three amino acid substitutions at different locations . This paper reviews the phenotypic, genetic and biochemical characteristics of IRT beta-lactamases in an attempt to shed light on the pressures that have contributed to their emergence. Protein Eng, 1999 Apr, 12(4), 313 - 8 Site-directed mutagenesis of residues 164, 170, 171, 179, 220, 237 and 242 in PER-1 beta-lactamase hydrolysing expanded-spectrum cephalosporins; Bouthors AT et al.; The class A beta-lactamase PER-1, which displays 26% identity with the TEM-type extended-spectrum beta-lactamases (ESBLs), is characterized by a substrate profile similar to that conferred by these latter enzymes . The role of residues Ala164, His170, Ala171, Asn179, Arg220, Thr237 and Lys242, found in PER-1, was assessed by site-directed mutagenesis . Replacement of Ala164 by Arg yielded an enzyme with no detectable beta-lactamase activity . Two other mutants, N179D and A164R+N179D, were also inactive . Conversely, a mutant with the A171E substitution displayed a substrate profile very similar to that of the wild-type enzyme . Moreover, the replacement of Ala171 by Glu in the A164R enzyme yielded a double mutant which was active, suggesting that Glu171 could compensate for the deleterious effect of Arg164 in the A164R+A171E enzyme . A specific increase in kcat for cefotaxime was observed with H170N, whereas R220L and T237A displayed a specific decrease in activity towards the same drug and a general increase in affinity towards cephalosporins . Finally, the K242E mutant displayed a kinetic behaviour very similar to that of PER-1 . Based on three-dimensional models generated by homology modelling and molecular dynamics, these results suggest novel structure-activity relationships in PER-1, when compared with those previously described for the TEM-type ESBLs. Biochemistry, 1999 May 4, 38(18), 5720 - 7 Structure of the SHV-1 beta-lactamase; Kuzin AP et al.; The X-ray crystallographic structure of the SHV-1 beta-lactamase has been established . The enzyme crystallizes from poly(ethylene glycol) at pH 7 in space group P212121 with cell dimensions a = 49.6 A, b = 55.6 A, and c = 87.0 A . The structure was solved by the molecular replacement method, and the model has been refined to an R-factor of 0.18 for all data in the range 8.0-1.98 A resolution . Deviations of model bonds and angles from ideal values are 0.018 A and 1.8 degrees, respectively . Overlay of all 263 alpha-carbon atoms in the SHV-1 and TEM-1 beta-lactamases results in an rms deviation of 1.4 A . Largest deviations occur in the H10 helix (residues 218-224) and in the loops between strands in the beta-sheet . All atoms in residues 70, 73, 130, 132, 166, and 234 in the catalytic site of SHV-1 deviate only 0.23 A (rms) from atoms in TEM-1 . However, the width of the substrate binding cavity in SHV-1, as measured from the 104-105 and 130-132 loops on one side to the 235-238 beta-strand on the other side, is 0.7-1.2 A wider than in TEM-1 . A structural analysis of the highly different affinity of SHV-1 and TEM-1 for the beta-lactamase inhibitory protein BLIP focuses on interactions involving Asp/Glu104. FEMS Microbiol Lett, 1999 Apr 15, 173(2), 459 - 65 Mutations in the ampC promoter of Escherichia coli isolates resistant to oxyiminocephalosporins without extended spectrum beta-lactamase production; Caroff N et al.; Escherichia coli strains showing an increased resistance to oxyiminocephalosporins without an extended spectrum beta-lactamase production have been screened for mutations in their ampC beta-lactamase gene promoter . Mutations were found by direct sequencing of seven promoters at positions -42, -32 (box -35), -18, -1, +5, +24 (attenuator), +31 (attenuator) and +58 . By using rapid and simple methods, three of these mutations (-42, -32 and +24), which could enhance transcription, were searched in 37 resistant and 25 sensitive isolates . The -42 mutation was present in 33 of the 37 promoters from the resistant isolates . The -32 and +24 mutations were present only in three and two promoters, respectively, and they were combined in the most resistant strain of the study . The +24 mutation was detected in another strain associated with a 1-bp insertion between the -35 and -10 conserved sequences . None of these mutations was detected in the ampC beta-lactamase from the sensitive isolates. Antimicrob Agents Chemother, 1999 May, 43(5), 1206 - 10 Carbapenem resistance in Escherichia coli associated with plasmid-determined CMY-4 beta-lactamase production and loss of an outer membrane protein; Stapleton PD et al.; Three cefoxitin-resistant Escherichia coli isolates from stool specimens of a patient with leukemia were either resistant, intermediate, or sensitive to imipenem . Conjugation experiments showed that cefoxitin resistance, but not imipenem resistance, was transferable . All isolates were shown by isoelectric focusing to produce two beta-lactamases with isoelectric points of 5.4 (TEM-1, confirmed by sequencing of a PCR product) and >8.5 (consistent with a class C beta-lactamase) . The gene coding for the unknown beta-lactamase was cloned and sequenced and revealed an enzyme which had 99.9% sequence identity with the plasmid-determined class C beta-lactamase CMY-2 . The cloned beta-lactamase gene differed from blaCMY-2 at one nucleotide position that resulted in an amino acid change, tryptophan to arginine at position 221 . We propose that this enzyme be designated CMY-4 . Both the imipenem-resistant and -intermediate isolates lacked a 38-kDa outer membrane protein (OMP) that was present in the imipenem-sensitive isolate . The lack of an OMP alone did not explain the difference in carbapenem susceptibilities observed . However, measurement of beta-lactamase activities (including measurements under conditions where TEM-1 beta-lactamase was inhibited) indicated that the imipenem-intermediate isolate expressed six- to eightfold less beta-lactamase than did the other isolates . This study illustrates that carbapenem resistance in E . coli can arise from high-level expression of plasmid-mediated class C beta-lactamase combined with an OMP deficiency . Furthermore, in the presence of an OMP deficiency, the level of expression of a plasmid-mediated class C beta-lactamase is an important factor in determining whether E . coli isolates are fully resistant to carbapenems. Biotechnol Prog, 1999 Mar-Apr, 15(2), 164 - 7 Secretory production of recombinant protein by a high cell density culture of a protease negative mutant Escherichia coli strain; Park SJ et al.; Several protease negative mutant strains including HM114, HM126, and HM130 as well as their parent strain KS272 were compared for their growth and secretory production of a model fusion protein, protein A-beta-lactamase . HM114, a strain deficient in two cell envelope proteases, grew slightly faster and produced more fusion protein than the other strains deficient in more proteases . HM114 was grown to a cell dry weight of 47.86 g/L in 29 h using pH-stat, fed-batch cultivation . The beta-lactamase activity was 11.25 x 10(4) U/L, which was 30% higher than that obtained with its parent strain KS272 . Up to 96% of protein A-beta-lactamase fusion protein could be recovered by a simple cold osmotic shock method . The specific beta-lactamase activity obtained with HM114 after fractionation was 4.5 times higher than that obtained with KS272. Antimicrob Agents Chemother, 1999 Apr, 43(4), 862 - 7 Ampicillin-sulbactam and amoxicillin-clavulanate susceptibility testing of Escherichia coli isolates with different beta-lactam resistance phenotypes; Oliver A et al.; The activities of ampicillin-sulbactam and amoxicillin-clavulanate were studied with 100 selected clinical Escherichia coli isolates with different beta-lactam susceptibility phenotypes by standard agar dilution and disk diffusion techniques and with a commercial microdilution system (PASCO) . A fixed ratio (2:1) and a fixed concentration (clavulanate, 2 and 4 micrograms/ml; sulbactam, 8 micrograms/ml) were used in the agar dilution technique . The resistance frequencies for amoxicillin-clavulanate with different techniques were as follows: fixed ratio agar dilution, 12%; fixed concentration 4-micrograms/ml agar dilution, 17%; fixed ratio microdilution, 9%; and disk diffusion, 9% . Marked discrepancies were found when these results were compared with those obtained with ampicillin-sulbactam (26 to 52% resistance), showing that susceptibility to amoxicillin-clavulanic acid cannot be predicted by testing the isolate against ampicillin-sulbactam . Interestingly, the discrimination between susceptible and intermediate isolates was better achieved with 4 micrograms of clavulanate per ml than with the fixed ratio . In contrast, amoxicillin susceptibility was not sufficiently restored when 2 micrograms of clavulanate per ml was used, particularly in moderate (mean beta-lactamase activity, 50.8 mU/mg of protein) and high-level (215 mU/mg) TEM-1 beta-lactamase producer isolates . Four micrograms of clavulanate per milliliter could be a reasonable alternative to the 2:1 fixed ratio, because most high-level beta-lactamase-hyperproducing isolates would be categorized as nonsusceptible, and low- and moderate-level beta-lactamase-producing isolates would be categorized as nonresistant . This approach cannot be applied to sulbactam, either with the fixed 2:1 ratio or with the 8-micrograms/ml fixed concentration, because many low-level beta-lactamase-producing isolates would be classified in the resistant category . These findings call for a review of breakpoints for beta-lactam-beta-lactamase inhibitors combinations. Biotechnol Bioeng, 1999 Jan 20, 62(2), 155 - 9 Site-protected fixation and immobilization of Escherichia coli cells displaying surface-anchored beta-lactamase; Freeman A et al.; Bacteria displaying heterologous receptors or enzymes on their surface hold great potential as whole-cell adsorbents and biocatalysts, respectively . For industrial applications, such surface-engineered cells need to be killed and chemically fixed to prevent disintegration and leakage of the displayed proteins under process conditions . It is also highly desirable to couple the chemically stabilized cells onto a solid support matrix for additional mechanical stability, flexibility in reactor choice, and easy separation from processed medium . Recently, we described the development of a readily scalable methodology for cell killing, fixation, and outer membrane stabilization via glutaraldehyde fixation followed by secondary crosslinking (Freeman, A., Abramov, S . and Georgiou, G . 1996 . Biotechnol . Bioeng . 52: 625-630) . Glutaraldehyde treatment was also found, however, to reduce the specific activity of a model enzyme, beta-lactamase displayed on the surface of E . coli . Here, we show that crosslinking carried out in the presence of beta-lactamase inhibitors, namely phenyl boronic acid or sodium borate, protects the active site from chemical modification resulting in up to threefold higher specific activities without affecting the cell-stabilizing effect of the glutaraldehyde treatment . To prepare an immobilized whole cell biocatalyst, residual unreacted surface aldehyde groups were employed to immobilize covalently the fixed bacteria onto chitosan-coated cellulose powder . The binding of the bacteria onto chitosan-coated cellulose was quantitative up to cell loading of 83 mg dry cell weight/g of support . Cell immobilization did not introduce mass transfer limitations and created only a modest reduction in Vmax . Thus, chemical crosslinking, affected in presence of reversible active-site inhibitors and coupled with cell immobilization on chitosan-coated cellulose represents a widely useful methodology for the process application of recombinant bacteria displaying surface-anchored heterologous proteins . Biotechnol Bioeng, 1998 Sep 20, 59(6), 659 - 65 Construction and characterization of F plasmid-based expression vectors; Jones KL et al.; A low-copy expression vector has been constructed from a 9 Kbp region of the Escherichia coli F plasmid containing the oriV and oriS origins of replication . This plasmid carries the beta-lactamase gene (Apr) and the araBAD promoter/araC regulator for arabinose-inducible gene expression . A derivative which carries a lacZ reporter gene was found to be stably maintained for at least 150 generations . A related multi-copy plasmid was stably maintained in arabinose-free medium, but no plasmid-bearing segregants remained after 60 generations when lacZ expression was induced . Induced expression resulted in 27% (multi-copy) and 12% (low-copy) decreases in growth rate . The uninduced levels of beta-galactosidase were 200 units (multi-copy) and 15 units (low-copy) . Anal Biochem, 1999 Apr 10, 269(1), 32 - 7 RNA-binding properties of in vitro expressed histidine-tagged RB69 RegA translational repressor protein; Allen SV et al.; To facilitate RNA-binding studies of the phage RB69 RegA translational repressor protein, regA was configured to add six histidines to the carboxyl end of the protein . In vitro transcription-translation from the T7 promoter on plasmid pSA1 yielded a RegA69-His6 protein that binds nickel-Sepharose and elutes with 0.5 M imidazole . The system was further modified to avoid cloning and the toxic effects of RegA on Escherichia coli by the polymerase chain reaction (PCR), producing linear templates with the configuration T7 promoter-TIR-regA-His6 . A translation initiation region was used that conforms to consensus E . coli and eukaryotic initiation sites and eliminates the target for RegA autogenous repression . RegA69-His6 synthesized in E . coli S30 or wheat germ extracts displayed RNA-binding properties similar to wild-type RB69 RegA . Specificity of RNA binding was demonstrated by in vitro repression of T4 gp44 and gp45 but not beta-lactamase, by differential binding to poly(U)- and poly(C)-agarose, and by site-specific binding to a 23-base gene 44 target RNA but not to mutant 44 RNA . Therefore, addition of the His6 tag to the C-terminus of RB69 RegA does not dramatically alter RNA binding, indicating that this region is not directly involved in site recognition . With access to several T4-like phage genomes and regA mutant sequences, in vitro synthesis of His-tagged proteins directly from linear PCR products provides a convenient and efficient system to study RegA and other interesting RNA-binding proteins . Rev Mal Respir, 1999 Feb, 16(1), 65 - 70 {Evolution of the management of lung diseases in general medicine in Bordeaux (1992-1995)}; Vernejoux JM et al.; Lower respiratory tract infections (LRTI) are very often managed by General Practitioners (GPs) . In France, the 1991 Lille Consensus Conference set out guidelines for the management of respiratory tract infections; in 1994, the Ministry of Health published Official Medical Recommendations (OMR) to be applied to seasonal respiratory infections . The aim of the study is to evaluate the impact of these OMR in 1995 on GPs' attitude when confronted with a community-acquired pneumonia in a previously healthy 40-year-old adult, with no sign of complications . Sixty seven GPs took, part in the same study by questionnaire in 1992 and 1995; we observed an increase in the prescription of aminopenicillin without a beta-lactamase inhibitor (41% in 1992 vs 66% in 1995; p = 0.009), and a reduction in both the use of aminopenicillin with a beta-lactamase inhibitor (35% in 1992 vs 11% in 1995; p = 0.002) and the concomitant prescription of cortico-steroids (43% in 1992 vs 14% vs 14% in 1995; p = 0.0009) . Between 1992 and 1995, general practitioners in the Bordeaux region have changed their therapeutic choices in community-acquired pneumonia . In 1995, antibiotic prescriptions followed consensus guidelines more closely. Int J Clin Pharmacol Ther, 1999 Feb, 37(2), 63 - 75 Pharmacokinetic properties of beta-lactamase inhibitors; de la Pena A et al.; OBJECTIVES: In recent years, the use of combinations of beta-lactam-antibiotics with beta-lactamase inhibitors has been proven to be a useful and effective strategy to improve upon the therapeutic value of beta-lactam antibiotics . The objective of this article is to evaluate the pharmacokinetic properties of three commercially available beta-lactamase inhibitors . MATERIALS AND METHODS: Based on published articles in the literature, the pharmacokinetic properties of the beta-lactamase inhibitors clavulanic acid, sulbactam and tazobactam are reviewed and compared . RESULTS: There are some considerable differences between these three compounds: Only clavulanic acid is orally bioavailable . Tazobactam and sulbactam are eliminated renally to a larger extent than clavulanic acid . Tazobactam's total body clearance is almost twice that of sulbactam, and the two drugs differ significantly in their degree of protein-binding . Furthermore, sulbactam has a larger volume of distribution than tazobactam . CONCLUSIONS: When choosing combinations of a beta-lactam antibiotic with a beta-lactamase inhibitor, it is important to make sure that the pharmacokinetic properties of drug and inhibitor are similar and remain similar under changing pathophysiological conditions . Hence, beta-lactam inhibitors should not be freely interchanged, but for each beta-lactam antibiotic the best partner needs to be identified . If this is done properly, fixed combinations of beta-lactam-antibiotic and beta-lactamase inhibitor are appropriate and convenient . This situation may represent one of the few cases in pharmacotherapy, where fixed combinations of two drugs are beneficial. Arch Pharm Res, 1999 Feb, 22(1), 68 - 71 Synthesis of 6-exomethylenepenams as beta-lactamase inhibitors; Im C et al.; The 6,6-dibromopenam (6) was treated with CH3MgBr and carbaldehyde 5 to afford the hydroxy compound 7, which was reacted with acetic anhydride to give acetoxy compound 8 . The deacetobromination of 8 with zinc and acetic acid gave 6-exomethylenepenams, E-isomer 10 and Z-isomer 9, which was oxidized to sulfone 11 by m-CPBA . The p-methoxybenzyl compounds were deprotected by AlCl3 and neutralized to give the sodium salts 12, 13 and 14. Protein Sci, 1999 Feb, 8(2), 404 - 9 pKa calculations for class A beta-lactamases: influence of substrate binding; Lamotte-Brasseur J et al.; Beta-Lactamases are responsible for bacterial resistance to beta-lactams and are thus of major clinical importance . However, the identity of the general base involved in their mechanism of action is still unclear . Two candidate residues, Glu166 and Lys73, have been proposed to fulfill this role . Previous studies support the proposal that Glu166 acts during the deacylation, but there is no consensus on the possible role of this residue in the acylation step . Recent experimental data and theoretical considerations indicate that Lys73 is protonated in the free beta-lactamases, showing that this residue is unlikely to act as a proton abstractor . On the other hand, it has been proposed that the pKa of Lys73 would be dramatically reduced upon substrate binding and would thus be able to act as a base . To check this hypothesis, we performed continuum electrostatic calculations for five wild-type and three beta-lactamase mutants to estimate the pKa of Lys73 in the presence of substrates, both in the Henri-Michaelis complex and in the tetrahedral intermediate . In all cases, the pKa of Lys73 was computed to be above 10, showing that it is unlikely to act as a proton abstractor, even when a beta-lactam substrate is bound in the enzyme active site . The pKa of Lys234 is also raised in the tetrahedral intermediate, thus confirming a probable role of this residue in the stabilization of the tetrahedral intermediate . The influence of the beta-lactam carboxylate on the pKa values of the active-site lysines is also discussed. J Mol Biol, 1999 Feb 5, 285(5), 2079 - 87 Crystal structure of the E166A mutant of extended-spectrum beta-lactamase Toho-1 at 1.8 A resolution; Ibuka A et al.; Bacterial resistance to beta-lactams is mainly due to the production of beta-lactamase . Especially through the production of extended-spectrum beta-lactamases (ESBLs), bacteria have acquired resistance not only to penicillins, but also to expanded-spectrum cephems . Here, we describe the crystal structure of the E166A mutant of class A beta-lactamase Toho-1 at 1.8 A resolution, the first reported tertiary structure of an ESBL . Instead of the wild-type enzyme, a mutant Toho-1, in which Glu166 was replaced with alanine, was used for this study, because of the strong tendency of the wild-type enzyme to form twinned crystals . The overall structure of Toho-1 is similar to the crystal structures of non-ESBLs, with no pronounced backbone rearrangement of the framework . However, there are some notable local changes . First, a difference in the disposition of an arginine residue, which is at position 244 in non-ESBLs but at position 276 in Toho-1 and other ESBLs, was revealed and the role of this arginine residue is discussed . Moreover, changes in the hydrogen-bonding pattern and in the formation of the hydrophobic core were also observed near the Omega loop . In particular, the lack of hydrogen bonds in the vicinity of the Omega loop could be a cause of the extended substrate specificity of Toho-1 . Through the generation of a model for the enzyme-substrate complex, a conformational change of Toho-1 occurring on complex formation is discussed based on the active-site cleft structure and the substrate profile . Antimicrob Agents Chemother, 1999 Feb, 43(2), 393 - 6 Diversification of Escherichia coli expressing an SHV-type extended-spectrum beta-lactamase (ESBL) during a hospital outbreak: emergence of an ESBL-hyperproducing strain resistant to expanded-spectrum cephalosporins; Palucha A et al.; Twelve SHV-type extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli lac mutant isolates were recovered in October 1997 from 11 patients of the neonatal ward in a Warsaw hospital . The outbreak was clonal; however, some of the isolates expressed a much higher level of resistance to several beta-lactam antibiotics, including expanded-spectrum cephalosporins . This phenotype has been attributed to beta-lactamase hyperproduction correlating with the multiplication of ESBL gene copies, as was demonstrated for representative isolates. Antimicrob Agents Chemother, 1999 Feb, 43(2), 367 - 70 Updated sequence information for TEM beta-lactamase genes; Goussard S et al.; The sequences of the promoter regions and of the structural genes for 13 penicillinase, extended-spectrum, and inhibitor-resistant TEM-type beta-lactamases have been determined, and an updated blaTEM gene nomenclature is proposed. J Biol Chem, 1999 Jan 22, 274(4), 2424 - 31 Constitutive calcium-independent release of Toxoplasma gondii dense granules occurs through the NSF/SNAP/SNARE/Rab machinery; Chaturvedi S et al.; The signals and the molecular machinery mediating release of dense matrix granules from pathogenic protozoan parasites are unknown . We compared the secretion of the endogenous dense granule marker GRA3 in Toxoplasma gondii with the release of a stably transfected foreign reporter, beta-lactamase, that localizes to parasite dense granules . Both proteins were released constitutively in a calcium-independent fashion, as shown using both intact and streptolysin O-permeabilized parasites . N-Ethylmaleimide and recombinant bovine Rab-guanine dissociation inhibitor inhibited beta-lactamase secretion in permeabilized parasites, whereas recombinant hamster N-ethylmaleimide-sensitive fusion protein and bovine alpha-SNAP augmented release . Guanosine 5'-3-O-(thio)triphosphate, but not cAMP, augmented secretion in the presence but not in the absence of ATP . The T . gondii NSF/SNAP/SNARE/Rab machinery participates in dense granule release using parasite protein components that can interact functionally with their mammalian homologues. Biochemistry, 1999 Jan 5, 38(1), 11 - 21 Biophysical characterization of the interaction of the beta-lactamase TEM-1 with its protein inhibitor BLIP; Albeck S et al.; BLIP is a secreted protein from Streptomyces clavuligerus that inhibits a wide range of beta-lactamases . Here we investigate the tight interaction of BLIP, expressed heterologousely in E . coli, with TEM-1 . Kinetic and thermodynamic constants were determined using methods with the proteins either in a homogeneous or in a heterogeneous phase . While values of Delta DeltaG(mut-wt) are similar whether measured by fluorescence quench, enzyme inhibition, or surface plasmon resonance, absolute values of DeltaG and kinetic constants vary . Association and dissociation rate constants of 10(5) M-1 s-1 and 10(-)4 s-1, respectively, and a nanomolar affinity were determined for the wild-type proteins . The highest affinity is measured at pH 7.5, with a decreasing association rate constant at higher pH values, and an increasing dissociation rate constant at lower pH values . The marginal effect of salt on the kinetics of binding, as well as the calculated surface potentials, suggests a limited role for electrostatic forces in guiding this reaction . Still, mutations of interfacial residues affect the rate of association significantly, so that an increase in the net negative charge on either protein reduces the association rate constant . We show that simple electrostatic rules can explain this behavior . BLIP inhibits the catalytic activity of TEM-1 by binding its active site . Yet, mutations of active site residues on TEM-1 only have a moderate though cooperative effect on the binding energy . This can be explained in light of the peripheral location of the active site in the interface between the two proteins. Nat Biotechnol, 1998 Dec, 16(13), 1329 - 33 A genome-wide functional assay of signal transduction in living mammalian cells; Whitney M et al.; We describe a genome-wide functional assay for rapid isolation of cell clones and genetic elements responsive to specific stimuli . A promoterless beta-lactamase reporter gene was transfected into a human T-cell line to generate a living library of reporter-tagged clones . When loaded with a cell-permeable fluorogenic substrate, the cell library simultaneously reports the expression of a large number of endogenous genes . Flow cytometry was used to recover individual clones whose reporter-tagged genes were either induced or repressed following T-cell activation . Responsive clones were expanded and analyzed pharmacologically to identify patterns of regulation associated with specific genes . Although demonstrated using T cells, the genomic assay could be applied to map downstream transcriptional consequences for any propagating cell line in response to any stimulus of interest. J Bacteriol, 1998 Dec, 180(24), 6625 - 34 Topological analysis of the aerobic membrane-bound formate dehydrogenase of Escherichia coli; Benoit S et al.; Besides formate dehydrogenase N (FDH-N), which is involved in the major anaerobic respiratory pathway in the presence of nitrate, Escherichia coli synthesizes a second isoenzyme, called FDH-O, whose physiological role is to ensure rapid adaptation during a shift from aerobiosis to anaerobiosis . FDH-O is a membrane-bound enzyme complex composed of three subunits, alpha (FdoG), beta (FdoH), and gamma (FdoI), which exhibit high sequence similarity to the equivalent polypeptides of FDH-N . The topology of these three subunits has been studied by using blaM (beta-lactamase) gene fusions . A collection of 47 different randomly generated Fdo-BlaM fusions, 4 site-specific fusions, and 3 sandwich fusions were isolated along the entire sequence of the three subunits . In contrast to previously reported predictions from sequence analysis, our data suggested that the alphabeta catalytic dimer is located in the cytoplasm, with a C-terminal anchor for beta protruding into the periplasm . As expected, the gamma subunit, which specifies cytochrome b, was shown to cross the cytoplasmic membrane four times, with the N and C termini exposed to the cytoplasm . Protease digestion studies of the 35S-labelled FDH-O heterotrimer in spheroplasts add further support to this model . Consistently, prior studies regarding the bioenergetic function of formate dehydrogenase provided evidence for a mechanism in which formate is oxidized in the cytoplasm. Dakar Med, 1997, 42(1), 6 - 10 {Broad-spectrum beta-lactamases: profile and perspectives after 3 years of systematic detection at the Main Hospital of Dakar (1 February 1992 to 1 February 1995)}; Ndoye B et al.; After having undertaken an estimation upon infections from bacteria generating extended broad spectrum Beta-lactamase at "Hospital Principal" of Dakar between 1992 to 1993 and after having drawn the attention of authorities of the Hospital to the hazard caused by this sort of infections, authors come back in doing a second estimation during the two following years . The results of this estimation show that measures preciously taken, not with standing an certain impact, are clearly unsufficient and have got a limited effect in the future . They suggest practical solutions in the long run, solutions that have already been taken at "Hospital Principal" of Dakar and deserve to be kept on and encouraged. Dev Biol, 1998 Nov 15, 203(2), 290 - 4 beta-lactamase as a marker for gene expression in live zebrafish embryos; Raz E et al.; In this report we describe the development of a sensitive assay for gene expression in zebrafish embryos using beta-lactamase as a reporter gene . We show that injection of a green fluorescent substrate for beta-lactamase allows the detection of reporter gene expression in live embryos . The beta-lactamase enzyme catalyzes the hydrolysis of the substrate, thereby disrupting fluorescence resonance energy transfer from the donor to the acceptor dye in the molecule . As a result, a blue fluorescent product is produced and retained specifically in cells within which the enzyme is expressed . beta-Lactamase is therefore suitable for monitoring spatially restricted patterns of gene expression in the early embryo . We suggest that this new reporter system provides a major advancement in sensitivity over the existing methods for monitoring gene expression in vivo during early embryogenesis . J Med Chem, 1998 Nov 5, 41(23), 4577 - 86 Structure-based enhancement of boronic acid-based inhibitors of AmpC beta-lactamase; Weston GS et al.; The expression of beta-lactamases is the most common form of bacterial resistance to beta-lactam antibiotics . To combat these enzymes, agents that inhibit (e.g . clavulanic acid) or evade (e.g . aztreonam) beta-lactamases have been developed . Both the beta-lactamase inhibitors and the beta-lactamase-resistant antibiotics are themselves beta-lactams, and bacteria have responded to these compounds by expressing variant enzymes resistant to inhibition (e.g . IRT-3) or that inactivate the beta-lactamase-resistant antibiotic (e.g . TEM-10) . Moreover, these compounds have increased the frequency of bacteria with intrinsically resistant beta-lactamases (e.g . AmpC) . In an effort to identify non-beta-lactam-based beta-lactamase inhibitors, we used the crystallographic structure of the m-aminophenylboronic acid-Escherichia coli AmpC beta-lactamase complex to suggest modifications that might enhance the affinity of boronic acid-based inhibitors for class C beta-lactamases . Several types of compounds were modeled into the AmpC binding site, and a total of 37 boronic acids were ultimately tested for beta-lactamase inhibition . The most potent of these compounds, benzo{b}thiophene-2-boronic acid (36), has an affinity for E . coli AmpC of 27 nM . The wide range of functionality represented by these compounds allows for the steric and chemical "mapping" of the AmpC active site in the region of the catalytic Ser64 residue, which may be useful in subsequent inhibitor discovery efforts . Also, the new boronic acid-based inhibitors were found to potentiate the activity of beta-lactam antibiotics, such as amoxicillin and ceftazidime, against bacteria expressing class C beta-lactamases . This suggests that boronic acid-based compounds may serve as leads for the development of therapeutic agents for the treatment of beta-lactam-resistant infections. J Med Chem, 1998 Oct 8, 41(21), 3972 - 5 Structure-based design of beta-lactamase inhibitors . 2 . Synthesis and evaluation of bridged sulfactams and oxamazins; Hubschwerlen C et al.; A series of bridged monocyclic beta-lactams activated by various groups on the beta-lactam nitrogen (X = OCH2CO2H, OSO3H) has been synthesized and evaluated . Among them, the bridged sulfactams (X = OSO3H) were found to be effective beta-lactamase inhibitors . They inhibit both class A and class C beta-lactamases. Antimicrob Agents Chemother, 1998 Sep, 42(9), 2319 - 25 Characterization of a PSE-4 mutant with different properties in relation to penicillanic acid sulfones: importance of residues 216 to 218 in class A beta-lactamases; Sabbagh Y et al.; Class A beta-lactamases are inactivated by the suicide inactivators sulbactam, clavulanic acid, and tazobactam . An examination of multiple alignments indicated that amino acids 216 to 218 differed among class A enzymes . By random replacement mutagenesis of codons 216 to 218 in PSE-4, a complete library consisting of 40,864 mutants was created . The library of mutants with mutations at positions 216 to 218 in PSE-4 was screened on carbenicillin and ampicillin with the inactivator sulbactam; a collection of 14 mutants was selected, and their bla genes were completely sequenced . Purified wild-type and mutant PSE-4 beta-lactamases were used to measure kinetic parameters . One enzyme, V216S:T217A:G218R, was examined for its peculiar pattern of inhibition . There was an increase in the Km from 68 microM for the wild type to 271 microM for the mutant for carbenicillin and 33 to 216 microM for ampicillin . Relative to the wild-type PSE-4 enzyme, 37- and 30-fold increases in Ki values were observed for the mutant enzyme for sulbactam and tazobactam, respectively . The results that were obtained suggested that positions 216 to 218 are important for interactions with penicillanic acid sulfone inhibitors . In contrast, V216 and A217 in the TEM-1 class A beta-lactamase do not tolerate amino acid residue substitutions . However, for the PSE-4 beta-lactamase, 11 of 14 mutants from the library of mutants with mutations at positions 216 to 218 whose sequences were determined had substitutions at position 216 (G, R, A, S) and position 217 (A, S) . The data showed the importance of residues 216 to 218 in their atomic interactions with inactivators in the PSE-4 beta-lactamase structure. Biochem J, 1998 Jul 15, 333 ( Pt 2), 395 - 400 Kinetic analysis of an inhibitor-resistant variant of the OHIO-1 beta-lactamase, an SHV-family class A enzyme; Lin S et al.; The Met69-->Ile mutant of the OHIO-1 beta-lactamase, an SHV-family enzyme, is resistant to inactivation by beta-lactamase inhibitors . Analysis of purified Met69-->Ile enzyme reveals that its isoelectric point (pI 7.0) and CD spectrum are identical with those of the OHIO-1 enzyme . Levels of beta-lactamase expression in Escherichia coli as determined by immunoblotting are similar for OHIO-1 and Met69-->Ile beta-lactamase . The kinetic constants of the Met69-->Ile enzyme compared with OHIO-1 are smaller for benzylpenicillin (Km = 6 microM compared with 17 microM; kcat = 234 s-1 compared with 345 s-1 respectively) and carbenicillin (Km = 3 microM compared with 17 microM; kcat = 131 s-1 compared with 320 s-1 respectively) . For the cephalosporins cephaloridine and 7-(thienyl- 2-acetamido)-3-{2-(4-N,N- dimethylaminophenylazo)pyridinium-methyl}-3-cephem-4-carboxylic acid (PADAC), a similar pattern is also seen (Km=38 microM compared with 96 microM and 6 microM compared with 75 microM respectively; kcat = 235 s-1 compared with 1023 s-1 and 9 s-1 compared with 50 s-1 respectively) . Consistent with minimum inhibitory concentrations that show resistance to beta-lactam beta-lactamase inhibitors, the apparent Ki values, turnover numbers and partition ratios (kcat/kinact) for the mechanism-based inactivators clavulanate, sulbactam and tazobactam are increased . The inactivation rate constants (kinact) are decreased . The difference in activation energy, a measurement of altered affinity for the wild-type and mutant enzymes leading to acylation of the active site, reveals small energy differences of less than 8.4 kJ/mol . In total, these results suggest that the Met-->Ile substitution at position 69 in the OHIO-1 beta-lactamase alters the active site, primarily affecting the interactions with beta-lactamase inhibitors. J Bacteriol, 1998 Sep, 180(18), 4821 - 7 Topological analysis of DcuA, an anaerobic C4-dicarboxylate transporter of Escherichia coli; Golby P et al.; Escherichia coli possesses three independent anaerobic C4-dicarboxylate transport systems encoded by the dcuA, dcuB, and dcuC genes . The dcuA and dcuB genes encode related integral inner-membrane proteins, DcuA and DcuB (433 and 446 amino acid residues), which have 36% amino acid sequence identity . A previous amino acid sequence-based analysis predicted that DcuA and DcuB contain either 12 or 14 transmembrane helices, with the N and C termini located in the cytoplasm or periplasm (S . Six, S . C . Andrews, G . Unden, and J . R . Guest, J . Bacteriol . 176:6470-6478, 1994) . These predictions were tested by constructing and analyzing 66 DcuA-BlaM fusions in which C terminally truncated forms of DcuA are fused to a beta-lactamase protein lacking the N-terminal signal peptide . The resulting topological model differs from those previously predicted . It has just 10 transmembrane helices and a central, 80-residue cytoplasmic loop between helices 5 and 6 . The N and C termini are located in the periplasm and the predicted orientation is consistent with the "positive-inside rule." Two highly hydrophobic segments are not membrane spanning: one is in the cytoplasmic loop; the other is in the C-terminal periplasmic region . The topological model obtained for DcuA can be applied to DcuA homologues in other bacteria as well as to DcuB . Overproduction of DcuA to 15% of inner-membrane protein was obtained with the lacUV5-promoter-based plasmid, pYZ4. Microbiology, 1998 Aug, 144 ( Pt 8), 2161 - 7 Cloning and heterologous expression of the gene for BLIP-II, a beta-lactamase-inhibitory protein from Streptomyces exfoliatus SMF19; Park HU et al.; A beta-lactamase-inhibitory protein (BLIP-II) was purified from the culture filtrate of Streptomyces exfoliatus SMF 19 and its N-terminal amino acid sequence was determined . A clone containing the gene encoding BLIP-II (bliB) was selected from a cosmid library by colony hybridization using an oligonucleotide probe based on the N-terminal amino acid sequence of BLIP-II . The bliB gene was isolated and sequenced . Analysis of the nucleotide sequence revealed that the gene consists of 1116 bp and encodes a mature protein of 332 amino acids preceded by a 40 amino acid signal sequence . bliB, expressed under the control of the T7 promoter in Escherichia coli, was accumulated in an inactive form in inclusion bodies, but beta-lactamase-inhibitory activity was recovered after refolding . In addition, bliB was heterologously expressed in Streptomyces lividans TK24 using the melC1 promoter . The BLIP-II protein produced in recombinant strains of S . lividans was secreted into the culture supernatant in a biologically active form. Biochemistry, 1998 Aug 18, 37(33), 11660 - 9 Identification of the binding surface on beta-lactamase for GroEL by limited proteolysis and MALDI-mass spectrometry; Gervasoni P et al.; Escherichia coli beta-lactamase, alone or as a complex with GroEL at 48 degreesC, was partially digested with trypsin, endoproteinase Glu-C, or thermolysin . Peptides were analyzed by matrix-assisted laser desorption and ionization mass spectrometry and aligned with the known sequence . From the protease cleavage sites which become protected upon binding and those which become newly accessible, a model of the complex is proposed in which the carboxy-terminal helix has melted, two loops form the binding interface and the large beta-sheet become partially uncovered by the slight dislocation of other structural elements . This explains how hydrophobic surface on the substrate protein can become accessible while scarcely disrupting the hydrogen bond network of the native structure . An analysis of the GroEL-bound peptides bound after digestion of the beta-lactamase showed no obvious sequence motifs, indicating that binding is provided by hydrophobic patches in the three-dimensional structure. FASEB J, 1998 Aug, 12(11), 1055 - 60 Functional mimicry: elicitation of a monoclonal anti-idiotypic antibody hydrolizing beta-lactams; Avalle B et al.; Antigen mimicry by anti-idiotypic antibodies is investigated as a reliable strategy to achieve molecular imprinting of an enzymatic activity . A monoclonal anti-idiotypic antibody (Ab2-9G4H9) was elicited by using a monoclonal antibody (Ab1-7AF9) specific for the beta-lactamase active site . Catalytic features of Ab2 were characterized with beta-lactamase substrates . The antibody combining site appeared to have retained a part of the catalytic specificity . The relevance of the idiotypic mimicry concept for the generation of catalytic antibodies was further demonstrated by eliciting a third generation antibody (Ab3), which was shown to recognize beta-lactamase: the complete internal image properties of Ab2 9G4H9, including binding and catalytic properties, were thus checked. Chemotherapy, 1998 Jul-Aug, 44(4), 260 - 4 In vitro activity of loracarbef against TEM extended-spectrum beta-lactamase-producing Escherichia coli strains and its interaction with the enzymes; Prinarakis EE et al.; The in vitro activity of loracarbef was evaluated against Escherichia coli strains producing TEM extended-spectrum beta-lactamases (ESBLs) . The MICs of loracarbef were lower than those of cefaclor and cephalothin . The antibiotic was active (MICs < or = 2 mg/l) against strains producing TEM-6, TEM-8, TEM-10, TEM-16, TEM-26 and TEM-28 beta-lactamases . The MICs for TEM-3-, TEM-5- and TEM-24-producing strains were higher (> or = 16 mg/l) . Hydrolysis studies indicated that the latter beta-lactamases inactivated loracarbef more efficiently than the remaining TEM-type ESBLs examined. Protein Expr Purif, 1998 Jul, 13(2), 184 - 90 Hyperexpression of rat spermatidal protein TP2 in Escherichia coli by codon optimization and engineering the vector-encoded 5' UTR; Meetei AR et al.; We have recently reported the cDNA cloning of rat spermatidal protein TP2 and its expression in Escherichia coli using pTrc 99A as the expression vector . However, the expression level was very low . We have now improved the expression of TP2 over fivefold by (1) optimizing the codons for lysine, arginine, proline, leucine, glycine, valine, threonine, alanine, and tyrosine and (2) by engineering the vector-encoded 5' UTR . The expressed protein was in the soluble phase and could be purified to homogeneity by successive chromatography on Zinc-NTA-agarose affinity matrix and heparin agarose . Serendipitously, we have also observed a concomitant hyperinduction of vector encoded beta-lactamase gene along with TP2 in the E . coli BL21 (DE3) cells . Antimicrob Agents Chemother, 1998 Jul, 42(7), 1542 - 8 Selection and characterization of beta-lactam-beta-lactamase inactivator-resistant mutants following PCR mutagenesis of the TEM-1 beta-lactamase gene; Vakulenko SB et al.; Mechanism-based inactivators of beta-lactamases are used to overcome the resistance of clinical pathogens to beta-lactam antibiotics . This strategy can itself be overcome by mutations of the beta-lactamase that compromise the effectiveness of their inactivation . We used PCR mutagenesis of the TEM-1 beta-lactamase gene and sequenced the genes of 20 mutants that grew in the presence of ampicillin-clavulanate . Eleven different mutant genes from these strains contained from 1 to 10 mutations . Each had a replacement of one of the four residues, Met69, Ser130, Arg244, and Asn276, whose substitutions by themselves had been shown to result in inhibitor resistance . None of the mutant enzymes with multiple amino acid substitutions generated in this study conferred higher levels of resistance to ampicillin alone or ampicillin with beta-lactamase inactivators (clavulanate, sulbactam, or tazobactam) than the levels of resistance conferred by the corresponding single-mutant enzymes . Of the four enzymes with just a single mutation (Ser130Gly, Arg244Cys, Arg244Ser, or Asn276Asp), the Asn276Asp beta-lactamase conferred a wild-type level of ampicillin resistance and the highest levels of resistance to ampicillin in the presence of inhibitors . Site-directed random mutagenesis of the Ser130 codon yielded no other mutant with replacement of Ser130 besides Ser130Gly that produced ampicillin-clavulanate resistance . Thus, despite PCR mutagenesis we found no new mutant TEM beta-lactamase that conferred a level of resistance to ampicillin plus inactivators greater than that produced by the single-mutation enzymes that have already been reported in clinical isolates . Although this is reassuring, one must caution that other combinations of multiple mutations might still produce unexpected resistance. Anal Biochem, 1998 Jul 1, 260(2), 160 - 5 In vitro measurement of beta-lactamase-catalyzed ampicillin hydrolysis by recombinant Escherichia coli extracts using quantitative high-performance liquid chromatography; Urbain JL et al.; We report a rapid and simple protocol for measuring the beta-lactamase activity from recombinant Escherichia coli cells transformed with any of the common plasmid vectors that provide ampicillin resistance through constitutive expression and periplasmic localization of the beta-lactamase TEM-1 . The hydrolytic enzyme was extracted from the E . coli periplasm and the beta-lactamase activity determined by measuring conversion of ampicillin to aminobenzyl-penicilloic acid using quantitative high-performance liquid chromatography . Under saturating conditions the in vitro assay was linear as a function of both incubation time and enzyme concentration . Application of this assay to investigate TEM-1 expression, from two different protein expression vector systems, demonstrated the potential importance of this assay in studies of recombinant protein expression and translocation . J Clin Microbiol, 1998 Jul, 36(7), 1977 - 83 Genetic diversity among strains of Moraxella catarrhalis: analysis using multiple DNA probes and a single-locus PCR-restriction fragment length polymorphism method; Walker ES et al.; Moraxella (Branhamella) catarrhalis, a causative agent of otitis media, sinusitis, and exacerbation of bronchitis, has acquired widespread ability to produce beta-lactamase and can be nosocomially transmitted . The typing methods used in epidemiological analyses of M . catarrhalis are not optimal for genetic analyses . Two methods, a multiple-locus Southern blot (SB) method and a single-locus PCR-restriction fragment length polymorphism (RFLP) method, were developed and used to assess genetic diversity and potential clinical and geographic relationships in M . catarrhalis . Nine randomly cloned M . catarrhalis DNA fragments were used as probes of SBs containing DNA from 54 geographically and clinically diverse strains . For comparison, a PCR-RFLP method was developed as a quick, inexpensive, and discriminating alternative . A highly variable 3.7-kb genomic region (M46) was cloned and sequenced, and 3.5 kb of the cloned DNA was targeted for PCR amplification . DNAs from the 54 strains were subjected to PCR-RFLP . SB analysis distinguished all strains that had no apparent epidemiological linkage (40 of 54), and PCR-RFLP distinguished fewer strains (21 of 54) . Epidemiologically linked strains appeared genetically identical by both methods . PCR-RFLP was compared to pulsed-field gel electrophoresis (PFGE) for 8 of the 54 strains and 23 additional strains . PCR-RFLP distinguished fewer strains than PFGE typing (16 of 31 versus 20 of 31 strains), but PCR-RFLP was more useful for inferring interstrain relatedness . Separate cluster analyses of multilocus SB and single locus PCR-RFLP data showed high genetic diversity within and across geographic locations and clinical presentations . The resultant dendrograms were not entirely concordant, but both methods often gave similar strain clusters at the terminal branches . High genetic diversity, nonconcordance of cluster analyses from different genetic loci, and shared genotypes among epidemiologically linked strains support a hypothesis of high recombination relative to spread of clones . Single-locus PCR-RFLP may be suitable for short-term epidemiological studies, but the SB data demonstrate that greater strain discrimination may be obtained by sampling variation at multiple genomic sites. FEBS Lett, 1998 Jun 12, 429(2), 162 - 6 Different degradation pathways for heterologous glycoproteins in yeast; Holkeri H et al.; Rat nerve growth factor receptor ectodomain (NGFRe) and Escherichia coli beta-lactamase were translocated into the yeast endoplasmic reticulum (ER), glycosylated, misfolded and rapidly degraded . NGFRe underwent ATP-dependent thermosensitive degradation independently of vesicular transport . Since no evidence for degradation by the cytoplasmic 26S proteosome complex could be obtained, NGFRe appeared to be degraded in the ER . Beta-lactamase exited the ER by vesicular traffic and was transported from the Golgi via the Vps10 receptor pathway to the vacuole for degradation . Machineries in the ER and the Golgi appear to recognize distinct structural features on misfolded heterologous proteins and guide them to different degradation pathways. East Afr Med J, 1998 Mar, 75(3), 175 - 9 Human antibody response to Moraxella catarrhalis antigens; Gabre-Selasie S; Moraxella catarrhalis was isolated from 68 of 200 (34%) sputum and 56 (28%) nasopharyngeal swab samples of patients with community-acquired pneumonia . Of the 68 pneumonia patients, 42 (61.8%) were males and 26 (38.2%) females . Fifty one of the 68 patients (75%) had chronic underlying diseases . beta-lactamase was produced by 37 (54.4%) of the 68 sputum samples and 32(57.1%) of the 56 nasopharyngeal isolates . In an ELISA using outer membrane protein antigens of M . catarrhalis against patient sera showed 40 of 68 (58.8%), and 43 of 68 (63.2%) significant increase in convalescent to acute sera when IgA, IgM and IgG3 were used respectively . In control sera only of 30(3.3%) and none showed significant antibody rise when IgA, IgM and IgG3 conjugates were used respectively (P < 0.05). J Cell Biol, 1998 Jun 15, 141(6), 1323 - 33 The protozoan parasite Toxoplasma gondii targets proteins to dense granules and the vacuolar space using both conserved and unusual mechanisms; Karsten V et al.; All known proteins that accumulate in the vacuolar space surrounding the obligate intracellular protozoan parasite Toxoplasma gondii are derived from parasite dense granules . To determine if constitutive secretory vesicles could also mediate delivery to the vacuolar space, T . gondii was stably transfected with soluble Escherichia coli alkaline phosphatase and E . coli beta-lactamase . Surprisingly, both foreign secretory reporters were delivered quantitatively into parasite dense granules and efficiently secreted into the vacuolar space . Addition of a glycosylphosphatidylinositol membrane anchor rerouted alkaline phosphatase to the parasite surface . Alkaline phosphatase fused to the transmembrane domain and cytoplasmic tail from the endogenous dense granule protein GRA4 localized to dense granules . The protein was secreted into a tuboreticular network in the vacuolar space, in a fashion dependent upon the cytoplasmic tail, but not upon a tyrosine-based motif within the tail . Alkaline phosphatase fused to the vesicular stomatitis virus G protein transmembrane domain and cytoplasmic tail localized primarily to the Golgi, although staining of dense granules and the intravacuolar network was also detected; truncating the cytoplasmic tail decreased Golgi staining and increased delivery to dense granules but blocked delivery to the intravacuolar network . Targeting of secreted proteins to T . gondii dense granules and the plasma membrane uses general mechanisms identified in higher eukaryotic cells but is simplified and exaggerated in scope, while targeting of secreted proteins beyond the boundaries of the parasite involves unusual sorting events. Biophys J, 1998 Jun, 74(6), 3256 - 63 Atomic force microscopy detects changes in the interaction forces between GroEL and substrate proteins; Vinckier A et al.; The structure of the Escherichia coli chaperonin GroEL has been investigated by tapping-mode atomic force microscopy (AFM) under liquid . High-resolution images can be obtained, which show the up-right position of GroEL adsorbed on mica with the substrate-binding site on top . Because of this orientation, the interaction between GroEL and two substrate proteins, citrate synthase from Saccharomyces cerevisiae with a destabilizing Gly-->Ala mutation and RTEM beta-lactamase from Escherichia coli with two Cys-->Ala mutations, could be studied by force spectroscopy under different conditions . The results show that the interaction force decreases in the presence of ATP (but not of ATPgammaS) and that the force is smaller for native-like proteins than for the fully denatured ones . It also demonstrates that the interaction energy with GroEL increases with increasing molecular weight . By measuring the interaction force changes between the chaperonin and the two different substrate proteins, we could specifically detect GroEL conformational changes upon nucleotide binding. J Biol Chem, 1998 May 15, 273(20), 12109 - 15 Characterization of the C-terminal propeptide involved in bacterial wall spanning of alpha-amylase from the psychrophile Alteromonas haloplanctis; Feller G et al.; The antarctic psychrophile Alteromonas haloplanctis secretes a Ca2+- and Cl--dependent alpha-amylase . The nucleotide sequence of the amy gene and the amino acid sequences of the gene products indicate that the alpha-amylase precursor is a preproenzyme composed by the signal peptide (24 residues), the mature alpha-amylase (453 residues, 49 kDa), and a long C-terminal propeptide or secretion helper (192 residues, 21 kDa) . In cultures of the wild-type strain, the 70-kDa precursor is secreted at the mid-exponential phase and is cleaved by a nonspecific protease into the mature enzyme and the propeptide . The purified C-terminal propeptide displays several features common to beta-pleated transmembrane proteins . It has no intramolecular chaperone function because active alpha-amylase is expressed by Escherichia coli in the absence of the propeptide coding region . In E . coli, the 70-kDa precursor is directed toward the supernatant . When the alpha-amylase coding region is excised from the gene, the secretion helper can still promote its own membrane spanning . It can also accept a foreign passenger, as shown by the extracellular routing of a beta-lactamase-propeptide fusion protein. Cell Mol Life Sci, 1998 Apr, 54(4), 341 - 6 The diversity, structure and regulation of beta-lactamases; Philippon A et al.; beta-Lactamase production is responsible for the appearance of a large number of pathogenic bacterial strains exhibiting a high degree of resistance to beta-lactam antibiotics . A large number of enzymes have been described with very diverse primary structures and catalytic profiles . Nevertheless, all known three-dimensional structures of active-site serine beta-lactamases exhibit a high degree of similarity with apparently equivalent chemical functionalities in the same strategic positions . These groups might not, however, play identical roles in the various classes of enzymes . Structural data have also been recently obtained for the zinc metallo-beta-lactamases, but the detailed catalytic mechanisms might also differ widely, depending on the enzyme studied . Similarly, the induction of the synthesis of beta-lactamases is now better understood, but many questions remain to be answered. Protein Eng, 1998 Feb, 11(2), 87 - 93 Recurrence quantification analysis in structure-function relationships of proteins: an overview of a general methodology applied to the case of TEM-1 beta-lactamase; Zbilut JP et al.; Protein structure-function relationships have been increasingly scrutinized by a variety of correlational and information theoretic measures . In an effort to extend this methodology, a technique originally developed in non-linear science, recurrence quantification analysis, was combined with traditional principal components analysis to study a large number (56) of TEM-1 beta-lactamase mutants . The hydrophobicity profiles corresponding to the primary structure of 13 naturally occurring mutations partially impairing function, together with 43 artificial non-tolerated mutations were subjected to discriminant analysis, derived from the results of recurrence quantification analysis, coupled to a principal exponents extraction . Eleven (85%) of the naturally occurring mutants and 36 (84%) of the artificial mutants were correctly classified (p < 0.0001) .