Beta lactamase

Proc Natl Acad Sci U S A . 2005 Jan 11; {Epub ahead of print}
Thermodynamic prediction of protein neutrality; Bloom JD et al.; We present a simple theory that uses thermodynamic parameters to predict the probability that a protein retains the wild-type structure after one or more random amino acid substitutions . Our theory predicts that for large numbers of substitutions the probability that a protein retains its structure will decline exponentially with the number of substitutions, with the severity of this decline determined by properties of the structure . Our theory also predicts that a protein can gain extra robustness to the first few substitutions by increasing its thermodynamic stability . We validate our theory with simulations on lattice protein models and by showing that it quantitatively predicts previously published experimental measurements on subtilisin and our own measurements on variants of TEM1 beta-lactamase . Our work unifies observations about the clustering of functional proteins in sequence space, and provides a basis for interpreting the response of proteins to substitutions in protein engineering applications.

Genomics Proteomics Bioinformatics, 2003 Aug, 1(3), 173 - 9
Integration of G-protein coupled receptor signaling pathways for activation of a transcription factor (EGR-3); Tan X et al.; We recently reported the use of a gene-trapping approach to isolate cell clones in which a reporter gene had integrated into genes modulated by T-cell activation . We have now tested a panel of clones from that report and identified the one that responds to a variety of G-protein coupled receptors (GPCR) . The beta-lactamase tagged EGR-3 Jurkat cell was used to dissect specific GPCR signaling in vivo . Three GPCRs were studied, including the chemokine receptor CXCR4 (Gi-coupled) that was endogenously expressed, the platelet activation factor (PAF) receptor (Gq-coupled), and beta2 adrenergic receptor (Gs-coupled) that was both stably transfected . Agonists for each receptor activated transcription of the beta-lactamase tagged EGR-3 gene . Induction of EGR-3 through CXCR4 was blocked by pertussis toxin and PD58059, a specific inhibitor of MEK (MAPK/ERK kinase) . Neither of these inhibitors blocked isoproterenol or PAF-mediated activation of EGR-3 . Conversely, beta2- and PAF-mediated EGR-3 activation was blocked by the p38, specific inhibitor SB580 . In addition, both beta2- and PAF-mediated EGR-3 activation could be synergistically activated by CXCR4 activation . This combined result indicates that EGR-3 can be activated through distinct signal transduction pathways by different GPCRs and that signals can be integrated and amplified to efficiently tune the level of activation.

Medicina (B Aires), 2004, 64(2), 113 - 9
{Survey of chest physicians regarding COPD diagnosis and treatment}; Sivori ML et al.; A survey on COPD diagnostic procedures, treatment and management was conducted in a group of 517 chest physicians randomized from a list of the 1121 affiliates to the Asociacion Argentina de Medicina Respiratoria . One hundred eighty-seven responses were obtained (36.2% of the questionnaires mailed) . They treat an average of 53.3 COPD patients every month . Twenty-four percent of them had mild, 41.8% moderate and 33.8% severe disease (GOLD criteria) . Only clinical criteria for diagnosis of COPD, clinical criteria + spirometry (S), and clinical criteria + S + chest X ray were used by 2.9, 23.4 and 73.7% of responders, respectively . Seventy percent of responders believed that chronic asthma without bronchodilator response must be included in the COPD definition . Only 14.1% of responders performed S in every office visit . Cardiac function was assessed using clinical criteria, electrocardiogram and echocardiogram by 90.6, 80.6 and 73.8% of responders, respectively, while 98.3% stated that they trained most of their patients in the inhalation technique . Metered Dose Inhaled was the first option for bronchodilators administration (64.8%) followed by nebulization (16.5%), dry powder inhalation (13.7%) and oral administration (4.8%) . First option for chronic therapy in severe COPD patients was the association of anticholinergic drug (AC) + short acting beta2-agonists (SABA) (65.5%), AC alone (18.8%), long acting beta2-agonists (LABA) (9.7%), inhaled corticosteroids (IC) (3.5%) and SABA alone (2.8%) . Corticosteroids and antibiotics were prescribed in severe COPD exacerbation by 92.5 and 70% of responders, respectively . First choice antibiotic formulation was beta-lactamics + beta-lactamase inhibitors in 39% of the responders followed by fluorquinolones in 23.7%, macrolides in 17.5% and beta-lactamics in 12.5% . Lastly, 12.7% of COPD patients received long-term domiciliary oxygen therapy . 59.3% of them were prescribed pulmonary rehabilitation, 94.1% vaccination against influenza and 91.4% pneumococcal vaccination . Thirty seven percent of the patients continued to smoke . Most of reponses regarding diagnosis and exacerbation treatment were in agreement with recommendations of international guidelines . For maintenance treatment the association of AC + SABA was commonly recommended as first option, whereas IC and LABA were rarely prescribed.

Acta Biochim Pol, 2004, 51(4), 925 - 31
Escherichia coli small heat shock proteins IbpA/B enhance activity of enzymes sequestered in inclusion bodies; Kuczynska-Wisnik D et al.; Escherichia coli small heat shock proteins, IbpA/B, function as molecular chaperones and protect misfolded proteins against irreversible aggregation . IbpA/B are induced during overproduction of recombinant proteins and bind to inclusion bodies in E . coli cells . We investigated the effect of DeltaibpA/B mutation on formation of inclusion bodies and biological activity of enzymes sequestered in the aggregates in E . coli cells . Using three different recombinant proteins: Cro-beta-galactosidase, beta-lactamase and rat rHtrA1 we demonstrated that deletion of the ibpA/B operon did not affect the level of produced inclusion bodies . However, in aggregates containing IbpA/B a higher enzymatic activity was detected than in the IbpA/B-deficient inclusion bodies . These results confirm that IbpA/B protect misfolded proteins from inactivation in vivo.

Proc Natl Acad Sci U S A, 2005 Jan 4, 102(1), 57 - 62 Epub 2004 Dec 23.
The modular architecture of protein-protein binding interfaces; Reichmann D et al.; Protein-protein interactions are essential for life . Yet, our understanding of the general principles governing binding is not complete . In the present study, we show that the interface between proteins is built in a modular fashion; each module is comprised of a number of closely interacting residues, with few interactions between the modules . The boundaries between modules are defined by clustering the contact map of the interface . We show that mutations in one module do not affect residues located in a neighboring module . As a result, the structural and energetic consequences of the deletion of entire modules are surprisingly small . To the contrary, within their module, mutations cause complex energetic and structural consequences . Experimentally, this phenomenon is shown on the interaction between TEM1-beta-lactamase and beta-lactamase inhibitor protein (BLIP) by using multiple-mutant analysis and x-ray crystallography . Replacing an entire module of five interface residues with Ala created a large cavity in the interface, with no effect on the detailed structure of the remaining interface . The modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved.

Antimicrob Agents Chemother, 2005 Jan, 49(1), 358 - 65
Molecular characterization of cefoxitin-resistant Escherichia coli from Canadian hospitals; Mulvey MR et al.; A study designed to gain baseline information on strains of Escherichia coli displaying resistance to cefoxitin in Canada is described . A total of 29,323 E . coli isolates were screened at 12 participating hospital sites as part of an extended-spectrum beta-lactamase surveillance initiative . A total of 411 clinically significant, nonrepeat isolates displaying reduced susceptibilities to the NCCLS-recommended beta-lactams were submitted to a central laboratory over a 1-year period ending on 30 September 2000 . Two hundred thirty-two isolates were identified as resistant to cefoxitin . All cefoxitin-resistant strains were subtyped by pulsed-field gel electrophoresis, and of these, 182 strains revealed a unique fingerprint and 1 strain was untypeable . PCR and sequence analysis of the ampC promoter region revealed 51 different promoter or attenuator variants and 14 wild-type promoters . Three promoter regions were interrupted by insertion elements, two contained IS10 elements, and one contained an IS911 variant . PCR and sequence analysis for the detection of acquired AmpC resistance (by the acquisition of ACT-1/MIR-1, CMY-2, or FOX) revealed that 25 strains contained CMY-2, including 7 of the strains found to have wild-type promoters . The considerable genetic variability in both the strain fingerprint and the promoter region suggests that AmpC-type resistance may emerge spontaneously by mutation of sensitive strains rather than by the spread of strains or plasmids in the hospital setting.

J Virol, 2005 Jan, 79(2), 918 - 26
Studies of ebola virus glycoprotein-mediated entry and fusion by using pseudotyped human immunodeficiency virus type 1 virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha; Yonezawa A et al.; The Ebola filoviruses are aggressive pathogens that cause severe and often lethal hemorrhagic fever syndromes in humans and nonhuman primates . To date, no effective therapies have been identified . To analyze the entry and fusion properties of Ebola virus, we adapted a human immunodeficiency virus type 1 (HIV-1) virion-based fusion assay by substituting Ebola virus glycoprotein (GP) for the HIV-1 envelope . Fusion was detected by cleavage of the fluorogenic substrate CCF2 by beta-lactamase-Vpr incorporated into virions and released as a result of virion fusion . Entry and fusion induced by the Ebola virus GP occurred with much slower kinetics than with vesicular stomatitis virus G protein (VSV-G) and were blocked by depletion of membrane cholesterol and by inhibition of vesicular acidification with bafilomycin A1 . These properties confirmed earlier studies and validated the assay for exploring other properties of Ebola virus GP-mediated entry and fusion . Entry and fusion of Ebola virus GP pseudotypes, but not VSV-G or HIV-1 Env pseudotypes, were impaired in the presence of the microtubule-disrupting agent nocodazole but were enhanced in the presence of the microtubule-stabilizing agent paclitaxel (Taxol) . Agents that impaired microfilament function, including cytochalasin B, cytochalasin D, latrunculin A, and jasplakinolide, also inhibited Ebola virus GP-mediated entry and fusion . Together, these findings suggest that both microtubules and microfilaments may play a role in the effective trafficking of vesicles containing Ebola virions from the cell surface to the appropriate acidified vesicular compartment where fusion occurs . In terms of Ebola virus GP-mediated entry and fusion to various target cells, primary macrophages proved highly sensitive, while monocytes from the same donors displayed greatly reduced levels of entry and fusion . We further observed that tumor necrosis factor alpha, which is released by Ebola virus-infected monocytes/macrophages, enhanced Ebola virus GP-mediated entry and fusion to human umbilical vein endothelial cells . Thus, Ebola virus infection of one target cell may induce biological changes that facilitate infection of secondary target cells that play a key role in filovirus pathogenesis . Finally, these studies indicate that pseudotyping in the HIV-1 virion-based fusion assay may be a valuable approach to the study of entry and fusion properties mediated through the envelopes of other viral pathogens.

FEMS Microbiol Lett, 2004 Dec 15, 241(2), 229 - 232
Overexpression system and biochemical profile of CTX-M-3 extended-spectrum beta-lactamase expressed in Escherichia coli; Perilli M et al.; An efficient over-expression system was developed for CTX-M-3 extended-spectrum-beta-lactamase . The recombinant enzyme was purified from 1 l of culture to yield 22 mg of pure enzyme . The N-terminal amino acid sequence was determined to be NH(2)-QTADVQ.. . Determination of kinetic parameters with the purified CTX-M-3 revealed efficient hydrolysis of penicillins and cephalosporins, while ceftazidime and aztreonam were very poor substrates . Clavulanic acid, sulbactam and especially tazobactam inhibited the CTX-M-3 enzyme.

Biochemistry, 2004 Dec 21, 43(50), 15737 - 45
An engineered disulfide bond between residues 69 and 238 in extended-spectrum beta-lactamase Toho-1 reduces its activity toward third-generation cephalosporins; Shimizu-Ibuka A et al.; Previous crystallographic structural analysis of extended-spectrum beta-lactamase Toho-1 predicted that the high flexibility of beta-strand B3, the region that contains a conserved KTG motif and forms one wall of the substrate-binding site, could be one of the key features contributing to Toho-1 activity toward third-generation cephalosporins . To investigate whether this possible flexibility really affects the substrate profile of this enzyme, two Toho-1 mutants have been produced, G238C and G238C/G239in, in which the glycine residue at position 238 was replaced with a cysteine and an additional glycine residue was inserted . Our intent was to introduce a disulfide bond between the cysteine residues at positions 69 and 238, and thus to lock the position of beta-strand B3 . The results of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) titration indicated formation of a new disulfide bridge in the G238C mutant, although disulfide bond formation was not confirmed in the G238C/G239in mutant . Kinetic analysis showed that the activity of the G238C mutant decreased drastically against third-generation cephalosporins, while its catalytic efficiency against penicillins and first-generation cephalosporins was almost identical to that of the wild-type enzyme . This result was consistent with the prediction that flexibility in beta-strand B3 was critical for activity against third-generation cephalosporins in Toho-1 . Furthermore, we have determined the crystal structure of the G238C mutant enzyme to analyze the structural changes in detail . The structural model clearly shows the introduction of a new disulfide bridge and that there is no appreciable difference between the overall structures of the wild-type enzyme and the G238C mutant, although the introduced disulfide bond slightly influenced the positions of Ser237 on beta-strand B3 and Asn170 on the Omega loop . The results of our kinetic and structural analyses suggest that the flexibility of beta-strand B3, as well as the positions of Ser237 and the Omega loop, is critical for the substrate specificity expansion of Toho-1.

J Med Chem, 2004 Dec 16, 47(26), 6556 - 68
Structure-activity relationship of 6-methylidene penems bearing tricyclic heterocycles as broad-spectrum beta-lactamase inhibitors: crystallographic structures show unexpected binding of 1,4-thiazepine intermediates; Venkatesan AM et al.; The design and synthesis of a series of seven tricyclic 6-methylidene penems as novel class A and C serine beta-lactamase inhibitors is described . These compounds proved to be very potent inhibitors of the TEM-1 and AmpC beta-lactamases and less so against the class B metallo-beta-lactamase CcrA . In combination with piperacillin, their in vitro activities enhanced susceptibility of all class C resistant strains from various bacteria . Crystallographic structures of a serine-bound reaction intermediate of 17 with the class A SHV-1 and class C GC1 enzymes have been established to resolutions of 2.0 and 1.4 A, respectively, and refined to R-factors equal 0.163 and 0.145 . In both beta-lactamases, a seven-membered 1,4-thiazepine ring has formed . The stereogenic C7 atom in the ring has the R configuration in the SHV-1 intermediate and has both R and S configurations in the GC1 intermediate . Hydrophobic stacking interactions between the tricyclic C7 substituent and a tyrosine side chain, rather than electrostatic or hydrogen bonding by the C3 carboxylic acid group, dominate in both complexes . The formation of the 1,4- thiazepine ring structures is proposed based on a 7-endo-trig cyclization.

Protein Eng Des Sel . 2004 Dec 1; {Epub ahead of print}
Construction of stabilized proteins by combinatorial consensus mutagenesis; Amin N et al.; We constructed stabilized variants of beta-lactamase (BLA) from E . cloacae by combinatorial recruitment of consensus mutations . By aligning the sequences of 38 BLA homologs, we identified 29 positions where the E . cloacae gene differs from the consensus sequence of lactamases and constructed combinatorial libraries using mixtures of mutagenic oligonucleotides encompassing all 29 positions . Screening of 90 random isolates from these libraries identified 15 variants with significantly increased thermostability . The stability of these isolates suggest that all tested mutations make additive contributions to protein stability . A statistical analysis of sequence and stability data identified 11 mutations that made stabilizing contributions and 8 mutations that destabilized the protein . A second generation library recombining these 11 stabilizing mutations led to the identification of BLA variants that showed further stabilization . The most stable variant had a mid-point of thermal denaturation (Tm) that was 9.1 degrees higher than the starting molecule and contained 8 consensus mutations . Incubation of three stabilized BLA variants with several proteases showed that all tested isolates have significantly increased resistance to proteolysis . Our data demonstrate that combinatorial consensus mutagenesis (CCM) allows the rapid generation of protein variants with improved thermal and proteolytic stability.

Phys Med Biol, 1994 Nov, 39(11), 1801 - 9
Dielectric studies of native, unfolded and intermediate forms of beta-lactamase; Bone S; Time-domain dielectric spectroscopy has been applied to native, intermediate and unfolded beta -lactamase structures both in the hydrated solid state and in solution . Gravimetric and dielectric measurements indicate that nearly three times as many water molecules are multiply hydrogen bonded to the unfolded compared with the native protein . By contrast, the primary hydration population of the state A intermediate is only 20% larger than that of the folded enzyme . Analysis of the beta -dispersions in terms of rotational relaxation of the protein's permanent dipole has indicated that there is no significant difference in the dimensions of the three structures . This is not consistent with the findings of previous studies employing different experimental techniques.

Chem Biol, 2004 Nov, 11(11), 1483 - 7
A molecular switch created by in vitro recombination of nonhomologous genes; Guntas G et al.; We have created a molecular switch by the in vitro recombination of nonhomologous genes and subjecting the recombined genes to evolutionary pressure . The gene encoding TEM1 beta-lactamase was circularly permuted in a random fashion and subsequently randomly inserted into the gene encoding Escherichia coli maltose binding protein . From this library, a switch (RG13) was identified in which its beta-lactam hydrolysis activity was compromised in the absence of maltose but increased 25-fold in the presence of maltose . Upon removal of maltose, RG13's catalytic activity returned to its premaltose level, illustrating that the switching is reversible . The modularity of RG13 was demonstrated by increasing maltose affinity while preserving switching activity . RG13 gave rise to a novel cellular phenotype, illustrating the potential of molecular switches to rewire the cellular circuitry.

Langmuir, 2004 Nov 23, 20(24), 10464 - 73
Immobilization of the enzyme beta-lactamase on biotin-derivatized poly(L-lysine)-g-poly(ethylene glycol)-coated sensor chips: a study on oriented attachment and surface activity by enzyme kinetics and in situ optical sensing; Zhen G et al.; Understanding the conformation, orientation, and specific activity of proteins bound to surfaces is crucial for the development and optimization of highly specific and sensitive biosensors . In this study, the very efficient enzyme beta-lactamase is used as a model protein . The wild-type form was genetically engineered by site-directed mutagenesis to introduce single cysteine residues on the surface of the enzyme . The cysteine thiol group is subsequently biotinylated with a dithiothreitol (DTT)-cleavable biotinylation reagent . beta-Lactamase is then immobilized site-specifically via the biotin group on neutral avidin-covered surfaces with the aim to control the orientation of the enzyme molecule at the surface and study its effect on enzymatic activity using Nitrocefin as the substrate . The DTT-cleavable spacer allows the release of the specifically bound enzyme from the surface . Immobilization of the enzyme is performed on a monolayer of the polycationic, biotinylated polymer PLL-g-PEG/PEG-biotin assembled on niobium oxide (Nb2O5) surfaces via neutral avidin as the docking site . Two different assembly protocols, the sequential adsorption of avidin and biotinylated beta-lactamase and the immobilization of preformed complexes of beta-lactamase and avidin, are compared in terms of immobilization efficiency . In situ optical waveguide lightmode spectroscopy and colorimetric analysis of enzymatic activity were used to distinguish between specific and unspecific enzyme adsorption, to sense quantitatively the amount of immobilized enzyme, and to determine Michaelis-Menten kinetics . All tested enzyme variants turned out to be active upon immobilization at the polymeric surface . However, the efficiency of immobilized enzymes relative to the soluble enzymes was reduced about sevenfold, mainly because of impaired substrate (Nitrocefin) diffusion or restricted accessibility of the active site . No significant effect of different enzyme orientations could be detected, probably because the enzymes were attached to the surface through long, flexible PEG chain linkers.

Nucleic Acids Res . 2004 Nov 10;32(20):e158.
Combinatorial codon-based amino acid substitutions; Yanez J et al.; Twenty Fmoc-protected trinucleotide phosphoramidites representing a complete set of codons for the natural amino acids were chemically synthesized for the first time . A pool of these reagents was incorporated into oligonucleotides at substoichiometric levels to generate two libraries of variants that randomly carry either few or many codon replacements on a region encoding nine amino acids of the bacterial enzyme TEM-1 beta-lactamase . Assembly of the libraries was performed in a completely automated mode through a simple modification of ordinary protocols . This technology eliminates codon redundancy, stop codons and enables complete exploration of sequence space for single, double and triple mutations throughout a protein region spanning several residues . Sequence analysis of many non-selected clones revealed a good incorporation of the trinucleotides, producing combinations of mutations quite different from those obtained using conventional degenerate oligonucleotides . Ceftazidime-selection experiments yielded several never before reported variants containing novel amino acid combinations in the beta-lactamase omega loop region.

Hum Mol Genet, 2005 Jan 1, 14(1), 19 - 38 Epub 2004 Nov 10.
The hereditary spastic paraplegia protein spastin interacts with the ESCRT-III complex-associated endosomal protein CHMP1B; Reid E et al.; Pure hereditary spastic paraplegia is characterized by length-dependent degeneration of the distal ends of long axons . Mutations in spastin are the most common cause of the condition . We set out to investigate the function of spastin using a yeast two-hybrid approach to identify interacting proteins . Using full-length spastin as bait, we identified CHMP1B, a protein associated with the ESCRT (endosomal sorting complex required for transport)-III complex, as a binding partner . Several different approaches confirmed the physiological relevance of the interaction in mammalian cells . Epitope-tagged CHMP1B and spastin showed clear cytoplasmic co-localization in Cos-7 and PC12 cells . CHMP1B and spastin interacted specifically in vitro and in vivo in beta-lactamase protein fragment complementation assays, and spastin co-immunoprecipitated with CHMP1B . The interaction was mediated by a region of spastin lying between residues 80 and 196 and containing a microtubule interacting and trafficking domain . Expression of epitope-tagged CHMP1B in mammalian cells prevented the development of the abnormal microtubule phenotype associated with expression of ATPase-defective spastin . These data point to a role for spastin in intracellular membrane traffic events and provide further evidence to support the emerging recognition that defects in intracellular membrane traffic are a significant cause of motor neuron pathology.

Biochemistry, 2004 Nov 9, 43(44), 14111 - 7
Inhibitor-resistant class A beta-lactamases: consequences of the Ser130-to-Gly mutation seen in Apo and tazobactam structures of the SHV-1 variant; Sun T et al.; A bacterial response to the clinical use of class A beta-lactamase inhibitors such as tazobactam and clavulanic acid is the expression of variant beta-lactamases with weaker binding affinities for these mechanism-based inhibitors . Some of these inhibitor-resistant variants contain a glycine mutation at Ser130, a conserved active site residue known to be adventitiously involved in the inhibition mechanism . The crystallographic structure of a complex of tazobactam with the Ser130Gly variant of the class A SHV-1 beta-lactamase has been determined to 1.8 A resolution . Two reaction intermediates are observed . The primary intermediate is an acyclic species bound to the reactive Ser70 . It is poorly primed for catalytic hydrolysis because its ester carbonyl group is completely displaced from the enzyme's oxyanion hole . A smaller fraction of the enzyme contains a Ser70-bound aldehyde resulting from hydrolytic loss of the triazoyl-sulfinyl amino acid moiety from the primary species . This first structure of a class A beta-lactamase lacking Ser130, the side chain of which functions in beta-lactam binding and possibly in catalysis, gives crystallographic evidence that the acylation step of beta-lactam turnover can occur without Ser130 . Unexpectedly, the crystal structure of the uncomplexed Ser130Gly enzyme, also determined to 1.8 A resolution, shows that a critical Glu166-activated water molecule is missing from the catalytic site . Comparison of this uncomplexed variant with the wild-type structure reveals that Ser130 is required for orienting the side chain of Ser70 and ensuring the hydrogen bonding of Ser70 to both Lys73 and the catalytic water molecule.

Antimicrob Agents Chemother, 2004 Nov, 48(11), 4482 - 4
Pharmacodynamics of ceftazidime plus the serine beta-lactamase inhibitor AM-112 against Escherichia coli containing TEM-1 and CTX-M-1 beta-lactamases; Bowker KE et al.; A strain of Escherichia coli containing TEM-1 and CTX-M-1 was tested in an in vitro pharmacokinetic model against ceftazidime with and without AM-112, a serine beta-lactamase inhibitor . Ceftazidime alone was less effective than ceftazidime plus AM-112, and a single dose was more effective than three fractionated doses.

Antimicrob Agents Chemother, 2004 Nov, 48(11), 4217 - 25
OXA-60, a chromosomal, inducible, and imipenem-hydrolyzing class D beta-lactamase from Ralstonia pickettii; Girlich D et al.; A chromosomally encoded oxacillinase, OXA-22, had been characterized from Ralstonia pickettii PIC-1 that did not explain by itself the resistance profile of this strain to beta-lactams . Thus, further analysis of the genetic background of this species led to the identification of another oxacillinase, OXA-60, that was expressed only after beta-lactam induction . This chromosomally encoded oxacillinase shared 19% amino acid identity with OXA-22 . It has a narrow-spectrum hydrolysis profile that includes imipenem . OXA-60-like enzymes were identified in several R . pickettii strains . Gene inactivation and induction studies of the bla(OXA-60) and bla(OXA-22) genes in R . pickettii identified the relative contribution of each oxacillinase to the resistance profile of R . pickettii to beta-lactams.

Bioorg Med Chem, 2004 Nov 15, 12(22), 5807 - 17
Novel imidazole substituted 6-methylidene-penems as broad-spectrum beta-lactamase inhibitors; Venkatesan AM et al.; Beta-lactamases are serine and metallo-dependent enzymes produced by the bacteria in defense against beta-lactam antibiotics . Production of class-A, class-B, and class-C enzymes by the bacteria make the use of beta-lactam antibiotics ineffective in certain cases . To overcome resistance to beta-lactam antibiotics, several beta-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam are widely used in the clinic in combination with beta-lactam antibiotics . However, single point mutations within these enzymes have allowed bacteria to overcome the inhibitory effect of the commercially approved beta-lactamase inhibitors . Although the commercially available beta-lactamase inhibitor/beta-lactam antibiotic combinations are effective against class-A producing bacteria and many extended spectrum beta-lactamase (ESBL's) producing bacteria they are less effective against class-C enzymes expressing bacteria . To circumvent this problem, based on modeling studies several novel imidazole substituted 6-methylidene-penem derivatives were synthesized and tested against various beta-lactamase producing isolates . The present paper deals with the synthesis and structure-activity relationships (SAR) of these compounds.

J Biol Chem, 2004 Dec 31, 279(53), 55728 - 36 Epub 2004 Oct 20.
Oligomerization of the {gamma}-aminobutyric acid transporter-1 is driven by an interplay of polar and hydrophobic interactions in transmembrane helix II; Korkhov VM et al.; The available evidence indicates that members of the neurotransmitter:sodium symporter family form constitutive oligomers . Their second transmembrane helix (TM2) contains a leucine heptad repeat proposed to be involved in oligomerization . In artificial transmembrane segments, interhelical interactions are stabilized by polar residues . We searched for these hydrogen bond donors in TM2 by mutating the five polar residues in TM2 of the gamma-aminobutyric acid transporter-1 (GAT1) . We tested the ability of the resulting mutants to oligomerize by fluorescence microscopy, Foerster resonance energy transfer, and beta-lactamase fragment complementation . Of all generated mutants, only Y86A- (but not Y86F-), E101A-, E101Q-, and E101D-GAT1 were judged by these criteria to be deficient in oligomerization and were retained intracellularly . The observations are consistent with a model where the leucine heptad repeat in TM2 drives a homophilic association that is stabilized by Tyr(86) and Glu(101); Tyr(86) participates in hydrophobic stacking . Glu(101) is in the a-position of the leucine heptad repeat (where positions 1-7 are denoted a-g, and each leucine is in the central d-position) . Thus, Glu(101) is in the position predicted for the hydrogen bond donor (i.e . sandwiched between Leu(97) and Leu(104), which are one helical turn above and below Glu(101)) . These key residues, namely Tyr(86) and Glu(101), are conserved in related transporters from archaeae to humans; they are therefore likely to support oligomeric assembly in transporter orthologs and possibly other proteins with multiple transmembrane segments.

Anal Biochem, 2004 Nov 15, 334(2), 344 - 55
A cell-based beta-lactamase reporter gene assay for the identification of inhibitors of hepatitis C virus replication; Zuck P et al.; This report describes the development, optimization, and implementation of a cell-based assay for high-throughput screening (HTS) to identify inhibitors to hepatitis C virus (HCV) replication . The assay is based on a HCV subgenomic RNA replicon that expresses beta-lactamase as a reporter for viral replication in enhanced Huh-7 cells . The drug targets in this assay are viral and cellular enzymes required for HCV replication, which are monitored by fluorescence resonance energy transfer using cell-permeable CCF4-AM as a beta-lactamase substrate . Digital image processing was used to visualize cells that harbor viral RNA and to optimize key assay development parameters such as transfection and culturing conditions to obtain a cell line which produced a robust assay window . Formatting the assay for compound screening was problematic due to small signal-to-background ratio and reduced potency to known HCV inhibitors . These technical difficulties were solved by using clavulanic acid, an irreversible inhibitor of beta-lactamase, to eliminate residual beta-lactamase activity after HCV replication was terminated, thus resulting in an improved assay window . HTS was carried out in 384-well microplate format, and the signal-to-background ratio and Z factor for the assay plates during the screen were approximately 13-fold and 0.5, respectively.

Nucleic Acids Res . 2004 Sep 30;32(17):e136.
Protein evolution by codon-based random deletions; Osuna J et al.; A method to delete in-phase codons throughout a defined target region of a gene has been developed . This approach, named the codon-based random deletion (COBARDE) method, is able to delete complete codons in a random and combinatorial mode . Robustness, automation and fine-tuning of the mutagenesis rate are essential characteristics of the method, which is based on the assembly of oligonucleotides and on the use of two transient orthogonal protecting groups during the chemical synthesis . The performance of the method for protein function evolution was demonstrated by changing the substrate specificity of TEM-1 beta-lactamase . Functional ceftazidime-resistant beta-lactamase variants containing several deleted residues inside the catalytically important omega-loop region were found . The results show that the COBARDE method is a useful new molecular tool to access previously unexplorable sequence space.

Antimicrob Agents Chemother, 2004 Oct, 48(10), 4050 - 3
AmpC beta-lactamase in an Escherichia coli clinical isolate confers resistance to expanded-spectrum cephalosporins; Mammeri H et al.; Cloning, sequencing, and biochemical analysis identified a novel AmpC-type beta-lactamase conferring resistance to extended-spectrum cephalosporins in an Escherichia coli clinical isolate . This enzyme, exhibiting 14 amino acid substitutions compared to a reference AmpC cephalosporinase of E . coli, hydrolyzed ceftazidime and cefepime significantly.

Antimicrob Agents Chemother, 2004 Oct, 48(10), 3980 - 8
Antibody mapping of the linear epitopes of CMY-2 and SHV-1 beta-lactamases; Hujer AM et al.; Knowledge of the amino acids that define recognition of anti-beta-lactamase antibodies is critical to the interpretation of sensitivity and specificity of these antibodies when they are used in a clinical or research setting . To this end, we mapped the epitopes of the CMY-2 and SHV-1 beta-lactamases by using the SPOT synthesis method . Eight linear epitopes in SHV-1 and seven linear epitopes in CMY-2 were identified by using anti-SHV-1 and anti-CMY-2 polyclonal antibodies, respectively . The epitopes of SHV-1 were mapped to amino acids at the Ambler positions ABL 28 to 38, 42 to 54, 88 to 100, 102 to 114, 170 to 182, 186 to 194, 202 to 210, and 276 to 288 . In the epitope spanning amino acids 102 to 114, alanine and X-Scan analysis demonstrated that D104, Y105, P107, and S109 are essential residues for antibody recognition . In the epitope containing amino acids 170 to 182, N170, L173, P174, G175, and D176 were immunodominant . In CMY-2 beta-lactamase, amino acids 4 to 16, 70 to 79, 211 to 223, 274 to 286, 289 to 298, 322 to 334, and 343 to 358 of the mature enzyme defined the major linear epitopes . A detailed analysis of the recognition sites that are located in an area analogous to the omega loop of class A beta-lactamases (V211 to V223) showed that the amino acids Q215 to E219 are important in antibody binding . Incubation of CMY-2 beta-lactamase with a 10-fold molar excess of anti-CMY-2 antibody for 60 min resulted in greater than 80% inhibition of nitrocefin hydrolysis . A 10-fold molar excess of anti-SHV-1 antibody reduced the activity of SHV-1 by 69% . Analysis of the CMY-2 and SHV-1 structures suggest that this reduction of hydrolytic activity may be due in part to the direct binding of antibodies to the omega loop, thereby hindering access of substrate to the active site.

Virology, 2004 Oct 10, 328(1), 36 - 44
HIV-1 virion fusion assay: uncoating not required and no effect of Nef on fusion; Cavrois M et al.; We recently described a sensitive and specific assay that detects the fusion of HIV-1 virions to a broad range of target cells, including primary CD4 cells . This assay involves the use of virions containing beta-lactamase-Vpr (BlaM-Vpr) and the loading of target cells with CCF2, a fluorogenic substrate of beta-lactamase . Since Vpr strongly associates with the viral core, uncoating of the viral particle might be required for effective cleavage of CCF2 by BlaM-Vpr . Here, we show that BlaM-Vpr within mature viral cores effectively cleaves CCF2, indicating that this assay measures virion fusion independently of uncoating . We also show that wildtype and Nef-deficient HIV-1 virions fuse with equivalent efficiency to HeLa-CD4 cells, SupT1 T cells, and primary CD4 T cells . Since Nef enhances cytoplasmic delivery of viral cores and increases viral infectivity, these findings indicate that Nef enhances an early post-fusion event in the multistep process of viral entry . Possible sites of Nef action include enlargement of the fusion pore, enhanced uncoating of viral particles, and more efficient passage of viral cores through the dense cortical actin network located immediately beneath the plasma membrane .

Int J Antimicrob Agents, 2004 Oct, 24(4), 320 - 6
Dissemination of Escherichia coli producing AmpC-type beta-lactamase (CMY-11) in Korea; Kim JY et al.; Among the 51 clinical isolates collected from a university hospital in Korea, nine isolates were resistant to cephamycins . Nine isolates were shown to produce CMY-11 and these also included three isolates producing TEM-1 . The results from ERIC-PCR revealed that dissemination of CMY-11 was due to outbreaks of resistant species and to the intra-species spread of resistance to cephamycins in Korea . CMY-11 beta-lactamase genes from nine clinical isolates that were responsible for resistance to cephamycins (cefoxitin and cefotetan), amoxicillin, cephalothin and amoxicillin-clavulanic acid, were cloned and characterised . A sequence identical to the common regions in In6, In7 and a novel integron from pSAL-1 was found upstream from bla(CMY-11) gene at nucleotide 1-71 . Eighteen nucleotides between position 71 and 72 were inserted into the bla(CMY-11) gene.

Bioorg Med Chem Lett, 2004 Oct 18, 14(20), 5117 - 20
Benzopyranones with retro-amide side chains as (inhibitory) beta-lactamase substrates; Adediran SA et al.; 3-(N-Benzylcarbamoyl)-7-carboxy-3, 4-dihydro-2H-1-benzo-pyran-2-one and its 8-carboxy analogue have been synthesized and evaluated as potential (inhibitory) substrates of beta-lactam-recognizing enzymes . These compounds are bicyclic delta-lactones with retro-amide (with respect to classical beta-lactams) side chains . They were found to be comparably effective as substrates of typical class A, C and D beta-lactamases as analogous benzopyranones bearing 'normal' amide side chains . The new 8-carboxy derivative, however, formed a much more (1000-fold) tightly-bound acyl-enzyme with a class C beta-lactamase than did its 'normal' analogue, and thus provides a structural lead to new inhibitors of this class of beta-lactamase.

Antimicrob Agents Chemother, 2004 Sep, 48(9), 3579 - 82
Biochemical characterization of laboratory mutants of extended-spectrum beta-lactamase TEM-60; Caporale B et al.; Three mutants of the extended-spectrum beta-lactamase TEM-60, the P51L, K104E, and S164R mutants, were constructed by site-directed mutagenesis . The kinetic parameters of the mutated enzymes and interactions of inhibitors were significantly different from those of TEM-60, revealing that the L51P mutation plays an important role in enzyme activity and stability in the TEM-60 background.

J Mol Biol, 2004 Aug 27, 341(5), 1295 - 315
Estimating the prevalence of protein sequences adopting functional enzyme folds; Axe DD; Proteins employ a wide variety of folds to perform their biological functions . How are these folds first acquired? An important step toward answering this is to obtain an estimate of the overall prevalence of sequences adopting functional folds . Since tertiary structure is needed for a typical enzyme active site to form, one way to obtain this estimate is to measure the prevalence of sequences supporting a working active site . Although the immense number of sequence combinations makes wholly random sampling unfeasible, two key simplifications may provide a solution . First, given the importance of hydrophobic interactions to protein folding, it seems likely that the sample space can be restricted to sequences carrying the hydropathic signature of a known fold . Second, because folds are stabilized by the cooperative action of many local interactions distributed throughout the structure, the overall problem of fold stabilization may be viewed reasonably as a collection of coupled local problems . This enables the difficulty of the whole problem to be assessed by assessing the difficulty of several smaller problems . Using these simplifications, the difficulty of specifying a working beta-lactamase domain is assessed here . An alignment of homologous domain sequences is used to deduce the pattern of hydropathic constraints along chains that form the domain fold . Starting with a weakly functional sequence carrying this signature, clusters of ten side-chains within the fold are replaced randomly, within the boundaries of the signature, and tested for function . The prevalence of low-level function in four such experiments indicates that roughly one in 10(64) signature-consistent sequences forms a working domain . Combined with the estimated prevalence of plausible hydropathic patterns (for any fold) and of relevant folds for particular functions, this implies the overall prevalence of sequences performing a specific function by any domain-sized fold may be as low as 1 in 10(77), adding to the body of evidence that functional folds require highly extraordinary sequences.

Acta Crystallogr D Biol Crystallogr, 1995 Sep, 51(Pt 5), 682 - 94
TEM1 beta-lactamase structure solved by molecular replacement and refined structure of the S235A mutant; Fonze E; beta-Lactamases are bacterial enzymes which catalyse the hydrolysis of the beta-lactam ring of penicillins, cephalosporins and related compounds, thus inactivating these antibiotics . The crystal structure of the TEM1 beta-lactamase has been determined at 1.9 A resolution by the molecular-replacement method, using the atomic coordinates of two homologous beta-lactamase refined structures which show about 36% strict identity in their amino-acid sequences and 1.96 A r.m.s . deviation between equivalent Calpha atoms . The TEM1 enzyme crystallizes in space group P2(1)2(1)2(1) and there is one molecule per asymmetric unit . The structure was refined by simulated annealing to an R-factor of 15.6% for 15 086 reflections with I >/= 2sigma(I) in the resolution range 5.0-1.9 A . The final crystallographic structure contains 263 amino-acid residues, one sulfate anion in the catalytic cleft and 135 water molecules per asymmetric unit . The folding is very similar to that of the other known class A beta-lactamases . It consists of two domains, the first is formed by a five-stranded beta-sheet covered by three alpha-helices on one face and one alpha-helix on the other, the second domain contains mainly alpha-helices . The catalytic cleft is located at the interface between the two domains . We also report the crystallographic study of the TEM S235A mutant . This mutation of an active-site residue specifically decreases the acylation rate of cephalosporins . This TEM S235A mutant crystallizes under the same conditions as the wild-type protein and its structure was refined at 2.0 A resolution with an R value of 17.6% . The major modification is the appearance of a water molecule near the mutated residue, which is incompatible with the OG 235 present in the wild-type enzyme, and causes very small perturbations in the interaction network in the active site.

J Clin Microbiol, 2004 Aug, 42(8), 3888 - 90
Mucoid nitrate-negative Moraxella nonliquefaciens from three patients with chronic lung disease; Davis JM et al.; Mucoid strains of Moraxella nonliquefaciens were recovered from the sputa of three indigenous Australians with chronic lung disease . These atypical strains failed to reduce nitrate, and one strain produced beta-lactamase . While the mucoid phenotype of M . nonliquefaciens has rarely been reported, the mucoid nitrate-negative biovar has never been previously reported.

Infection, 2004 Aug, 32(4), 246 - 8
Failure of ceftriaxone in an intravenous drug user with invasive infection due to Ralstonia pickettii; Zellweger C et al.; We report a case of septic arthritis due to Ralstonia pickettii in an intravenous drug user with unfavorable clinical course under antibiotic therapy with ceftriaxone despite in vitro susceptibility to the drug . The treatment failure may have been due to a discrepancy between in vitro and in vivo susceptibility of R . pickettii, or to resistance development mediated by a recently described inducible beta-lactamase.

J Bacteriol, 2004 Aug, 186(16), 5486 - 95
Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 beta-lactamase as a new fluorescence-based reporter; Charpentier X et al.; Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) strains are human and animal pathogens that inject effector proteins into host cells via a type III secretion system (TTSS) . Cif is an effector protein which induces host cell cycle arrest and reorganization of the actin cytoskeleton . Cif is encoded by a lambdoid prophage present in most of the EPEC and EHEC strains . In this study, we analyzed the domain that targets Cif to the TTSS by using a new reporter system based on a translational fusion of the effector proteins with mature TEM-1 beta-lactamase . Translocation was detected directly in living host cells by using the fluorescent beta-lactamase substrate CCF2/AM . We show that the first 16 amino acids (aa) of Cif were necessary and sufficient to mediate translocation into the host cells . Similarly, the first 20 aa of the effector proteins Map, EspF, and Tir, which are encoded in the same region as the TTSS, mediated secretion and translocation in a type III-dependent but chaperone-independent manner . A truncated form of Cif lacking its first 20 aa was no longer secreted and translocated, but fusion with the first 20 aa of Tir, Map, or EspF restored both secretion and translocation . In addition, the chimeric proteins were fully able to trigger host cell cycle arrest and stress fiber formation . In conclusion, our results demonstrate that Cif is composed of a C-terminal effector domain and an exchangeable N-terminal translocation signal and that the TEM-1 reporter system is a convenient tool for the study of the translocation of toxins or effector proteins into host cells.

Curr Med Chem, 2004 Jul, 11(14), 1813 - 35
Strategies for the stereocontrolled formation of oxygen analogues of penicillins and cephalosporins; Lysek R et al.; The synthesis of oxacephalotin and oxacephamandol, which are more active than natural, sulfur-containing congeners, and the isolation of clavulanic acid, a potent inhibitor of beta-lactamase enzymes, directed attention of many academic and industrial laboratories the synthesis of oxygen analogues of penicillins and cephalosporins . The present review focuses attention on the problem of stereocontrol in the formation of a desired configuration of the bridgehead carbon atom in the title compounds . Five feasible synthetic methods leading to the basic skeletons of clavams and 5-oxacephams are discussed . Three of them involve the nucleophilic substitution at C-4 of the azetidin- 2-ones performed as inter- or intramolecular process and the remaining two involve cycloaddition reactions between ketenes and iminoethers, or between vinyl ethers and isocyanates . Owing to the general application, stereospecificity and high asymmetric induction, the last method seems to be most advantageous . The weak point of the nucleophilic substitution methodology is that a nucleophile approaches the 3-substituted azetidin-2-one ring preferentially anti to the existing substituent and in the case where there is no substituent at C-3, that the stereoselectivity of formation of the new chirality center at C-4 is low . All discussed methods are illustrated by the examples taken from the literature.

J Chem Phys, 2004 May 1, 120(17), 8039 - 52
Adapting the nudged elastic band method for determining minimum-energy paths of chemical reactions in enzymes; Xie L et al.; Optimization of reaction paths for enzymatic systems is a challenging problem because such systems have a very large number of degrees of freedom and many of these degrees are flexible . To meet this challenge, an efficient, robust and general approach is presented based on the well-known nudged elastic band reaction path optimization method with the following extensions: (1) soft spectator degrees of freedom are excluded from path definitions by using only inter-atomic distances corresponding to forming/breaking bonds in a reaction; (2) a general transformation of the distances is defined to treat multistep reactions without knowing the partitioning of steps in advance; (3) a multistage strategy, in which path optimizations are carried out for reference systems with gradually decreasing rigidity, is developed to maximize the opportunity of obtaining continuously changing environments along the path . We demonstrate the applicability of the approach using the acylation reaction of type A beta-lactamase as an example . The reaction mechanism investigated involves four elementary reaction steps, eight forming/breaking bonds . We obtained a continuous minimum energy path without any assumption on reaction coordinates, or on the possible sequence or the concertedness of chemical events . We expect our approach to have general applicability in the modeling of enzymatic reactions with quantum mechanical/molecular mechanical models .

J Antimicrob Chemother, 2004 Aug, 54(2), 348 - 53 Epub 2004 Jul 14.
Analysis of sequence variation among smeDEF multi drug efflux pump genes and flanking DNA from defined 16S rRNA subgroups of clinical Stenotrophomonas maltophilia isolates; Gould VC et al.; OBJECTIVES: To determine the level of variation in the smeDEF efflux pump and smeT transcriptional regulator genes among three defined 16S rRNA sequence subgroups of clinical Stenotrophomonas maltophilia isolates . METHODS: smeDEF sequencing used a PCR genome walking approach . Determination of the sequence surrounding smeDEF used a flanking primer PCR method and specific primers anchored in smeD or smeF together with random primers . RESULTS: smeDEF is chromosomal and located in the same position in the chromosome in all three subgroups of isolates . Flanking smeD is a gene, smeT, encoding a putative transcriptional repressor for smeDEF . Variation at these loci among the isolates is considerably lower (up to 10%) than at intrinsic beta-lactamase loci (up to 30%) in the same isolates, implying greater functional constraint . The smeD-smeT intergenic region contains a highly conserved section, which maps with previously predicted promoter/operator regions, and a hypervariable untranslated region, which can be used to subgroup clinical isolates . CONCLUSIONS: These data provide further evidence that it is possible to group clinical isolates of the inherently variable species, S . maltophilia, based on genotypic properties . Isolate D457, in which most work concerning smeDEF expression has been performed, does not fall into S . maltophilia subgroup A, which is the most typical.

J Gen Virol, 2004 Jul, 85(Pt 7), 1867 - 75
Dominant negative effect of wild-type NS5A on NS5A-adapted subgenomic hepatitis C virus RNA replicon; Graziani R et al.; An efficient model is currently used to study hepatitis C virus (HCV) replication in cell culture . It involves transfection in Huh7, a hepatoma-derived cell line, of an antibiotic (neomycin) selectable HCV subgenomic replicon encoding the non-structural (NS) proteins from NS3 to NS5B . However, strong and sustained replication is achieved only on the appearance of adaptive mutations in viral proteins . The most effective of these adaptive mutations are concentrated mainly in NS5A, not only into the original Con1 but also in the recently established HCV-BK and HCV-H77 isolate-derived replicons . This suggests that the expression of wild-type (wt) NS5A may not allow efficient HCV RNA replication in cell culture . With the use of a beta-lactamase reporter gene as a marker for HCV replication and TaqMan RNA analysis, the replication of different HCV replicons in cotransfection experiments was investigated . Comparing wt with NS5A-adapted replicons, the strong evidence accumulated showed that the expression of wt NS5A was actually able to inhibit the replication of NS5A-adapted replicons . This feature was characterized as a dominant negative effect . Interestingly, an NS5B (R2884G)-adapted replicon, containing a wt NS5A, was dominant negative on an NS5A-adapted replicon but was not inhibited by the original Con1 replicon . In conclusion, these studies revealed that the original wt Con1 replicon is not only incompetent for replication in cell culture, but is also able to interfere with NS5A-adapted replicons.

Appl Biochem Biotechnol, 2004 May, 117(2), 115 - 22
The "Bringer" strategy: a very fast and highly efficient method for construction of mutant libraries by error-prone polymerase chain reaction of ring-closed plasmids; Bichet A et al.; Random mutagenesis constitutes a keystone in many strategies of directed evolution of biocatalysts and is often done by error-prone polymerase chain reaction (epPCR) . Traditionally, the epPCR-generated DNA fragments are then subcloned into an expression vector to obtain a mutant library, which in turn is transformed into a suited host and screened for mutants that display the desired property . However, the vast majority of epPCR-generated fragments generally are lost during the subcloning step, making it the bottleneck in the mutant library construction procedure . Here we report a rapid and convenient strategy based on the epPCR amplification of a ring-closed expression plasmid that contains the gene of interest; after a DpnI digest the product of the epPCR reaction constitutes the mutant library and can be used directly for screening procedures . Primers binding to the beta-lactamase gene were chosen to allow application of the strategy to as broad a range of target plasmids as possible . The functionality of this approach was demonstrated by mutating the alpha-peptide coding region of the lacZ gene.

J Biol Chem, 2004 Aug 6, 279(32), 33630 - 8 Epub 2004 May 24.
Probing the specificity of the subclass B3 FEZ-1 metallo-beta-lactamase by site-directed mutagenesis; Mercuri PS et al.; The subclass B3 FEZ-1 beta-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the beta-lactam bond in penicillins, cephalosporins, and carbapenems . FEZ-1 has been extensively studied using kinetic, computational modeling and x-ray crystallography . In an effort to probe residues potentially involved in substrate binding and zinc binding, five site-directed mutants of FEZ-1 (H121A, Y156A, S221A, N225A, and Y228A) were prepared and characterized using metal analyses and steady state kinetics . The activity of H121A is dependent on zinc ion concentration . The H121A monozinc form is less active than the dizinc form, which exhibits an activity similar to that of the wild type enzyme . Tyr156 is not essential for binding and hydrolysis of the substrate . Substitution of residues Ser221 and Asn225 modifies the substrate profile by selectively decreasing the activity against carbapenems . The Y228A mutant is inhibited by the product formed upon hydrolysis of cephalosporins . A covalent bond between the side chain of Cys200 and the hydrolyzed cephalosporins leads to the formation of an inactive and stable complex.

Infect Immun, 2004 Jun, 72(6), 3336 - 43
Recombinant Mycobacterium bovis BCG expressing the Sm14 antigen of Schistosoma mansoni protects mice from cercarial challenge; Varaldo PB et al.; The Sm14 antigen of Schistosoma mansoni was cloned and expressed in Mycobacterium bovis BCG as a fusion with the Mycobacterium fortuitum beta-lactamase protein under the control of its promoter, pBlaF*; the protein was localized in the bacterial cell wall . The rBCG-Sm14 strain was shown to be relatively stable in cultured murine and bovine monocytes in terms of infectivity, bacterial persistence, and plasmid stability . The immunization of mice with rBCG-Sm14 showed no induction of anti-Sm14 antibodies; however, splenocytes of immunized mice released increased levels of gamma interferon upon stimulation with recombinant Sm14 (rSm14), indicating an induction of a Th1-predominant cellular response against Sm14 . Mice immunized with one or two doses of rBCG-Sm14 and challenged with live S . mansoni cercaria showed a 48% reduction in worm burden, which was comparable to that obtained by immunization with three doses of rSm14 purified from Escherichia coli . The data presented here further enhance the status of Sm14 as a promising candidate antigen for the control of schistosomiasis and indicate that a one-dose regimen of rBCG-Sm14 could be considered a convenient means to overcome many of the practical problems associated with the successful implementation of a multiple-dose vaccine schedule in developing countries.

J Bacteriol, 2004 Jun, 186(11), 3431 - 8
Two oligopeptide-permease-encoding genes in the clavulanic acid cluster of Streptomyces clavuligerus are essential for production of the beta-lactamase inhibitor; Lorenzana LM et al.; orf7 (oppA1) and orf15 (oppA2) are located 8 kb apart in the clavulanic acid gene cluster of Streptomyces clavuligerus and encode proteins which are 48.0% identical . These proteins show sequence similarity to periplasmic oligopeptide-binding proteins . Mutant S . clavuligerus oppA1::acc, disrupted in oppA1, lacks clavulanic acid production . Clavulanic acid production is restored by transformation with plasmid pIJ699-oppA1, which carries oppA1, but not with the multicopy plasmid pIJ699-oppA2, which carries oppA2 . The mutant S . clavuligerus oppA2::aph also lacks clavulanic acid production, shows a bald phenotype, and overproduces holomycin (5) . Clavulanic acid production at low levels is restored in the oppA2-disrupted mutants by transformation with plasmid pIJ699-oppA2, but it is not complemented by the multicopy plasmid pIJ699-oppA1 . Both genes encode oligopeptide permeases with different substrate specificities . The disrupted S . clavuligerus oppA2::aph is not able to grow on RPPGFSPFR (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; bradykinin), but both mutants grow on VAPG (Val-Ala-Pro-Gly) as the only nitrogen source, indicating differences in the peptide bound by the proteins encoded by both genes . The null S . clavuligerus oppA1::acc and S . clavuligerus oppA2::aph mutants are more resistant to the toxic tripeptide phosphinothricyl-alanyl-alanine (also named bialaphos) than the wild-type strain, suggesting that this peptide might be transported by these peptide-binding proteins.

Acc Chem Res, 2004 May, 37(5), 297 - 303
Beta-sultams-mechanism of reactions and use as inhibitors of serine proteases; Page MI; beta-Sultams are reactive sulfonyl analogues of beta-lactams and show enormous rate enhancements over analogous reactions of sulfonamides . N-Acyl beta-sultams undergo S-N rather than C-N fission, although alpha-alkenyl substituents direct nucleophilic attack to the acyl center . They also inactivate serine enzymes such as elastase and beta-lactamase by sulfonylation of the active site serine . Structure-activity relationships are used to identify differences in transition state structures.

J Biomol Screen, 2004 Apr, 9(3), 186 - 95
Miniaturization of whole live cell-based GPCR assays using microdispensing and detection systems; Kornienko O et al.; Cell-based beta-lactamase reporter gene assays designed to measure the functional responses of G-protein-coupled receptors (GPCRs) were miniaturized to less than 2 microL total assay volume in a 3456-well microplate . Studies were done to evaluate both receptor agonists and antagonists . The pharmacology of agonists and antagonists for target GPCRs originally developed in a 96-well format was recapitulated in a 3456-well microplate format without compromising data quality or EC(50)/IC(50) precision . These assays were employed in high-throughput screening campaigns, allowing the testing of more than 150,000 compounds in 8 h . The instrumentation used and practical aspects of the assay development are discussed.

J Clin Microbiol, 2004 May, 42(5), 2203 - 6
Use of beta-lactamase inhibitors in disk tests to detect plasmid-mediated AmpC beta-lactamases; Black JA et al.; Seeking a simple disk test for detection of organisms producing plasmid-mediated AmpC beta-lactamases, we evaluated the diagnostic utility of the beta-lactamase inhibitors 48-1220 (Ro 48-1220) and LN-2-128 . Using NCCLS disk methodology, inhibition zone diameters were determined for five beta-lactam antibiotics tested alone and in combination with 20 microg of either 48-1220 or LN-2-128 . Using an increase of > or =4 mm in zone diameter in the presence of an inhibitor as a positive test, cefotetan with LN-2-128 and 48-1220 was adequate for the detection of organisms producing plasmid-mediated AmpCs (specificity of 90% and sensitivity of 100%).

Nucleic Acids Res, 2004 Apr 28, 32(8), 2336 - 41 Print 2004.
A novel replicating circular DNAzyme; Chen F et al.; 10-23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage . However, the dependence on exogenous delivery limits its applications . The objective of this work is to establish a replicating DNAzyme in bacteria using a single-stranded DNA vector . By cloning the 10-23 DNAzyme into the M13mp18 vector, we constructed two circular DNAzymes, C-Dz7 and C-Dz482, targeting the beta-lactamase mRNA . These circular DNAzymes showed in vitro catalytic efficiencies (kcat/K(M)) of 7.82 x 10(6) and 1.36 x 10(7) M(-1) x min(-1), respectively . Their dependence on divalent metal ions is similar to that found with linear 10-23 DNAzyme . Importantly, the circular DNAzymes were not only capable of replicating in bacteria but also exhibited high activities in inhibiting beta-lactamase and bacterial growth . This study thus provides a novel strategy to produce replicating DNAzymes which may find widespread applications.

Folia Microbiol (Praha), 2004, 49(1), 71 - 4
Double-disk synergy test positivity in Stenotrophomonas maltophilia clinical strains; Hejnar P et al.; The double-disk synergy test (DDST) using Mueller-Hinton agar and antibiotic disks with centrally positioned disks of amoxicillin-clavulanate, ampicillin-sulbactam, and piperacillin-tazobactam and, at a center-to-center distance of 25-30 mm, 2-4 disks with 10 various beta-lactam antibiotics per one plate was performed in 58 clinical isolates of Stenotrophomonas maltophilia to determine the effectivity of 3 beta-lactamase inhibitors . When tested with clavulanate as the central beta-lactamase inhibitor synergic action on tested strains was the most frequent with aztreonam (81.0% of strains), cefoperazone (63.8%), and cefepime (60.3%) . With sulbactam the synergic action, i.e . DDST positivity, was high in the case of cefoperazone (15.5%), ampicillin, aztreonam and piperacillin (8.6% each); with tazobactam it was the most frequent with aztreonam (53.4%), cefoperazone (44.8%) and cefepime (37.9%) . No synergy was demonstrated after application of meropenem regardless of the kind of beta-lactamase inhibitor used . In 58 strains of S . maltophilia, 55 different profiles of DDST positivity were found . The results confirm that clavulanate is the most effective inhibitor of S . maltophilia beta-lactamases . The utilization of DDST (performed in the recommended way) for the typization of strains Stenotrophomonas species and for the estimation of potential effectiveness combinations of beta-lactams with beta-lactamase inhibitors for the therapy of stenotrophomonade infections was suggested.

Antimicrob Agents Chemother, 2004 May, 48(5), 1670 - 5
Genetic and biochemical characterization of a chromosome-encoded carbapenem-hydrolyzing ambler class D beta-lactamase from Shewanella algae; Heritier C et al.; A chromosome-encoded beta-lactamase gene from Shewanella algae clinical isolate KB-1 was cloned and expressed in Escherichia coli . It encoded the Ambler class D enzyme OXA-55, sharing less than 55% identity with any other oxacillinases . Although conferring a narrow-spectrum beta-lactam resistance phenotype, OXA-55 had carbapenem-hydrolyzing activity that mirrored the reduced susceptibility to imipenem observed in S . algae KB-1 . Very similar oxacillinases were found in other S . algae isolates.

Antimicrob Agents Chemother, 2004 May, 48(5), 1454 - 60
Role of a mutation at position 167 of CTX-M-19 in ceftazidime hydrolysis; Kimura S et al.; CTX-M-19 is a recently identified ceftazidime-hydrolyzing extended-spectrum beta-lactamase, which differs from the majority of CTX-M-type beta-lactamases that preferentially hydrolyze cefotaxime but not ceftazidime . To elucidate the mechanism of ceftazidime hydrolysis by CTX-M-19, the beta-lactam MICs of a CTX-M-19 producer, and the kinetic parameters of the enzyme were confirmed . We reconfirmed here that CTX-M-19 is also stable at a high enzyme concentration in the presence of bovine serum albumin (20 micro g/ml) . Under this condition, we obtained more accurate kinetic parameters and determined that cefotaxime (k(cat)/K(m), 1.47 x 10(6) s(-1) M(-1)), cefoxitin (k(cat)/K(m), 62.2 s(-1) M(-1)), and aztreonam (k(cat)/K(m), 1.34 x 10(3) s(-1) M(-1)) are good substrates and that imipenem (k(+2)/K, 1.20 x 10(2) s(-1) M(-1)) is a poor substrate . However, CTX-M-18 and CTX-M-19 exhibited too high a K(m) value (2.7 to 5.6 mM) against ceftazidime to obtain their catalytic activity (k(cat)) . Comparison of the MICs with the catalytic efficiency (k(cat)/K(m)) of these enzymes showed that some beta-lactams, including cefotaxime, ceftazidime, and aztreonam showed a similar correlation . Using the previously reported crystal structure of the Toho-1 beta-lactamase, which belongs to the CTX-M-type beta-lactamase group, we have suggested characteristic interactions between the enzymes and the beta-lactams ceftazidime, cefotaxime, and aztreonam by molecular modeling . Aminothiazole-bearing beta-lactams require a displacement of the aminothiazole moiety due to a severe steric interaction with the hydroxyl group of Ser167 in CTX-M-19, and the displacement affects the interaction between Ser130 and the acidic group such as carboxylate and sulfonate of beta-lactams.

J Mol Biol, 2004 Feb 20, 336(3), 763 - 74
Effect of crowding on protein-protein association rates: fundamental differences between low and high mass crowding agents; Kozer N et al.; Physiological media constitutes a crowded environment that serves as the field of action for protein-protein interaction in vivo . Measuring protein-protein interaction in crowded solutions can mimic this environment . In this work we follow the process of protein-protein association and its rate constants (k(on)) of the beta-lactamase (TEM)-beta-lactamase inhibitor protein (BLIP) complex in crowded solution using both low and high molecular mass crowding agents . In all crowded solutions (0-40% (w/w) of ethylene glycol (EG), poly(ethylene glycol) (PEG) 200, 1000, 3350, 8000 Da Ficoll-70 and Haemaccel the measured absolute k(on), but not k(off) values, were found to be slower as compared to buffer . However, there is a fundamental difference between low and high mass crowding agents . In the presence of low mass crowding agents and Haemaccel k(on) depends inversely on the solution viscosity . In high mass polymer solutions k(on) changes only slightly, even at viscosities 12-fold higher than water . The border between low and high molecular mass polymers is sharp and is dictated by the ratio between the polymer length (L) and its persistence length (Lp) . Polymers that are long enough to form a flexible coil (L/Lp > 2) behave as high molecular mass polymers and those who are unable to do so (L/Lp < 2) behave as low molecular mass polymers . We concluded that although polymers solution are crowded, this property is not uniform; i.e . there are areas in the solution that contain bulk water, and in these areas proteins can diffuse and associate almost as if they were in diluted environment . This porous medium may be taken as mimicking some aspects of the cellular environment, where many of the macromolecules are organized along membranes and the cytoskeleton . To determine the contribution of electrostatic attraction between proteins in crowded milieu, we followed k(on) of wt-TEM and three BLIP analogs with up to 100-fold increased values of k(on) due to electrostatic steering . Faster associating BLIP variants keep their relative advantage in all crowded solutions, including Haemaccel . This result suggests that faster associating protein complexes keep their advantage also in complex environment.

Assay Drug Dev Technol, 2003 Dec, 1(6), 789 - 800
A beta-lactamase-dependent Gal4-estrogen receptor beta transactivation assay for the ultra-high throughput screening of estrogen receptor beta agonists in a 3456-well format; Peekhaus NT et al.; Estrogen action is mediated via two estrogen receptor (ER) subtypes, ERalpha and ERbeta . Selective ER modulators with balanced high affinity for ERalpha and ERbeta have been developed as therapeutics for the treatment of a variety of diseases, including hormone-responsive breast cancer and osteoporosis . Recent data based primarily on the evaluation of ER-knockout mice have revealed that ERalpha and ERbeta may regulate separate and distinct biological processes . The identification of ERbeta specific ligands could further enhance our understanding of ERbeta biology . In addition, compounds targeting ERbeta may prove useful as therapeutic agents with activity profiles distinguishable from that of estradiol . To discover novel selective ligands for ERbeta, we developed and characterized a cell-based Gal4-ERbeta beta-lactamase reporter gene assay (GERTA) in CHO cells for the ligand-induced activation of the human ERbeta . The sensitivity and selectivity of this assay were found to be comparable to those of an ER ligand-binding assay . The assay was optimized for screening in an ultra high throughput 3456-well nanoplate format and was successfully used to screen a large compound collection for ERbeta agonists . Compounds identified in a primary screen were tested in an in vitro ligand-binding assay to characterize further the selectivity and potency for ERbeta.

Assay Drug Dev Technol, 2003 Dec, 1(6), 777 - 87
Miniaturization of cell-based beta-lactamase-dependent FRET assays to ultra-high throughput formats to identify agonists of human liver X receptors; Chin J et al.; Activation of liver X receptors (LXRs) induces reverse cholesterol transport and increases high-density lipoprotein cholesterol in vivo . Here, we describe novel, functional, homogeneous cell-based fluorescence resonance energy transfer assays for identifying agonists of LXRs using beta-lactamase as the reporter gene . Stable Chinese hamster ovary cell lines expressing LXRalpha-GAL4 or LXRbeta-GAL4 fusion proteins that regulate beta-lactamase transcription from upstream 7 x UAS GAL4 DNA binding sequences were generated and characterized . Synthetic and natural ligands of LXR dose-dependently activated the expression of beta-lactamase in a subtype-specific manner . These assays were used to demonstrate that a 1-pyridyl hydantoin small molecule LXR synthetic ligand specifically activates LXRalpha receptors . The beta-lactamase assays were optimized for cell density, dimethyl sulfoxide sensitivity, and time of agonist stimulation . Clonal LXRbeta-GAL4-beta-lactamase cells were miniaturized into an ultra high throughput (3456-well nanoplates) screening format.

Assay Drug Dev Technol, 2003 Dec, 1(6), 767 - 76
A one-arm homologous recombination approach for developing nuclear receptor assays in somatic cells; Qureshi SA et al.; Drug discovery is in need of technologies that enable investigators to develop cell-based assays that accurately reflect the functional consequence of small molecule intervention on biological processes . Here, we describe a strategy that uses both one-arm homologous recombination and the beta-lactamase (BLA) reporter system, a sensitive and robust transcriptional reporter for gene activation . We demonstrate that this powerful approach can be utilized for developing cell-based assays for the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) in HEK293 somatic cells . Specifically, one-arm homologous recombination was used to introduce the GAL4 DNA-binding domain (GAL4DBD) in the GR and MR genomic loci such that a chimeric GAL4DBD-GR (ligand-binding domain) {GAL4DBD-GR(LBD)} and GAL4DBD-MR(LBD) transcript is produced from the strong CMV promoter in HEK293 cells previously stably transfected with the UAS(GAL4)-BLA reporter construct . Dexamethasone- and aldosterone-responding BLA-positive cells were isolated by fluorescence-activated cell sorting, and then further expanded into separate cell lines . The sensitivity and robustness of the resulting GR and MR assays are demonstrated by the fact that the addition of dexamethasone and aldosterone to the two transgenic clonal cell lines for 16 h results in high Z' values (>0.8) and EC(50) values of 1 and 0.3 nM, respectively . These assays illustrate the flexibility of this technology to generate high-performance cellular assays for nuclear receptor targets without the need for target-specific cDNA.

Methods Mol Biol, 2004, 261, 411 - 26
Mapping biochemical networks with protein-fragment complementation assays; Remy I et al.; Cellular biochemical machineries, what we call pathways, consist of dynamically assembling and disassembling macromolecular complexes . Although our models for the organization of biochemical machines are derived largely from in vitro experiments, do they reflect their organization in intact, living cells? We have developed a general experimental strategy that addresses this question by allowing the quantitative probing of molecular interactions in intact, living cells . The experimental strategy is based on protein-fragment complementation assays (PCA), a method whereby protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction . A biochemical machine or pathway is defined by grouping interacting proteins into those that are perturbed in the same way by common factors (hormones, metabolites, enzyme inhibitors, and so on) . In this chapter we review some of the essential principles of PCA and provide details and protocols for applications of PCA, particularly in mammalian cells, based on three PCA reporters, dihydrofolate reductase, green fluorescent protein, and beta-lactamase.

J Mol Biol, 2004 Mar 5, 336(5), 1283 - 91
Allosteric inhibition through core disruption; Horn JR et al.; Although inhibitors typically bind pre-formed sites on proteins, it is theoretically possible to inhibit by disrupting the folded structure of a protein or, in the limit, to bind preferentially to the unfolded state . Equilibria defining how such molecules act are well understood, but structural models for such binding are unknown . Two novel inhibitors of beta-lactamase were found to destabilize the enzyme at high temperatures, but at lower temperatures showed no preference for destabilized mutant enzymes versus stabilized mutants . X-ray crystal structures showed that both inhibitors bound to a cryptic site in beta-lactamase, which the inhibitors themselves created by forcing apart helixes 11 and 12 . This opened up a portion of the hydrophobic core of the protein, into which these two inhibitors bind . Although this binding site is 16 A from the center of the active site, the conformational changes were transmitted through a sequence of linked motions to a key catalytic residue, Arg244, which in the complex adopts conformations very different from those in catalytically competent enzyme conformations . These structures offer a detailed view of what has heretofore been a theoretical construct, and suggest the possibility for further design against this novel site.

Biochemistry, 2004 Mar 30, 43(12), 3570 - 81
Correlation between catalytic efficiency and the transcription read-out in chemical complementation: a general assay for enzyme catalysis; Sengupta D et al.; High-throughput assays for enzyme catalysis that can be applied to a broad range of chemical reactions are key to advances in directed evolution and proteomics . Recently, we reported such a general assay, chemical complementation, which links enzyme catalysis to reporter gene transcription in vivo using the yeast three-hybrid assay . In this proof-of-principle experiment, it was shown that a wild-type beta-lactamase enzyme could be isolated from a pool of inactive mutants using a lacZ screen . Ideally, however, such an assay should be able to distinguish enzymes based on their catalytic activity . Thus, here, we set out to determine if the catalytic efficiency of an enzyme variant does in fact correlate with its level of transcription activation in the chemical complementation assay . First, the reaction mechanism for the cleavage of the beta-lactam substrate used in the chemical complementation proof-of-principle experiment was determined . Then a series of beta-lactamase variants was designed to span several orders of magnitude in k(cat)/K(m) . The activity of each variant was determined both in vitro using purified enzyme and in vivo in the chemical complementation transcription assay . Beta-lactamase variants spanning three-orders of magnitude in k(cat)/K(m) could be distinguished in the assay, and the catalytic efficiency of each variant correlated with its level of transcription activation in vivo . These results establish the suitability of chemical complementation for the directed evolution of enzymes with improvements in catalytic activity and for profiling the relative substrate specificities of a group of enzymes in proteomics applications.

Oligonucleotides, 2003, 13(6), 427 - 33
The translation start codon region is sensitive to antisense PNA inhibition in Escherichia coli; Dryselius R et al.; Antisense peptide nucleic acids (PNA) can inhibit bacterial gene expression with gene and sequence specificity . Using attached carrier peptides that aid cell permeation, the antisense effects when targeting essential genes are sufficient to prevent growth and even kill bacteria . However, many design uncertainties remain, including the difficult question of target sequence selection . In this study, we synthesized 90 antisense peptide-PNAs to target sequences in a head to tail manner across the entire length of the mRNA encoding beta-lactamase . The results from this scan pointed to the start codon region as most sensitive to inhibition . To confirm and refine the result, a higher-resolution scan was conducted over the start codon region of the beta-lactamase gene and the essential Escherichia coli acpP gene . For both genes, the start codon region, including the Shine-Dalgarno motif, was sensitive, whereas antisense agents targeted outside of this region were largely ineffective . These results are in accord with natural antisense mechanisms, which typically hinder the start codon region, and the sensitivity of this region should hold true for most bacterial genes as well as for other RNase H-independent antisense agents that rely on a steric blocking mechanism . Therefore, although other design parameters are also important, the start codon region in E . coli mRNA is the most reliable target site for antisense PNAs.

Clin Microbiol Infect, 2004 Mar, 10(3), 234 - 41
Mechanisms of reduced susceptibility to amoxycillin-clavulanic acid in Escherichia coli strains from the health region of Tortosa (Catalonia, Spain); Perez-Moreno MO et al.; This study investigated the mechanisms involved in reduced susceptibility to amoxycillin-clavulanic acid and the prevalence of enzymes compatible with inhibitor-resistant TEM (IRT) beta-lactamases produced by Escherichia coli isolates from patients in north-eastern Spain . The resistance mechanisms of 158 strains showing resistance or intermediate resistance to amoxycillin-clavulanic acid among 1122 ampicillin-resistant clinical isolates of E . coli were assessed on the basis of their beta-lactam resistance phenotypes . beta-Lactamases produced by strains showing resistant phenotypes suggestive of inhibitor-resistant penicillinase production were characterised by their isoelectric point . Specific activity and the concentration of clavulanic acid required to inhibit beta-lactamase activity by 50% (IC50) were determined in strains harbouring enzymes that focused at pI 5.2 or 5.4 in order to achieve presumptive identification of IRT beta-lactamases . Resistance phenotypes were consistent with overproduction of TEM-1, TEM-2 or SHV-1 beta-lactamases in 56 strains, with AmpC cephalosporinase hyperproduction in 46 strains, and with production of inhibitor-resistant penicillinases in 49 strains . Of the latter isolates, 17 produced moderately high or high levels of enzymes co-focusing with TEM-1, 17 produced enzymes co-focusing with OXA-1 (n = 12) or with PSE-1 (n = 5), either alone or in association with TEM-1, while only 15 produced enzymes with a phenotype characteristic of IRT beta-lactamases . It was concluded that resistance to amoxycillin-clavulanic acid in E . coli isolates from this area was mainly associated with presumptive overproduction of TEM-1, TEM-2 or SHV-1 beta-lactamases (46%) or of AmpC cephalosporinase (29%), while the occurrence of enzymes categorised as IRT beta-lactamases was unusual (9.5%).

J Infect Chemother, 2004 Feb, 10(1), 62 - 4
The combination of aztreonam and cefozopran against Stenotrophomonas maltophilia; Kataoka D et al.; Aztreonam is suited for combination chemotherapy because it could be a potent Beta-lactamase inhibitor . We designed a study to show the inhibitory activity of aztreonam, using Stenotrophomonas maltophilia, which produces both carbapenemase L1 and penicillinase L2 . Aztreonam showed considerable synergy with cefpirome and contributed to a decrease in minimum inhibitory concentrations of cefozopran . In further examinations, the mean viable bacterial counts in cultures treated with aztreonam-cefozopran were 1 log lower than those in cultures treated with cefozopran alone . These results confirm that inhibition of penicillinase L2 occurred . We hope that a combination chemotherapy using aztreonam and cefozopran will be used to prevent the emergence of penicillinase-producers.

Protein Eng, 2003 Dec, 16(12), 1025 - 34
Using a residue clash map to functionally characterize protein recombination hybrids; Saraf MC et al.; In this article, we introduce a rapid, protein sequence database-driven approach to characterize all contacting residue pairs present in protein hybrids for inconsistency with protein family structural features . This approach is based on examining contacting residue pairs with different parental origins for different types of potentially unfavorable interactions (i.e . electrostatic repulsion, steric hindrance, cavity formation and hydrogen bond disruption) . The identified clashing residue pairs between members of a protein family are then contrasted against functionally characterized hybrid libraries . Comparisons for five different protein recombination studies available in the literature: (i) glycinamide ribonucleotide transformylase (GART) from Escherichia coli (purN) and human (hGART), (ii) human Mu class glutathione S-transferase (GST) M1-1 and M2-2, (iii) beta-lactamase TEM-1 and PSE-4, (iv) catechol-2,3-oxygenase xylE and nahH, and (v) dioxygenases (toluene dioxygenase, tetrachlorobenzene dioxygenase and biphenyl dioxygenase) reveal that the patterns of identified clashing residue pairs are remarkably consistent with experimentally found patterns of functional crossover profiles . Specifically, we show that the proposed residue clash maps are on average 5.0 times more effective than randomly generated clashes and 1.6 times more effective than residue contact maps at explaining the observed crossover distributions among functional members of hybrid libraries . This suggests that residue clash maps can provide quantitative guidelines for the placement of crossovers in the design of protein recombination experiments.

Antimicrob Agents Chemother, 2004 Mar, 48(3), 1032 - 3
In vitro evolution predicts that the IMP-1 metallo-beta-lactamase does not have the potential to evolve increased activity against imipenem; Hall BG; In vitro evolution was used to predict whether the IMP-1 metallo-beta-lactamase has the potential to evolve an increased ability to confer resistance to imipenem . Screening of eight libraries containing 9.8 x 10(6) +/- 1.4 x 10(6) (mean +/- standard error) variants per library, with an average of 1.2 mutations per variant, detected no increased resistance to imipenem . The results predict, with >99.9% confidence, that even under intense selection the IMP-1 beta-lactamase will not evolve to confer increased resistance to imipenem.

Methods Mol Biol, 2004, 263, 333 - 44
Fluorescence resonance energy transfer-based HIV-1 virion fusion assay; Cavrois M et al.; The fluorescence resonance energy transfer (FRET)-based HIV-1 virion fusion assay exploits the incorporation of beta-lactamase-Vpr chimeric proteins into HIV-1 virions and their subsequent delivery into the cytoplasm of target cells as a marker of fusion . This transfer can be monitored by the enzymatic cleavage of the CCF2-AM dye, a fluorescent substrate of beta-lactamase (BlaM), loaded into the target cells . BlaM cleavage of the beta-lactam ring in CCF2-AM prevents the FRET between the coumarin and fluorescein moieties of the dye . This cleavage changes the fluorescence emission spectrum of CCF2-AM from green (520 nm) to blue (447 nm), and thus permits detection of fusion by fluorescence microscopy, flow cytometry, or UV photometry . This assay is simple and rapid to perform, and exhibits high sensitivity and specificity . Importantly, it can be applied to study HIV-1 virion fusion in primary cells and can be combined with immunostaining for subset discrimination in heterogeneous target cell populations . Finally, the assay can also be adapted to study fusion mediated by the envelope proteins from other viruses through the construction of HIV-1 pseudotypes.

Biochemistry, 2004 Feb 17, 43(6), 1715 - 23
Folding of an abridged beta-lactamase; Santos J et al.; The effects of C-terminal truncation on the equilibrium folding transitions and folding kinetics of B . licheniformis exo small beta-lactamase (ES-betaL) have been measured . ES-betaL lacking 19 residues (ES-betaL(C)(Delta)(19)) has no enzymic activity . Deletion of the last 14 residues produces ES-betaL(C)(Delta)(14), which is 0.1% active . The enzyme lacking nine residues (ES-betaL(C)(Delta)(9)) is nearly fully active, has native optical and hydrodynamic properties, and is protease resistant, a distinguishing feature of the wild-type enzyme . Although ES-betaL(C)(Delta)(9) folds properly, it does so 4 orders of magnitude slower than ES-betaL, making possible the isolation and characterization of a compact intermediate state (I(P) ES-betaL(C)(Delta)(9)) . Based on the analysis of folding rates and equilibrium constants, we propose that equilibrium between I(P) ES-betaL(C)(Delta)(9) and other intermediate slow folding . Residues removed in ES-betaL(C)(Delta)(9) and ES-betaL(C)(Delta)(14) are helical and firmly integrated into the enzyme body through many van der Waals interactions involving residues distant in sequence . The results suggest that the deleted residues play a key role in the folding process and also the existence of a modular organization of the protein matrix, at the subdomain level . The results are compared with other examples of this kind in the folding literature.

J Biol Chem, 2004 May 7, 279(19), 19494 - 501 Epub 2004 Feb 02.
Tazobactam inactivation of SHV-1 and the inhibitor-resistant Ser130 -->Gly SHV-1 beta-lactamase: insights into the mechanism of inhibition; Pagan-Rodriguez D et al.; The increasing number of bacteria resistant to combinations of beta-lactam and beta-lactamase inhibitors is creating great difficulties in the treatment of serious hospital-acquired infections . Understanding the mechanisms and structural basis for the inactivation of these inhibitor-resistant beta-lactamases provides a rationale for the design of novel compounds . In the present work, SHV-1 and the Ser(130) --> Gly inhibitor-resistant variant of SHV-1 beta-lactamase were inactivated with tazobactam, a potent class A beta-lactamase inhibitor . Apoenzymes and inhibited beta-lactamases were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), digested with trypsin, and the products resolved using LC-ESI/MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry . The mass increases observed for SHV-1 and Ser(130) --> Gly (+ Delta 88 Da and + Delta 70 Da, respectively) suggest that fragmentation of tazobactam readily occurs in the inhibitor-resistant variant to yield an inactive beta-lactamase . These two mass increments are consistent with the formation of an aldehyde (+ Delta 70 Da) and a hydrated aldehyde (+ Delta 88 Da) as stable products of inhibition . Our results reveal that the Ser --> Gly substitution at amino acid position 130 is not essential for enzyme inactivation . By examining the inhibitor-resistant Ser(130) --> Gly beta-lactamase, our data are the first to show that tazobactam undergoes fragmentation while still attached to the active site Ser(70) in this enzyme . After acylation of tazobactam by Ser(130) --> Gly, inactivation proceeds independent of any additional covalent interactions.

J Mol Biol, 2004 Feb 6, 336(1), 263 - 73
Creation of an allosteric enzyme by domain insertion; Guntas G et al.; Two allosteric enzymes have been created by the covalent linkage of non-interacting, monomeric proteins with the prerequisite effector-binding and catalytic functionalities, respectively . This was achieved through a combinatorial process called random domain insertion . The fragment of the TEM-1 beta-lactamase gene coding for the mature protein lacking its signal sequence was randomly inserted into the Escherichia coli maltose-binding protein (MBP) gene to create a domain insertion library . This library's diversity derived both from the site of insertion and from a distribution of tandem duplications or deletions of a portion of the MBP gene at the insertion site . From a library of approximately 2 x 10(4) in-frame fusions, approximately 800 library members conferred a phenotype to E.coli cells that was consistent with the presence of bifunctional fusions that could hydrolyze ampicillin and transport maltose in E.coli . Partial screening of this bifunctional sublibrary resulted in the identification of two enzymes in which the presence of maltose modulated the rate of nitrocefin hydrolysis . For one of these enzymes, the presence of maltose increased k(cat) by 70% and k(cat)/K(m) by 80% and resulted in kinetic parameters that were almost identical to TEM-1 beta-lactamase . Such an increase in activity was only observed with maltooligosaccharides whose binding to MBP is known to induce a conformational change . Modulation of the rate of nitrocefin hydrolysis could be detected at maltose concentrations less than 1 microM . Intrinsic protein fluorescence studies were consistent with a conformational change being responsible for the modulation of activity.

Vaccine, 2004 Jan 26, 22(5-6), 740 - 6
Adjuvant activity of Mycobacterium bovis BCG expressing CRM197 on the immune response induced by BCG expressing tetanus toxin fragment C; Mazzantini RP et al.; In order to develop a combined recombinant Mycobacterium bovis BCG (rBCG) vaccine against diphtheria, pertussis and tetanus (DPT), we have constructed different strains of rBCG expressing tetanus toxin fragment C (FC), driven by the up-regulated M . fortuitum beta-lactamase promoter, pBlaF* . Tetanus toxin FC was expressed in comparable levels in native form or in fusion with the beta-lactamase exportation signal sequence; however, in both constructs it was localized to the cytosol . Immunization of mice with rBCG-FC or its combination with rBCG expressing CRM197, induced anti-tetanus toxin antibodies with a Th2 immunoglobulin profile . Administration of a subimmunizing dose of the diphtheria-tetanus toxoid vaccine showed that rBCG-FC primed mice for production of an intense humoral response . Interestingly, the combination of rBCG-FC and rBCG-CRM197 reduced the time required for maturation of the immune response and increased anti-tetanus toxin antibody levels, suggesting adjuvant properties for rBCG-CRM197; this combination induced 75% protection in mice challenged with 100 minimum lethal doses (MLD) of tetanus toxin . Antisera from guinea pigs immunized with this combination were shown to neutralize tetanus toxin and diphtheria toxin . Our results suggest reciprocal adjuvant effects of rBCG-FC and rBCG-CRM197, which may contribute to induction of a more effective immune response against both diseases.

Appl Environ Microbiol, 2004 Jan, 70(1), 494 - 7
Improved method for polynucleotide probe-based cell sorting, using DNA-coated microplates; Zwirglmaier K et al.; We developed an improved method for cultivation-independent sorting of bacterial cells . The technique is based on labeling the target cells by in situ hybridization with polynucleotide transcript probes . Due to the probes' length, part of the probe remains outside the cell and can subsequently be used to capture the cells . Target cells are immobilized during a second hybridization step in microplates that are coated with DNA that is complementary to the probe sequence . The method was applied successfully to artificial mixtures of cells with polynucleotide probes targeting either rRNA, a plasmid-borne beta-lactamase gene, or a chromosome-borne glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene . Cells could be separated based on phylogenetic parameters (using rRNA-targeted probes) as well as on other DNA-encoded traits.

Chem Commun (Camb), 2003 Dec 21, (24), 2998 - 9
Colloidal stable silica encapsulated nano-magnetic composite as a novel bio-catalyst carrier; Gao X et al.; A colloidal stable silica-encapsulated magnetic nano-composite of a controlled dimension is, for the first time, employed to carry beta-lactamase via chemical linkage on the silica overlayer: activity study reflects that this new type of immobilisation allows site (enzyme) isolation, accessibility as good as free enzyme and recovery & reusability upon application of magnetic separation.

Yi Chuan Xue Bao, 2003 Aug, 30(8), 730 - 6
{Construction and verification of the effectiveness of pMBL: a cloning vector of exported proteins encoding genes}; Yu XP et al.; The beta-lactamase was used as the reporter of expression and transmembrane secretion in this paper . A fragment of Amp resistance gene encoding the mature part of beta-lactamase (delta P delta SP Amp, i.e . without promoter and signal peptide coding sequences) was amplified from pUC18 vector . The upstream primer has BglII, BclI, BamHI in three reading frame respectively, in order to in frame fuse and express target genes together with the downstream reporter in finally constructed vector . Meanwhile, pET-28 was digested with the restriction enzymes BglII and Bst1107 I . The 2.8 kb fragment with replication origin, Kan resistance gene and MCS was recovered, filled, self-ligated and resulted in a plasmid pKan-B . The Bgl II site on pKan-B was then filled and the plasmid pKan was obtained . The delta P delta SP Amp gene, which was first cloned into pGEM-T-EASY vector, was inserted into pKan between EcoR I and XbaI sites . A plasmid pMBL-E was selected, with which the bacteria host could grew on Kan plate but not on plate with both Amp and Kan . An EcoRI site beside HindIII on the plasmid pMBL-E was then filled, and the plasmid pMBL, a cloning vector of the exported proteins encoding genes was finally obtained . Both results of the restriction enzyme digestion and sequencing demonstrated the correctness of the construction . The Tet resistance gene, a transmemebrane protein encoding gene, was applied to verify the effectiveness of the reporter in the vector . Cut with EcoRI and BamHI, a 375 bp fragment including promoter and 96 animo acids coding sequence (including signal peptide) of Tet was obtained from pBR322 vector . The fragment was then ligated to the vector pMBL which had been cut with both enzymes of EcoRI and BglI, or EcoRI and BclI, or EcoRI and BamHI (as 0, +1, +2 respectively of the beta-lactamase gene reading frame) . Kan and Amp double resistant colonies only grew with the EcoRI and BglII combination (0 position) . Restriction enzyme digestion and sequencing results of the recombinant plasmid showed that Tet resistance gene, which promoted the expression and transmembrane secretion of downstream beta-lactamase, was inserted in a correct reading frame into the vector . Thus, the results verified the effectiveness of the constructed vector pMBL, which may be used effectively to clone genes encoding exported proteins with promoters and signal peptide sequences.

Curr Opin Struct Biol, 2003 Dec, 13(6), 709 - 15
Sub-Angstrom resolution enzyme X-ray structures: is seeing believing?
Vrielink A, Sampson N.
Recent technical advances in crystallographic analysis, particularly highly focused and high brilliance synchrotron beam lines, have significantly improved the resolutions that are attainable for many macromolecular crystal structures . The Protein Data Bank contains an increasing number of atomic resolution structures, which are providing a wealth of structural information that was not previously visible in lower resolution electron density maps . Here, we review the importance of visualizing hydrogen atoms and multiple sidechain conformations or anisotropy, as well as substrate strain, at sub-Angstrom resolution . The additional structural features that are visible in the electron density maps as a result of atomic resolution data provide a better understanding of the catalytic mechanisms of cholesterol oxidase, ribonuclease A, beta-lactamase, serine proteases, triosephosphate isomerase and endoglucanase.

Biochemistry, 2003 Dec 16, 42(49), 14483 - 91
Thermodynamic cycle analysis and inhibitor design against beta-lactamase; Roth TA et al.; Beta-lactamases are the most widespread resistance mechanism to beta-lactam antibiotics, such as the penicillins and cephalosporins . Transition-state analogues that bind to the enzymes with nanomolar affinities have been introduced in an effort to reverse the resistance conferred by these enzymes . To understand the origins of this affinity, and to guide design of future inhibitors, double-mutant thermodynamic cycle experiments were undertaken . An unexpected hydrogen bond between the nonconserved Asn289 and a key inhibitor carboxylate was observed in the X-ray crystal structure of a 1 nM inhibitor (compound 1) in complex with AmpC beta-lactamase . To investigate the energy of this hydrogen bond, the mutant enzyme N289A was made, as was an analogue of 1 that lacked the carboxylate (compound 2) . The differential affinity of the four different protein and analogue complexes indicates that the carboxylate-amide hydrogen bond contributes 1.7 kcal/mol to overall binding affinity . Synthesis of an analogue of 1 where the carboxylate was replaced with an aldehyde led to an inhibitor that lost all this hydrogen bond energy, consistent with the importance of the ionic nature of this hydrogen bond . To investigate the structural bases of these energies, X-ray crystal structures of N289A/1 and N289A/2 were determined to 1.49 and 1.39 A, respectively . These structures suggest that no significant rearrangement occurs in the mutant versus the wild-type complexes with both compounds . The mutant enzymes L119A and L293A were made to investigate the interaction between a phenyl ring in 1 and these residues . Whereas deletion of the phenyl itself diminishes affinity by 5-fold, the double-mutant cycles suggest that this energy does not come through interaction with the leucines, despite the close contact in the structure . The energies of these interactions provide key information for the design of improved inhibitors against beta-lactamases . The high magnitude of the ion-dipole interaction between Asn289 and the carboxylate of 1 is consistent with the idea that ionic interactions can provide significant net affinity in inhibitor complexes.

J Biol Chem, 2004 Feb 20, 279(8), 6650 - 7 Epub 2003 Dec 05.
Identification of an additional interaction domain in transmembrane domains 11 and 12 that supports oligomer formation in the human serotonin transporter; Just H et al.; Na+/Cl--dependent neurotransmitter transporters form constitutive oligomers . The topological arrangement is not known, but a leucine heptad repeat in transmembrane domain (TM) 2 and a glycophorin-like motif in TM6 have been proposed to stabilize the oligomer . To determine the topology, we generated versions of the human serotonin transporter (hSERT) that carried cyan or yellow fluorescent proteins at their amino and/or carboxyl terminus . Appropriate pairs were coexpressed to measure fluorescence resonance energy transfer (FRET) . Donor photobleaching FRET microscopy was employed to deduce the following arrangement: within the monomer, the amino and carboxyl termini are in close vicinity . In addition, in the oligomer, the carboxyl termini are closer to each other than the amino termini . Hence, a separate interaction domain (i.e . distinct from TM2 and TM6) must reside in the carboxyl-terminal half of hSERT . This was confirmed by expressing the amino- and carboxyl-terminal halves of hSERT . These were retained intracellularly; they also retained the coexpressed full-length transporter by forming export-deficient oligomers and, when cotransfected in all possible combinations, supported FRET . Hence, both the carboxyl and amino termini contain elements that drive oligomerization . By employing fragments comprising two neighboring TM helices, we unequivocally identified TM11/12 as a new contact site by donor photobleaching FRET and beta-lactamase protein fragment complementation assay . TM1/2 was also found to self-associate . Thus, oligomerization of hSERT involves at least two discontinuous interfaces . The currently identified interaction sites drive homophilic interactions . This is consistent with assembly of SERT oligomers in an array-like structure containing multimers of dimers.

FEMS Microbiol Lett, 2003 Nov 21, 228(2), 187 - 91
Comparison of two RT-PCR methods for quantifying ampC specific transcripts in Escherichia coli strains; Corvec S et al.; In Escherichia coli, beta-lactam resistance usually depends on beta-lactamase production . AmpC chromosomal cephalosporinase hyperproduction is generally due to mutations in the ampC gene promoter . In order to study ampC expression in E . coli clinical strains, we have compared two methods: conventional and real-time reverse transcription-polymerase chain reaction (RT-PCR) . With both methods, ampC mRNA was found to be greatly increased in strains presenting -42 or -32 mutations in the ampC promoter, and moderately increased when a -11 mutation was present in the Pribnow box . Real-time RT-PCR represents a powerful tool combining amplification, fluorescent detection and analysis.

J Biol Chem, 2004 Feb 13, 279(7), 5298 - 305 Epub 2003 Nov 21.
Molecular characterization of a phospholipase D generating anandamide and its congeners; Okamoto Y et al.; Anandamide (N-arachidonoylethanolamine) is known to be an endogenous ligand of cannabinoid and vanilloid receptors . Its congeners (collectively referred to as N-acylethanolamines) also show a variety of biological activities . These compounds are principally formed from their corresponding N-acyl-phosphatidylethanolamines by a phosphodiesterase of the phospholipase D-type in animal tissues . We purified the enzyme from rat heart, and by the use of the sequences of its internal peptides cloned its complementary DNAs from mouse, rat, and human . The deduced amino acid sequences were composed of 393-396 residues, and showed that the enzyme has no homology with the known phospholipase D enzymes but is classified as a member of the zinc metallohydrolase family of the beta-lactamase fold . As was overexpressed in COS-7 cells, the recombinant enzyme generated anandamide and other N-acylethanolamines from their corresponding N-acyl-phosphatidylethanolamines at comparable rates . In contrast, the enzyme was inactive with phosphatidylcholine and phosphatidylethanolamine . Assays of the enzyme activity and the messenger RNA and protein levels revealed its wide distribution in murine organs with higher contents in the brain, kidney, and testis . These results confirm that a specific phospholipase D is responsible for the generation of N-acylethanolamines including anandamide, strongly suggesting the physiological importance of lipid molecules of this class.

J Biol Chem, 2004 Feb 13, 279(7), 5685 - 92 Epub 2003 Nov 17.
Crystal structure and mechanistic implications of N2-(2-carboxyethyl)arginine synthase, the first enzyme in the clavulanic acid biosynthesis pathway; Caines ME et al.; The initial step in the biosynthesis of the clinically important beta-lactamase inhibitor clavulanic acid involves condensation of two primary metabolites, D-glyceraldehyde 3-phosphate and L-arginine, to give N2-(2-carboxyethyl)arginine, a beta-amino acid . This unusual N-C bond forming reaction is catalyzed by the thiamin diphosphate (ThP2)-dependent enzyme N2-(2-carboxyethyl)arginine synthase . Here we report the crystal structure of N2-(2-carboxyethyl)arginine synthase, complexed with ThP2 and Mg2+, to 2.35-A resolution . The structure was solved in two space groups, P2(1)2(1)2(1) and P2(1)2(1)2 . In both, the enzyme is observed in a tetrameric form, composed of a dimer of two more tightly associated dimers, consistent with both mass spectrometric and gel filtration chromatography studies . Both ThP2 and Mg2+ cofactors are present at the active site, with ThP2 in a "V" conformation as in related enzymes . A sulfate anion is observed in the active site of the enzyme in a location proposed as a binding site for the phosphate group of the d-glyceraldehyde 3-phosphate substrate . The mechanistic implications of the active site arrangement are discussed, including the potential role of the aminopyrimidine ring of the ThP2 . The structure will form a basis for future mechanistic and structural studies, as well as engineering aimed at production of alternative beta-amino acids.

Biochemistry, 2003 Nov 25, 42(46), 13386 - 92
Following the reactions of mechanism-based inhibitors with beta-lactamase by Raman crystallography; Helfand MS et al.; The reactions between three clinically relevant inhibitors, tazobactam, sulbactam, and clavulanic acid, and SHV beta-lactamase (EC 3.5.2.6) have been followed in single crystals using a Raman microscope . The data are far superior to those obtained for the enzyme in aqueous solution and allow us to identify species on the reaction pathway and to measure the rates of the accumulation and decay of these species . A key intermediate on the reaction pathway is an acyl enzyme formed between Ser70 and the lactam ring's C=O group . By using the E166A deacylation deficient variant of the enzyme, we were able to focus on the process of acyl enzyme formation . The Raman data show that all three inhibitors form an enamine-type acyl enzyme reaching maximal populations at 10, 22, and 29 min for sulbactam, clavulanic acid, and tazobactam, respectively . The enamine intermediate exhibits a characteristic and relatively intense band near 1595 cm(-1) due to a stretching motion of the O=C-C=C-NH moiety that shifts to lower frequency upon NH <--> ND exchange . This feature was used to follow the kinetics of enamine buildup and decay in the crystal . Quantum mechanical calculations support the assignment of the 1595 cm(-1) band, as well as several other bands, to a trans-enamine species . The Raman data also demonstrate that the lactam ring opens prior to enamine formation since the lactam ring carbonyl (C=O) peak disappears prior to the appearance of the enamine 1595 cm(-1) band . Tazobactam appears to form approximately twice as much enamine intermediate as sulbactam and clavulanic acid, which correlates with its superior performance in the clinic, a finding that may bear on future drug design.

Biochemistry, 2003 Nov 18, 42(45), 13152 - 9
Inhibition of class A and class C beta-lactamases by penems: crystallographic structures of a novel 1,4-thiazepine intermediate; Nukaga M et al.; A new beta-lactamase inhibitor, a methylidene penem having a 5,6-dihydro-8H-imidazo{2,1-c}{1,4}oxazine heterocyclic substituent at the C6 position with a Z configuration, irreversibly inhibits both class A and class C serine beta-lactamases with IC(50) values of 0.4 and 9.0 nM for TEM-1 and SHV-1 (class A), respectively, and 4.8 nM in AmpC (class C) beta-lactamases . The compound also inhibits irreversibly the class C extended-spectrum GC1 beta-lactamase (IC(50) = 6.2 nM) . High-resolution crystallographic structures of a reaction intermediate of (5R)-(6Z)-6-(5,6-dihydro-8H-imidazo{2,1-c}{1,4}oxazin-2-ylmethylene)-7-oxo-4-thia-1-azabicyclo{3.2.0}hept-2-ene-3-carboxylic acid 1 with the SHV-1 beta-lactamase and with the GC1 beta-lactamase have been determined by X-ray diffraction to resolutions of 1.10 and 1.38 A, respectively . The two complexes were refined to crystallographic R-factors (R(free)) of 0.141 (0.186) and 0.138 (0.202), respectively . Cryoquenching of the reaction of 1 with each beta-lactamase crystal produced a common, covalently bound intermediate . After acylation of the serine, a nucleophilic attack by the departing thiolate on the C6' atom yielded a novel seven-membered 1,4-thiazepine ring having R stereochemistry at the new C7 moiety . The orientation of this ring in each complex differs by a 180 degrees rotation about the bond to the acylated serine . The acyl ester bond is stabilized to hydrolysis through resonance stabilization with the dihydrothiazepine ring and by low occupancy or disorder of hydrolytic water molecules . In the class A complex, the buried water molecule on the alpha-face of the ester bond appears to be loosely bound or absent . In the class C complex, a water molecule on the beta-face is disordered and poorly activated for hydrolysis . Here, the acyl intermediate is unable to assist its own hydrolysis, as is thought to occur with many class C substrates.

J Clin Microbiol, 2003 Nov, 41(11), 5310 - 2
Presumed endocarditis caused by BRO beta-lactamase-producing Moraxella lacunata in an infant with Fallot's tetrad; Nagano N et al.; A case of presumed endocarditis caused by Moraxella lacunata in a 15-month-old male infant with Fallot's tetrad is described . This infection may have occurred as the result of transmission of this organism between the father and his son . This is the first report of BRO beta-lactamase-producing M . lacunata causing presumed endocarditis.

J Biol Chem, 2004 Jan 9, 279(2), 920 - 7 Epub 2003 Oct 22.
Metal binding Asp-120 in metallo-beta-lactamase L1 from Stenotrophomonas maltophilia plays a crucial role in catalysis; Garrity JD et al.; Metallo-beta-lactamase L1 from Stenotrophomonas maltophilia is a dinuclear Zn(II) enzyme that contains a metal-binding aspartic acid in a position to potentially play an important role in catalysis . The presence of this metal-binding aspartic acid appears to be common to most dinuclear, metal-containing, hydrolytic enzymes; particularly those with a beta-lactamase fold . In an effort to probe the catalytic and metal-binding role of Asp-120 in L1, three site-directed mutants (D120C, D120N, and D120S) were prepared and characterized using metal analyses, circular dichroism spectroscopy, and presteady-state and steady-state kinetics . The D120C, D120N, and D120S mutants were shown to bind 1.6 +/- 0.2, 1.8 +/- 0.2, and 1.1 +/- 0.2 mol of Zn(II) per monomer, respectively . The mutants exhibited 10- to 1000-fold drops in kcat values as compared with wild-type L1, and a general trend of activity, wild-type > D120N > D120C and D120S, was observed for all substrates tested . Solvent isotope and pH dependence studies indicate one or more protons in flight, with pKa values outside the range of pH 5-10 (except D120N), during a rate-limiting step for all the enzymes . These data demonstrate that Asp-120 is crucial for L1 to bind its full complement of Zn(II) and subsequently for proper substrate binding to the enzyme . This work also confirms that Asp-120 plays a significant role in catalysis, presumably via hydrogen bonding with water, assisting in formation of the bridging hydroxide/water, and a rate-limiting proton transfer in the hydrolysis reaction.

J Biol Chem, 2003 Dec 26, 278(52), 52724 - 9 Epub 2003 Oct 08.
Understanding resistance to beta-lactams and beta-lactamase inhibitors in the SHV beta-lactamase: lessons from the mutagenesis of SER-130; Helfand MS et al.; Bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations by single amino acid mutations in class A beta-lactamases threatens our most potent clinical antibiotics . In TEM-1 and SHV-1, the common class A beta-lactamases, alterations at Ser-130 confer resistance to inactivation by the beta-lactamase inhibitors, clavulanic acid, and tazobactam . By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 beta-lactamase that result in resistance to these inhibitors . Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G beta-lactamase expressed in Escherichia coli . Kinetic analysis of the S130G beta-lactamase demonstrated a significant elevation in apparent Km and a reduction in kcat/Km for ampicillin . Marked increases in the dissociation constant for the preacylation complex, KI, of clavulanic acid (SHV-1, 0.14 microm; S130G, 46.5 microm) and tazobactam (SHV-1, 0.07 microm; S130G, 4.2 microm) were observed . In contrast, the k(inact)s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam . Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar . Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for beta-lactams and beta-lactamase inhibitors . These results underscore the mechanistic versatility of class A beta-lactamases and have implications for the design of novel beta-lactamase inhibitors.

Chem Biol, 2003 Sep, 10(9), 787 - 97
The process of structure-based drug design; Anderson AC; The field of structure-based drug design is a rapidly growing area in which many successes have occurred in recent years . The explosion of genomic, proteomic, and structural information has provided hundreds of new targets and opportunities for future drug lead discovery . This review summarizes the process of structure-based drug design and includes, primarily, the choice of a target, the evaluation of a structure of that target, the pivotal questions to consider in choosing a method for drug lead discovery, and evaluation of the drug leads . Key principles in the field of structure-based drug design will be illustrated through a case study that explores drug design for AmpC beta-lactamase.

J Med Chem, 2003 Oct 9, 46(21), 4477 - 86
Identification and prediction of promiscuous aggregating inhibitors among known drugs; Seidler J et al.; Some small molecules, often hits from screening, form aggregates in solution that inhibit many enzymes . In contrast, drugs are thought to act specifically . To investigate this assumption, 50 unrelated drugs were tested for promiscuous inhibition via aggregation . Each drug was tested against three unrelated model enzymes: beta-lactamase, chymotrypsin, and malate dehydrogenase, none of which are considered targets of these drugs . To be judged promiscuous, the drugs had to inhibit all three enzymes, do so in a time-dependent manner, be sensitive to detergent and to enzyme concentration, and form particles detectable by light scattering . Of the 50 drugs tested, 43 were nonpromiscuous by these criteria . Surprisingly, four of the drugs showed promiscuous, aggregation-based inhibition at concentrations below 100 microM: clotrimazole, benzyl benzoate, nicardipine, and delavirdine . Three other drugs also behaved as aggregation-based inhibitors, but only at high concentrations (about 400 microM) . To investigate possible structure-activity relationships among promiscuous drugs, five analogues of the antifungal clotrimazole were studied . Three of these, miconazole, econazole, and sulconazole, were promiscuous but the other two, fluconazole and ketoconazole, were not . Using recursive partitioning, these experimental results were used to develop a model for predicting aggregate-based promiscuity . This model correctly classified 94% of 111 compounds-47 aggregators and 64 nonaggregators-that have been studied for this effect . To evaluate the model, it was used to predict the behavior of 75 drugs not previously investigated for aggregation . Several preliminary points emerge . Most drugs are not promiscuous, even at high concentrations . Nevertheless, at high enough concentrations (20-400 microM), some drugs can aggregate and act promiscuously, suggesting that aggregation may be common among small molecules at micromolar concentrations, at least in biochemical buffers.

Mol Genet Genomics, 2003 Dec, 270(4), 337 - 46 Epub 2003 Sep 19.
Site-directed mutagenesis of conserved inverted repeat sequences in the xylanase C promoter region from Streptomyces sp . EC3; Giannotta F et al.; Streptomyces sp . EC3, a strain which was originally isolated from cattle manure compost, was shown to possess a strong xylanolytic activity . One of the genes responsible for this activity, xlnC, encodes a secreted xylanase . In the native strain, as in the heterologous host S . lividans, expression of xlnC was detectable in the presence of xylan but not in the presence of glucose . Induction by xylan was shown to take place at the transcriptional level . The transcriptional start site of xlnC was mapped and likely -35 (5'-TTGACA-3') and -10 (5'-GAGAAC-3') motifs were identified . In order to localise putative conserved regulatory sequences, the promoter regions of xylanase-encoding genes from various Streptomyces species were aligned . This alignment revealed the existence of three sets of quite well conserved palindromic AT rich sequences called boxes 1, 2 and 3 . Box 3 (5'-CGAAA N TTTCG-3') is the farthest away from the promoter region (150-200 bp) . A shorter version of this palindrome (5'-GAAA NN TTTC-3') or (5'-CGAAA-3') constitutes box 1, which is located just upstream of the putative -35 promoter sequence . Box 2, located 5-7 bp upstream of box 1, comprises a shorter palindrome than box 3, with inverted polarity {5'-(G/C)TTTC (N) GAAA(G/C)-3'} . The putative regulatory role of the conserved inverted repeats in boxes 2 and 3 in the promoter region of the xlnC gene from Streptomyces sp . EC3, was assessed . These boxes were modified by site-directed mutagenesis, and the mutant promoter regions, as well as the wild-type promoter region, were separately fused to a beta-lactamase reporter gene . Analysis of the expression patterns of these fusions in cultures grown in the presence of glucose, xylan or both carbon sources demonstrated that these motifs were cis -acting negative regulatory elements, each playing a specific role in the regulation of xlnC expression . Box 3 was shown to be critical for the establishment of repression of xlnC expression by glucose, whereas box 2 was shown to play an important role in the induction of xlnC expression by xylan.

J Med Chem, 2003 Sep 25, 46(20), 4265 - 72
A specific mechanism of nonspecific inhibition; McGovern SL et al.; Promiscuous small molecules plague screening libraries and hit lists . Previous work has found that several nonspecific compounds form submicrometer aggregates, and it has been suggested that this aggregate species is responsible for the inhibition of many different enzymes . It is not understood how aggregates inhibit their targets . To address this question, biophysical, kinetic, and microscopy methods were used to study the interaction of promiscuous, aggregate-forming inhibitors with model proteins . By use of centrifugation and gel electrophoresis, aggregates and protein were found to directly interact . This is consistent with a subsequent observation from confocal fluorescence microscopy that aggregates concentrate green fluorescent protein . beta-Lactamase mutants with increased or decreased thermodynamic stability relative to wild-type enzyme were equally inhibited by an aggregate-forming compound, suggesting that denaturation by unfolding was not the primary mechanism of interaction . Instead, visualization by electron microscopy revealed that enzyme associates with the surface of inhibitor aggregates . This association could be reversed or prevented by the addition of Triton X-100 . These observations suggest that the aggregates formed by promiscuous compounds reversibly sequester enzyme, resulting in apparent inhibition . They also suggest a simple method to identify or reverse the action of aggregate-based inhibitors, which appear to be widespread.

Bioconjug Chem, 2003 Sep-Oct, 14(5), 860 - 9
Improved yield and stability of L49-sFv-beta-lactamase, a single-chain antibody fusion protein for anticancer prodrug activation, by protein engineering; McDonagh CF et al.; The L49 single-chain Fv fused to beta-lactamase (L49-sFv-bL) combined with the prodrug C-Mel is an effective anticancer agent against tumor cells expressing the p97 antigen . However, large-scale production of L49-sFv-bL from refolded E . coli inclusion bodies has been problematic due to inefficient refolding and instability of the fusion protein . Sequence analysis of the L49-sFv framework regions revealed three residues in the framework regions at positions L2, H82B, and H91, which are not conserved for their position, occurring in <1% of sequences in Fv sequence databases . One further unusual residue, found in <3% of variable sequences, was observed at position H39 . Each unusual residue was mutated to a conserved residue for its position and tested for refolding yield from inclusion bodies following expression in E . coli . The three V(H) single mutants showed improvement in the yield of active protein and were combined to form double and triple mutants resulting in a 7-8-fold increased yield compared to the parental protein . In an attempt to further improve yield, the orientation of the triple mutant was reversed to create a bL-L49-sFv fusion protein resulting in a 3-fold increase in expressed inclusion body protein and producing a 20-fold increase in the yield of purified protein compared to the parental protein . The triple mutants in both orientations displayed increased stability in murine plasma and binding affinity was not affected by the introduced mutations . Both triple mutants also displayed potent in vitro cytotoxicity and in vivo antitumor activity against p97 expressing melanoma cells and tumor xenografts, respectively . These results show that a rational protein-engineering approach improved the yield, stability, and refolding characteristics of L49-sFv-bL while maintaining binding affinity and therapeutic efficacy.

Biochemistry, 2003 Sep 16, 42(36), 10634 - 43
Crystal structure of extended-spectrum beta-lactamase Toho-1: insights into the molecular mechanism for catalytic reaction and substrate specificity expansion; Ibuka AS et al.; The crystallographic structure of the class A beta-lactamase Toho-1, an extended-spectrum beta-lactamase with potent activity against expanded-spectrum cephems, has been determined at 1.65 A resolution . The result reveals that the Lys73 side chain can adopt two alternative conformations . The predominant conformation of Lys73 is different from that observed in the E166A mutant, indicating that removal of the Glu166 side chain changes the conformation of the Lys73 side chain and thus the interaction between Lys73 and Glu166 . The Lys73 side chain would play an important role in proton relay, switching its conformation from one to the other depending on the circumstances . The electron density map also implies possible rotation of Ser237 . Comparison of the Toho-1 structure with the structure of other class A beta-lactamases shows that the hydroxyl group of Ser237 is likely to rotate through interaction with the carboxyl group of the substrate . Another peculiarity is the existence of three sulfate ions positioned in or near the substrate-binding cavity . One of these sulfate ions is tightly bound to the active center, while the other two are held by a region of positive charge formed by two arginine residues, Arg274 and Arg276 . This positively charged region is speculated to represent a pseudo-binding site of the beta-lactam antibiotics, presumably catching the methoxyimino group of the third-generation cephems prior to proper binding in the substrate-binding cleft for hydrolysis . This high-resolution structure, together with detailed kinetic analysis of Toho-1, provides a new hypothesis for the catalytic mechanism and substrate specificity of Toho-1.

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Sep 5, 794(2), 227 - 36
Simultaneous determination of ticarcillin and clavulanate in rabbit serum and tissue cage fluid by liquid chromatography; Li C et al.; A gradient elution HPLC method with a wavelength switch technique was developed to simultaneously analyze the beta-lactam ticarcillin and the beta-lactamase inhibitor clavulanate in rabbit serum and tissue cage fluid (TCF) . A C18 reversed-phase column with a programmable UV detector changing the wavelength from 218 to 254 nm at 9 min was used for chromatographic separation . The mobile phase consisted of acetonitrile, phosphate buffer and tetrabutylammonium hydrogen sulfate by following a gradient elution program at a flow-rate of 1 ml/min . Sample processing was carried out with liquid-liquid extraction . Good linearity, recoveries, precision and accuracy were obtained . The ranges of the standard curves were 1-100 microg/ml for ticarcillin, and 0.2-20 microg/ml for clavulanate . This assay has been successfully applied to analyze ticarcillin and clavulanate in rabbit serum and tissue cage fluid samples from a pharmacokinetic study.

FEMS Microbiol Lett, 2003 Aug 29, 225(2), 183 - 8
A detailed kinetic study of Mox-1, a plasmid-encoded class C beta-lactamase; Alba J et al.; Surveys of beta-lactamases in different parts of the world show an important increase in class C beta-lactamases, thus the study of these enzymes is becoming an important issue . We created an overproduction system for Mox-1, a plasmid class C beta-lactamase, by cloning the gene encoding this enzyme, and placing it under the control of a T7 promoter, using vector pET 28a . The enzyme, purified by ion exchange chromatography, was used to obtain the molecular mass (38246), the N-terminal sequence (GEASPVDPLRPVV), and pI (8.9), and to perform a detailed kinetic study . Cephalotin was used as reporter substrate in the case of poor substrates . The kinetic study showed that benzylpenicillin, cephalotin, cefcapene and moxalactam were good substrates for Mox-1 (k(cat)/K(m) values >2.5 x 10(6) M(-1) s(-1)) . On the other hand, ceftazidime and cefepime were poor substrates for this enzyme (K(m) values >200 microM) . Clavulanic acid had no inhibitory effect on Mox-1 (K(m)=30.2 mM), however aztreonam behaved as an inhibitor of Mox-1 (K(i)=2.85 microM).

Antimicrob Agents Chemother, 2003 Sep, 47(9), 2958 - 61
Effects of Ser130Gly and Asp240Lys substitutions in extended-spectrum beta-lactamase CTX-M-9; Aumeran C et al.; In CTX-M-9 extended-spectrum beta-lactamases (ESBLs), an S130G mutation induced a 40- to 650-fold increase in 50% inhibitory concentrations but decreased hydrolytic activity against cefotaxime . A D240K mutation did not modify enzymatic efficiency against ceftazidime . Residue K240 could interact with Q270 and therefore not with ceftazidime, in contrast with what was observed with certain TEM/SHV-type ESBLs.

J Biol Chem, 2003 Nov 14, 278(46), 45706 - 12 Epub 2003 Aug 21.
Determinants of binding affinity and specificity for the interaction of TEM-1 and SME-1 beta-lactamase with beta-lactamase inhibitory protein; Zhang Z et al.; The hydrolysis of beta-lactam antibiotics by class A beta-lactamases is a common cause of bacterial resistance to these agents . The beta-lactamase inhibitory protein (BLIP) is able to bind and inhibit several class A beta-lactamases, including TEM-1 beta-lactamase and SME-1 beta-lactamase . Although the TEM-1 and SME-1 enzymes share 33% amino acid sequence identity and a similar fold, they differ substantially in surface electrostatic properties and the conformation of a loop-helix region that BLIP binds . Alanine-scanning mutagenesis was performed to identify the residues on BLIP that contribute to its binding affinity for each of these enzymes . The results indicate that the sequence requirements for binding are similar for both enzymes with most of the binding free energy provided by two patches of aromatic residues on the surface of BLIP . Polar residues such as several serines in the interface do not make significant contributions to affinity for either enzyme . In addition, the specificity of binding is significantly altered by mutation of two charged residues, Glu73 and Lys74, that are buried in the structure of the TEM-1.BLIP complex as well as by residues located on two loops that insert into the active site pocket . Based on the results, a E73A/Y50A double mutant was constructed that exhibited a 220,000-fold change in binding specificity for the TEM-1 versus SME-1 enzymes.

Proc Natl Acad Sci U S A, 2003 Aug 19, 100(17), 9727 - 32 Epub 2003 Aug 08.
Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I; Camps M et al.; We present a system for random mutagenesis in Escherichia coli for the evolution of targeted genes . To increase error rates of DNA polymerase I (Pol I) replication, we introduced point mutations in three structural domains that govern Pol I fidelity . Expression of error-prone Pol I in vivo results in strong mutagenesis of a target sequence encoded in a Pol I-dependent plasmid (8.1 x 10-4 mutations per bp, an 80,000-fold increase), with a preference for plasmid relative to chromosome sequence . Mutagenesis is maximal in cultures maintained at stationary phase . Mutations are evenly distributed and show a variety of base pair substitutions, predominantly transitions . Mutagenesis extends at least 3 kb beyond the 400-500 nt reportedly synthesized by Pol I . We demonstrate that our error-prone Pol I can be used to generate enzymes with distinct properties by generating TEM-1 beta-lactamase mutants able to hydrolyze a third-generation lactam antibiotic, aztreonam . Three different mutations contribute to aztreonam resistance . Two are found in the extended-spectrum beta-lactamases most frequently identified in clinical isolates, and the third (G276R) has not been previously described . Our system of targeted mutagenesis in E . coli should have an impact on enzyme-based applications in areas such as synthetic chemistry, gene therapy, and molecular biology . Given the structural conservation between polymerases, this work should also provide a reference for altering the fidelity of other polymerases.

FEMS Microbiol Lett, 2003 Aug 8, 225(1), 149 - 53
Characterization of In111, a class 1 integron that carries the extended-spectrum beta-lactamase gene blaIBC-1; Vourli S et al.; A class 1 integron, In111, carried by a self-transferable plasmid from an Escherichia coli clinical strain was characterized . The variable region of In111 constituted an array of gene cassettes encoding the extended-spectrum beta-lactamase IBC-1, the aminoglycoside-modifying enzymes AAC(6')-Ib and ANT(3")-Ia, dihydrofolate reductase I and a putative polypeptide (SMR-2) sharing similarity with the Qac transporters . Transcription of the gene cassettes was driven by a hybrid-type P1 promoter located in a typical 5' conserved segment (CS) . The 3'CS included sulI, qacEDelta1, orf5 and orf6 . In111 was bounded on the right by an inversely oriented IRt . The 5'CS was preceded by an intact IS26 element followed by an aphA1 gene.

Genome Res, 2003 Aug, 13(8), 1938 - 43 Epub 2003 Jul 17.
E . coli selection of human genes encoding secreted and membrane proteins based on cDNA fusions to a leaderless beta-lactamase reporter; Tan R et al.; Although several signal peptide-trapping methods have been devised and used to detect signal sequences, none have relied on using E.coli to identify eukaryotic proteins with signal peptides . Here, we describe a system for selecting human secreted and membrane proteins in E . coli followed by the direct validation of secretion in human cells . The method is based on cDNA fusions to a leaderless beta-lactamase reporter gene to isolate clones encoding signal peptides of human genes . We found that beta-lactamase fusion proteins carrying a eukaryotic signal peptide at its N-terminus were able to direct their export into the periplasm in E . coli to confer survival upon challenge with carbenicillin . When libraries constructed from 5' end-enriched cDNAs fused to beta-lactamase were screened in E.coli, approximately 0.5%-1% of the cDNAs are selected, and over half of the surviving clones were found to encode for secreted fusion proteins when tested in human cells . These clones were sequenced and shown to represent human genes encoding signal peptides of secreted and membrane proteins . We conclude that this is an efficient and effective strategy to easily enrich cDNA libraries for the identification of novel genes likely to encode secreted enzymes, growth factors, and receptors.

FEMS Microbiol Lett, 2003 Jul 15, 224(1), 139 - 42
Identification of strong promoter elements of Mycobacterium smegmatis and their utility for foreign gene expression in mycobacteria; Spratt JM et al.; The isolation of elements driving high-level expression of foreign genes in mycobacteria would significantly aid characterization of mycobacterial antigens and recombinant vaccine development . Mycobacterium smegmatis is a widely employed host for recombinant mycobacterial gene expression . This report describes the identification of strong promoter elements of M . smegmatis . Fluorescence-activated cell sorting was employed to isolate DNA fragments permitting high-level expression of the Aequorea victoria green fluorescent protein within recombinant M . smegmatis . Ten postulated M . smegmatis promoters were identified which showed activity two to six times that of the strong beta-lactamase promoter of Mycobacterium fortuitum . The utility of one of these promoters for the over-expression of foreign genes in mycobacteria was demonstrated by the efficient purification of the Mycobacterium leprae 35-kDa antigen from recombinant M . smegmatis.

J Biochem Biophys Methods, 2003 Jun 30, 56(1-3), 177 - 88
Effect of column dimensions and flow rates on size-exclusion refolding of beta-lactamase; Harrowing SR et al.; We have investigated the effect of changing the column diameter and length on the size exclusion chromatography (SEC) refolding of beta-lactamase from Escherichia coli-derived inclusion bodies (IBs) . Inclusion bodies were recovered and solubilised in 6 M GdnHCl and 5 mM DTT . Up to 16 mg of denatured, solubilised beta-lactamase was loaded onto size exclusion columns packed with Sephacryl S-300 media (fractionation range: 10(4)-1.5 x 10(6) Da) . beta-Lactamase was refolded by eluting the loaded sample with 1 M urea in 0.05 M phosphate buffer, pH 7 at 23 degrees C . The following columns were studied: 26 x 400, 16 x 400 and 26 x 200 mm, with a range of mobile phase flow rates from 0.33 to 4.00 ml/min . beta-Lactamase was successfully refolded in all three columns and at all flow rates studied . The beta-lactamase activity peak coincided with the major protein peak . Reducing the column diameter had little effect on refolding performance . The enzyme activity recovered was relatively independent of the mobile phase linear velocity . Reducing the column length gave a poorer resolution of the protein peaks, but the enzyme activity peaks were well resolved . Calculation of the partition coefficients for beta-lactamase activity showed that the 26 x 400 column gave the greatest refolding performance.

J Infect Chemother, 2003 Jun, 9(2), 183 - 6
Clinical effect of ampicillin with beta-lactamase inhibitor (sulbactam/ampicillin) on community-acquired pneumonia in the elderly; Okimoto N et al.; Sulbactam/ampicillin (SBT/ABPC) was administered to 83 patients aged over 75 years with community-acquired bacterial pneumonia (mild, n = 43; moderate, n = 40), and its clinical effect was reviewed . It was effective in 37 of the 43 patients with mild disease (efficacy rate, 86.0%), in 27 of the 40 patients with moderate disease (efficacy rate, 67.5%), and overall in 64 of the 83 patients (efficacy rate, 77.1%) . Side effects included drug eruption in 1 patient (1.2%) and abnormal laboratory findings in 11 (13.3%), all of which were mild . Based on the above, SBT/ABPC may be recommended as the first-choice drug for community-acquired bacterial pneumonia in the elderly.

J Biol Chem, 2003 Aug 15, 278(33), 30927 - 35 Epub 2003 May 22.
Anticodon recognition in evolution: switching tRNA specificity of an aminoacyl-tRNA synthetase by site-directed peptide transplantation; Brevet A et al.; The highly conserved aspartyl-, asparaginyl-, and lysyl-tRNA synthetases compose one subclass of aminoacyl-tRNA synthetases, called IIb . The three enzymes possess an OB-folded extension at their N terminus . The function of this extension is to specifically recognize the anticodon triplet of the tRNA . Three-dimensional models of bacterial aspartyl- and lysyl-tRNA synthetases complexed to tRNA indicate that a rigid scaffold of amino acid residues along the five beta-strands of the OB-fold accommodates the base U at the center of the anticodon . The binding of the adjacent anticodon bases occurs through interactions with a flexible loop joining strands 4 and 5 (L45) . As a result, a switching of the specificity of lysyl-tRNA synthetase from tRNALys (anticodon UUU) toward tRNAAsp (GUC) could be attempted by transplanting the small loop L45 of aspartyl-tRNA synthetase inside lysyl-tRNA synthetase . Upon this transplantation, lysyl-tRNA synthetase loses its capacity to aminoacylate tRNALys . In exchange, the chimeric enzyme acquires the capacity to charge tRNAAsp with lysine . Upon giving the tRNAAsp substrate the discriminator base of tRNALys, the specificity shift is improved . The change of specificity was also established in vivo . Indeed, the transplanted lysyl-tRNA synthetase succeeds in suppressing a missense Lys --> Asp mutation inserted into the beta-lactamase gene . These results functionally establish that sequence variation in a small peptide region of subclass IIb aminoacyl-tRNA synthetases contributes to specification of nucleic acid recognition . Because this peptide element is not part of the core catalytic structure, it may have evolved independently of the active sites of these synthetases.

Genome Res, 2003 May, 13(5), 980 - 90
Selecting open reading frames from DNA; Zacchi P et al.; We describe a method to select DNA encoding functional open reading frames (ORFs) from noncoding DNA within the context of a specific vector . Phage display has been used as an example, but any system requiring DNA encoding protein fragments, for example, the yeast two-hybrid system, could be used . By cloning DNA fragments upstream of a fusion gene, consisting of the beta-lactamase gene flanked by lox recombination sites, which is, in turn, upstream of gene 3 from fd phage, only those clones containing DNA fragments encoding ORFs confer ampicillin resistance and survive . After selection, the beta-lactamase gene can be removed by Cre recombinase, leaving a standard phage display vector with ORFs fused to gene 3 . This vector has been tested on a plasmid containing tissue transglutaminase . All surviving clones analyzed by sequencing were found to contain ORFs, of which 83% were localized to known genes, and at least 80% produced immunologically detectable polypeptides . Use of a specific anti-tTG monoclonal antibody allowed the identification of clones containing the correct epitope . This approach could be applicable to the efficient selection of random ORFs representing the coding potential of whole organisms, and their subsequent downstream use in a number of different systems.

Appl Biochem Biotechnol, 2003 Spring, 105 -108, 689 - 703
Automated filter paper assay for determination of cellulase activity; Decker SR et al.; Recent developments in molecular breeding and directed evolution have promised great developments in industrial enzymes as demonstrated by exponential improvements in beta-lactamase and green fluorescent protein (GFP) . Detection of and screening for improved enzymes are relatively easy if the target enzyme is expressible in a suitable high-throughput screening host and a clearly defined and usable screen or selection is available, as with GFP and beta-lactamase . Fungal cellulases, however, are difficult to measure and have limited expressibility in heterologous hosts . Furthermore, traditional cellulase assays are tedious and time-consuming . Multiple enzyme components, an insoluble substrate, and generally slow reaction rates have plagued cellulase researchers interested in creating cellulase mixtures with increased activities and/or enhanced biochemical properties . Although the International Union of Pure and Applied Chemists standard measure of cellulase activity, the filter paper assay (FPA), can be reproduced in most laboratories with some effort, this method has long been recognized for its complexity and susceptibility to operator error . Our current automated FPA method is based on a Cyberlabs C400 robotics deck equipped with customized incubation, reagent storage, and plate-reading capabilities that allow rapid evaluation of cellulases acting on cellulose and has a maximum throughput of 84 enzyme samples per day when performing the automated FPA.

Rinsho Byori, 2003 Mar, 51(3), 201 - 7
{Detection of novel nucleotide substitutions resulting amino acid substitutions in TEM type beta-lactamase gene isolated from a clinically isolated extended spectrum beta-lactamases (ESBLs) productive Escherichia coli}; Yasuhara T et al.; We screened Escherichia coli (E . coli) from specimens submitted to the Showa University hospital clinical laboratory that showed an advanced resistance to third generation cephems (Cefotaxime or Ceftazidime), as candidate extended spectrum beta-lactamases(ESBLs) producing strains . Among the candidates, two strains showed the characteristics of class A ESBLs in their susceptibility to Cefmetazole, a second generation cephem, and their resistance was inhibited by the addition of clavulanic acid . Further, we detected the TEM-type gene in one of the two E . coli strains . By determining the nucleotide sequence of the whole coding region of the TEM gene, two nucleotide substitutions with amino acid substitutions 82Val-->Ile, and 182Ala-->Val were identified.

J Mol Biol, 2003 Apr 18, 328(1), 289 - 301
Ultrahigh resolution structure of a class A beta-lactamase: on the mechanism and specificity of the extended-spectrum SHV-2 enzyme; Nukaga M et al.; Bacterial beta-lactamases hydrolyze beta-lactam antibiotics such as penicillins and cephalosporins . The TEM-type class A beta-lactamase SHV-2 is a natural variant that exhibits activity against third-generation cephalosporins normally resistant to hydrolysis by class A enzymes . SHV-2 contains a single Gly238Ser change relative to the wild-type enzyme SHV-1 . Crystallographic refinement of a model including hydrogen atoms gave R and R(free) of 12.4% and 15.0% for data to 0.91 A resolution . The hydrogen atom on the O(gamma) atom of the reactive Ser70 is clearly seen for the first time, bridging to the water molecule activated by Glu166 . Though hydrogen atoms on the nearby Lys73 are not seen, this observation of the Ser70 hydrogen atom and the hydrogen bonding pattern around Lys73 indicate that Lys73 is protonated . These findings support a role for the Glu166-water couple, rather than Lys73, as the general base in the deprotonation of Ser70 in the acylation process of class A beta-lactamases . Overlay of SHV-2 with SHV-1 shows a significant 1-3 A displacement in the 238-242 beta-strand-turn segment, making the beta-lactam binding site more open to newer cephalosporins with large C7 substituents and thereby expanding the substrate spectrum of the variant enzyme . The OH group of the buried Ser238 side-chain hydrogen bonds to the main-chain CO of Asn170 on the Omega loop, that is unaltered in position relative to SHV-1 . This structural role for Ser238 in protein-protein binding makes less likely its hydrogen bonding to oximino cephalosporins such as cefotaxime or ceftazidime .

FEMS Microbiol Lett, 2003 Mar 28, 220(2), 177 - 80
A novel sequence framework (bla(TEM-1G)) encoding the parental TEM-1 beta-lactamase; Pomba-Feria C et al.; A novel parental bla(TEM) gene (bla(TEM-1G)), encoding a TEM-1 beta-lactamase (pI of 5.4) produced by the uropathogenic Escherichia coli strain FMV194 was isolated from a dog . We report PCR-restriction fragment length polymorphism analysis and nucleotide sequencing of this gene . The bla(TEM-1G) sequence was identical to the bla(TEM-1C) gene framework in the coding and promoter (P3) regions, except for a silent G(604)-->T mutation in the coding region . Molecular phylogenetic analysis of parental bla(TEM) genes indicated two distinct groups, one comprising bla(TEM-1F) and bla(TEM-2) . The other group comprises bla(TEM-1C) which is the probable ancestor of bla(TEM-1A), bla(TEM-1D) and bla(TEM-1G) . The bla(TEM-1G) gene has the same framework as a gene encoding an inhibitor-resistant TEM beta-lactamase produced by an E . coli strain of human origin . Thus, parental bla(TEM) genes encoding beta-lactamases in E . coli strains isolated from different host species, in this case human and canine, may be phylogenetically very close.

Anal Biochem, 2003 Mar 1, 314(1), 16 - 29
Development of an intact cell reporter gene beta-lactamase assay for G protein-coupled receptors for high-throughput screening; Kunapuli P et al.; G protein-coupled receptors (GPCRs) are involved in a large variety of physiological disorders, and are thus important pharmaceutical drug targets . Here, we describe the development and characterization of a beta-lactamase reporter gene assay as a functional readout for the ligand-induced activation of the human bradykinin B1 receptor, expressed recombinantly in CHO cells . The beta-lactamase reporter gene assay provides high sensitivity due to the absence of endogenous beta-lactamase activity in mammalian cells . The cell-permeable fluorogenic substrate allows single-cell cloning of cells expressing functional BK1 receptors . Pharmacological characterization reveals comparable sensitivity and potency of known BK1 receptor agonists and antagonists between the beta-lactamase assay, competition-binding assay, and other direct measurements of second messengers . The beta-lactamase assay has been optimized for cell density, time of agonist stimulation, and DMSO sensitivity . This CHO-hBK1-beta-lactamase assay is well suited to automation and miniaturization required for high-throughput screening.

J Microbiol Methods, 2003 Apr, 53(1), 37 - 42
Surprisingly fast disappearance of beta-lactam selection pressure in cultivation as detected with novel biosensing approaches; Korpimaki T et al.; Tetracycline and beta-lactam resistances among others are used as selection markers in the production of recombinant proteins . The beta-lactam resistance is based on degradation, i.e . the selection pressure gradually disappears from the culture, whereas tetracycline resistance is based on active efflux . We have studied the kinetics of the stability of antibiotic selection pressure in culture using a simple model system (pBR322 in Escherichia coli) . Concentrations of ampicillin, carbenicillin and tetracycline were measured with novel sensor cells developed in our lab . These cells are specifically induced to produce light in the presence of the drugs and here their performance was shown to be excellent in monitoring antibiotic concentrations in cell culture . The sensor cells are cheap to produce and use and a high number of samples can be analysed simultaneously . To our surprise, ampicillin and carbenicillin were completely degraded after 2.5-3.0 h of culture, although it has been widely claimed that especially carbenicillin is a good selective agent, whereas tetracycline was stable in culture . beta-lactamase activity in culture was found to correlate with the kinetics of ampicillin degradation.

Protein Eng, 2002 Dec, 15(12), 1025 - 30
A universal, vector-based system for nucleic acid reading-frame selection; Lutz S et al.; The identification of a nucleic acid sequence's correct reading frame has important implications for homology-independent protein engineering techniques such as incremental truncation and SCRATCHY . We report the development and experimental implementation of a general in-frame selection system, pSALect, a plasmid vector that utilizes two marker sequences flanking the DNA of interest . This dual selection approach overcomes inconsistencies observed with traditional C-terminally fused reporter proteins . In the pSALect vector, sequences of interest are positioned between an N-terminal Tat-signal sequence and a C-terminal beta-lactamase reporter . In-frame selection of the resulting three-domain protein is performed by growing colonies on ampicillin-containing plates, requiring full-length translation in order to link covalently the signal sequence to the lactamase for export into the periplasm . This dual selection scheme has been validated successfully using defined sequences of glycinamide ribonucleotide formyltransferases (GARTs) from Escherichia coli and human and, in contrast to C-terminal fusion systems, proved effective when applied towards the selection of in-frame constructs in an incremental truncation library.

J Virol, 2003 Mar, 77(5), 2928 - 35
Persistent replication of hepatitis C virus replicons expressing the beta-lactamase reporter in subpopulations of highly permissive Huh7 cells; Murray EM et al.; Progress toward development of better therapies for the treatment of hepatitis C virus (HCV) infection has been hampered by poor understanding of HCV biology and the lack of biological assays suitable for drug screening . Here we describe a powerful HCV replication system that employs HCV replicons expressing the beta-lactamase reporter (bla replicons) and subpopulations of Huh7 cells that are more permissive (or "enhanced") to HCV replication than naive Huh7 cells . Enhanced cells represent a small fraction of permissive cells present among naive Huh7 cells that is enriched during selection with replicons expressing the neomycin phosphotransferase gene (neo replicons) . The level of permissiveness of cell lines harboring neo replicons can vary greatly, and the enhanced phenotype is usually revealed upon removal of the neo replicon with inhibitors of HCV replication . Replicon removal is responsible for increased permissiveness, since this effect could be reproduced either with alpha interferon or with an HCV NS5B inhibitor . Moreover, adaptive mutations present in the replicon genome used during selection do not influence the permissiveness of the resulting enhanced-cell population, suggesting that the mechanisms governing the permissiveness of enhanced cells are independent from viral adaptation . Because the beta-lactamase reporter allows simultaneous quantitation of replicon-harboring cells and reporter activity, it was possible to investigate the relationship between genome replication activity and the frequency with which transfected genomes can establish persistent replication . Our study demonstrates that differences in the replication potential of the viral genome are manifested primarily in the frequency with which persistent replication is established but modestly affect the number of replicons observed per replicon-harboring cell . Replicon copy number was found to vary over a narrow range that may be defined by a minimal number required for persistent maintenance and a maximum that is limited by the availability of essential host factors.

J Am Chem Soc, 2003 Jan 22, 125(3), 672 - 84
Insights into the acylation mechanism of class A beta-lactamases from molecular dynamics simulations of the TEM-1 enzyme complexed with benzylpenicillin; Diaz N et al.; Herein, we present results from molecular dynamics MD simulations ( approximately 1 ns) of the TEM-1 beta-lactamase in aqueous solution . Both the free form of the enzyme and its complex with benzylpenicillin were studied . During the simulation of the free enzyme, the conformation of the Omega loop and the interresidue contacts defining the complex H-bond network in the active site were quite stable . Most interestingly, the water molecule connecting Glu166 and Ser70 does not exchange with bulk solvent, emphasizing its structural and catalytic relevance . In the presence of the substrate, Ser130, Ser235, and Arg244 directly interact with the beta-lactam carboxylate via H-bonds, whereas the Lys234 ammonium group has only an electrostatic influence . These interactions together with other specific contacts result in a very short distance ( approximately 3 A) between the attacking hydroxyl group of Ser70 and the beta-lactam ring carbonyl group, which is a favorable orientation for nucleophilic attack . Our simulations also gave insight into the possible pathways for proton abstraction from the Ser70 hydroxyl group . We propose that either the Glu166 carboxylate-Wat1 or the substrate carboxylate-Ser130 moieties could abstract a proton from the nucleophilic Ser70.

Microb Drug Resist, 2002 Winter, 8(4), 329 - 33
A clinical strain of Escherichia coli possessing CMY-2 plasmid-mediated amp C beta-lactamase: an emerging concern in pediatrics?
Hoyen CM, Hujer AM, Hujer KM, Marshall SH, Carias L, Toltzis P, Rice LB, Bonomo RA.
A 5-year-old child was colonized by an isolate of Escherichia coli that transferred resistance to third-generation cephalosporins and cefoxitin . This resistance phenotype was encoded on a >75-kb plasmid pLRM 22 . The transferable plasmid contained both blaCMY-2 and blaTEM-1b . Increasing reports of CMY-2 beta-lactamase in clinical isolates in children raise concerns about the empiric use of third-generation cephalosporins in this patient group.

Microb Drug Resist, 2002 Winter, 8(4), 267 - 72
Molecular characterization of amoxicillin-clavulanate resistance in a clinical isolate of Escherichia coli; Bellaaj A et al.; The resistance phenotype of the clinical isolate of Escherichia coli 1941 was characterized by high-level resistance to penicillins and to combinations amoxicillin-ticarcillin/clavulanate and ampicillin/sulbactam . This resistance was carried by the conjugative plasmid pEC1941 that encoded a beta-lactamase activity . The purified enzyme focused at pI 5.4 and was strongly inhibited in vitro by clavulanic acid (IC50 = 0.09 microM) . Nucleotide sequence analysis revealed identity between the plasmid borne blaTEM gene of E . coli 1941 and the blaTEM-1B gene, except for a single C-to-T substitution at position 32 in the promoter region leading to the overlapping promoters Pa and Pb . No alterations in the expression of outer membrane porins OmpC and OmpF have been detected . These findings show that the resistance of E . coli 1941 to the combinations of beta-lactams with beta-lactamase inhibitors is related to high-level production of TEM-1 enzyme expressed from the strong promoters Pa and Pb.

J Synchrotron Radiat, 2003 Jan 1, 10(Pt 1), 64 - 8 Epub 2002 Dec 24.
Simulating the XANES of metalloenzymes - a case study; Mijovilovich A et al.; The analysis of XANES patterns is very indicative for screening samples . Powerful X-ray absorption spectroscopy data-analysis programs can simulate these patterns . Here, a case study on two structural motifs is presented: a non-heme Fe site (2-His-1-carboxylate motif) and the metallo beta-lactamase dinuclear Zn site . Simulations of the edge shapes for different structural models will be compared with experimental results, pointing out limitations and challenges . The influence of single neighbouring atoms in the first and second shell on the resulting XANES pattern is discussed . Insights into catalytic mechanisms and the requirements for future theory development are addressed.

Invest Ophthalmol Vis Sci, 2003 Jan, 44(1), 37 - 43
Localization and activity of membrane dipeptidase in bovine ciliary epithelium; Ikeda H et al.; PURPOSE: To investigate the localization and the activity of membrane dipeptidase (MDP) in the bovine eye . METHODS: A monoclonal antibody (mAb), 49C mAb, raised against bovine ciliary process was used to examine the localization of MDP . Conversion of leukotriene (LT)D4 to LTE4 was evaluated by enzyme-linked immunosorbent assay for LTE4 . Hydrolytic activity (beta-lactamase activity) was evaluated with a fluorometric assay . To clarify the contribution of MDP to conversion of LTD4 and beta-lactamase activity, we separated MDP from other enzymes by 49C mAb-conjugated gel . RESULTS: The antigenic molecule of 49C mAb was shown to be MDP by amino acid sequencing . MDP was immunohistochemically detected in the ciliary pigmented and nonpigmented epithelial cells . Conversion of LTD4 to LTE4 in the ciliary process was much greater than that of the neural retina (NR) . beta-Lactamase activity in the ciliary process was apparent, but that in the NR or the retinal pigment epithelium was negligible . Approximately 100% of beta-lactamase activity in the ciliary process was catalyzed by the 49C mAb-bound fraction . Conversion of LTD4 was catalyzed by the 49C mAb-bound fraction (55% of total activity) and by the unbound fraction (45% of total activity) . CONCLUSIONS: This study produced the first evidence of the presence of MDP in ciliary epithelial cells . The ciliary epithelium converts LTD4 to LTE4 and shows beta-lactamase activity . Conversion of LTD4 is catalyzed by at least two enzymes, and a major part of the conversion is induced by MDP.

Antimicrob Agents Chemother, 2002 Dec, 46(12), 4038 - 40
Chromosome-encoded Ambler class A beta-lactamase of Kluyvera georgiana, a probable progenitor of a subgroup of CTX-M extended-spectrum beta-lactamases; Poirel L et al.; A chromosome-encoded beta-lactamase gene, cloned and expressed in Escherichia coli from Kluyvera georgiana reference strain CUETM 4246-74 (DSM 9408), encoded the extended-spectrum beta-lactamase KLUG-1, which shared 99% amino acid identity with the plasmid-mediated beta-lactamase CTX-M-8 . This work provides further evidence that Kluyvera spp . may be the progenitor(s) of CTX-M-type beta-lactamases.

Antimicrob Agents Chemother, 2002 Dec, 46(12), 4035 - 7
Promoters P3, Pa/Pb, P4, and P5 upstream from bla(TEM) genes and their relationship to beta-lactam resistance; Lartigue MF et al.; Using an isogenic system, we have determined the impact that the four promoters known to control bla(TEM) gene expression have on beta-lactamase activity . For both TEM-1 and TEM-30, this activity gradually increased in relation to the presence of promoters P3, Pa/Pb, and P4 upstream of the corresponding gene . Promoter P5, only found upstream of the bla(TEM-1B) gene, was related to the highest expression of this gene.

Antimicrob Agents Chemother, 2002 Dec, 46(12), 3991 - 4
Prevalence of clinical isolates of Escherichia coli producing inhibitor-resistant beta-lactamases at a University Hospital in Barcelona, Spain, over a 3-year period; Miro E et al.; About 7% of 7,252 nonduplicated clinical Escherichia coli strains from a Spanish hospital showed reduced susceptibility to amoxicillin-clavulanate . Of these, 0.37% produced the IRTs TEM-30, TEM-31, TEM-33, TEM-34, TEM-37, TEM-40, TEM-51, and TEM-54; 5.3% were probable class C beta-lactamase overproducers; 0.8% were probable TEM-1 hyperproducers; 0.18% produced OXA-30; 0.15% overexpressed SHV-1; and 0.03% produced a PSE-1 enzyme.

Antimicrob Agents Chemother, 2002 Dec, 46(12), 3978 - 80
Structural basis for imipenem inhibition of class C beta-lactamases; Beadle BM et al.; To determine how imipenem inhibits the class C beta-lactamase AmpC, the X-ray crystal structure of the acyl-enzyme complex was determined to a resolution of 1.80 A . In the complex, the lactam carbonyl oxygen of imipenem has flipped by approximately 180 degrees compared to its expected position; the electrophilic acyl center is thus displaced from the point of hydrolytic attack . This conformation resembles that of imipenem bound to the class A enzyme TEM-1 but is different from that of moxalactam bound to AmpC.

J Bacteriol, 2002 Dec, 184(23), 6434 - 6
Substrate specificity of the AmpG permease required for recycling of cell wall anhydro-muropeptides; Cheng Q et al.; AmpG was originally identified as a gene required for induction of beta-lactamase . Subsequently, we found AmpG to be a permease required for recycling of murein tripeptide and uptake of anhydro-muropeptides . We have now studied the specificity of the AmpG permease . The principal requirement is for the presence of the disaccharide, N-acetylglucosaminyl-beta-1,4-anhydro-N-acetylmuramic acid (GlcNAc-anhMurNAc) . These unique substrates for AmpG, which contain murein peptides linked to GlcNAc-anhMurNAc, are produced by turnover of the cell wall during logarithmic growth . AmpG permease is sensitive to carbonylcyanide m-chlorophenylhydrazone, demonstrating that AmpG permease is a single-component permease and that transport is dependent on the proton motive force.

Therapie, 2002 May-Jun, 57(3), 214 - 28
{Development of surgical antibioprophylaxis kits: evaluation of the impact on prescribing habits}; Aouizerate P et al.; In our hospital, surgical antibioprophylaxis (ATBP) was too often administered too late, thus raising the infectious risk . Antibiotic stocks of the anaesthesia department were also systematically used, instead of nominal prescriptions of these drugs . The pharmacy could neither charge antibiotics to each surgical department nor quantify and differentiate ATBP from curative antibiotic therapy . The pharmacy and anaesthesia departments therefore set out to standardize surgical ATBP, in order to adapt this treatment to each surgical indication, and particularly in the case of allergy to beta-lactamase antibiotics (second line treatment kits) . Consequently, prescription forms were developed and supplied to each surgery department, as well as ATBP kits . The kits were prepared and distributed by the pharmacy, and comprised boxes containing antibiotics in sufficient quantities to respect the protocols approved by the French Society of Anaesthesia and Resuscitation (SFAR) . A protocol describing prescriptions, dispensation and administration has been presented to physicians and nurses . Fifteen surgical departments were included in our study and 30 different kits were prepared . From 1998 to 2001, 5586 surgical operations required administration of a kit (second line treatment kits in 5% of cases): 1848 (33%) in visceral surgery; 764 (13.8%) in urology; 802 (14%) in orthopaedics; 13 (0.2%) in vascular and thoracic surgery; 1236 (22%) in ear-nose-throat (ENT), periodontics and ophtalmology, and 923 (17%) in gynaecology and obstetrics . 93% of filled prescriptions forms were spontaneously returned to the pharmacy, the others were obtained during the renewal of kit stocks . The cost (over 4 years) of ATBP was quantified: 157,871 F for the 15 departments included, 26,123 F in visceral surgery, 13,520 F in urology, 73,741 F in orthopaedics, 569 F in vascular surgery, 39,720 F in ENT/ophthalmology/periodontics and 4,198 F in gynaecology and obstetrics . According to the Altemeier classification, 2226 class I, 3151 class II, and 209 class III surgical operations were performed . Since the kits have been brought into use, the committee for the protection against nosocomial infections (CLIN) has observed a reduction in the incidence of post-operative infections, according to the Altemeier classification: from 1.6% to 0.5% in class I, from 6.5% to 4.3% in class II, and from 11% to 8.5% in class III . The difference was statistically significant only for classes I (p < 0.01) and II (p < 0.001), and unchanged for class III (p = 0.3) . No analysis was carried out for class IV (curative treatments) . Both nurses and physicians have greatly appreciated the implementation of this organization . The advantage in terms of post-operative infections, administration exhaustiveness and stock management is obvious . The prescribed kits were systematically appropriate for the surgical interventions . In orthopaedics, cefamandole was used over 24 h (188 kits) in ligament plasty and osteotomy, or for 48 h (499 kits) in prosthetic surgery; 24 amoxicillin/clavulanic acid (first line) and 9 clindamycin/gentamicin (second line) single dose kits have been prescribed in traumatic indications . In ophthalmology, kits were only prescribed in endophtalmitis (24 ofloxacin/fosfomycin single amount kits), implant replacement or cornea graft (1076 ofloxacin 24 h kits) and cataract surgery in diabetic patients (12 ofloxacin single amount kits) . In ENT and periodontics, 124 surgical operations required cefazolin single dose kits . In vascular surgery, 5 pefloxacin/gentamicin 48 h kits and 1 amoxicillin/clavulanic acid 48 h kit were used in contaminated limb amputation, 1 cefamandole 48 h kit in class I surgery and 1 vancomycin 24 h kit (betalactamase antibiotic allergy); in thoracic surgery, 1 cefamandole 24 h kit was used for a thoracic wound . In visceral surgery, 9 different kits have been used, depending on the opening (class II) or not (class I) of the digestive tract . 797 cefazolin (first line) and 68 clindamycin/gentamicin (second line) single dose kits were used in class I surgery, and 689 amoxicillin/clavulanic acid single dose (SD) kits in class II surgery . Specific protocols consisted of 18 ceftriaxone/metronidazole and 48 metronidazole/gentamicin SD kits in oesophagus surgery, 11 ceftriaxone and 17 gentamicin SD kits in biliary endoscopy, 137 metronidazole SD kits in proctology and 34 amoxicillin/gentamicin 6 h kits for prevention of endocarditis . In urology, 133 cefotaxime and 20 pefloxacin/gentamicin SD kits were precribed in renal lithiasis, 102 amoxicillin/clavulanic acid SD kits in cystectomy, 27 amoxicillin/gentamicin 6 h kits in endocarditis prevention and 58 cefamandole SD kits in all other indications . In gynaecology and obstetrics, 534 cefazoline and 19 clindamycin/gentamicin (second line) SD kits were used, and 370 doxycyclin SD kits were prescribed in pregnancy termination . Some departments (orthopaedics and visceral surgery) adapted the protocols to their needs, specifically with regard to treatment duration . However, these situations were quickly corrected . A constant follow-up and update of this system, associated with routine audits, should allow the maintenance and possibly the improvement of these results, hence shortening treatment duration.

Nat Immunol, 2002 Dec, 3(12), 1200 - 7 Epub 2002 Nov 04.
Identification of diversified genes that contain immunoglobulin-like variable regions in a protochordate; Cannon JP et al.; The evolutionary origin of adaptive immune receptors is not understood below the phylogenetic level of the jawed vertebrates . We describe here a strategy for the selective cloning of cDNAs encoding secreted or transmembrane proteins that uses a bacterial plasmid (Amptrap) with a defective beta-lactamase gene . This method requires knowledge of only a single target motif that corresponds to as few as three amino acids; it was validated with major histocompatibility complex genes from a cartilaginous fish . Using this approach, we identified families of genes encoding secreted proteins with two diversified immunoglobulin-like variable (V) domains and a chitin-binding domain in amphioxus, a protochordate . Thus, multigenic families encoding diversified V regions exist in a species lacking an adaptive immune response.

Proc Natl Acad Sci U S A, 2002 Nov 12, 99(23), 15142 - 7 Epub 2002 Nov 01.
Time-lapse imaging of a dynamic phosphorylation-dependent protein-protein interaction in mammalian cells; Spotts JM et al.; The ability to make sensitive measurements of protein-protein interaction kinetics in single neurons is critical for understanding the molecular and cellular basis of neuronal function . We have developed a reporter technology based on the differential induction of Escherichia coli TEM-1 beta-lactamase (Bla) enzymatic activity that can function as a sensor of the interaction state of two target proteins within single neurons in vivo . To modulate Bla enzymatic activity, we first split the enzyme into two separate, complementary protein fragments that we identified by using a functional screening approach based on circular permutation of the Bla enzyme . The split enzyme was then brought together by the phosphorylation-dependent association of the kinase inducible domain of the cAMP response element binding protein (CREB) and the KIX domain of the CREB binding protein . Using an intracellular substrate whose fluorescence spectrum changes after hydrolysis by Bla, we performed time-lapse ratiometric imaging measurements of Bla enzymatic induction after association of the CREB and CREB binding protein interaction domains . This approach permits direct imaging of protein-protein interactions in single cells with high signal discrimination.

Curr Opin Chem Biol, 2002 Oct, 6(5), 583 - 9
New reactions in clavulanic acid biosynthesis; Townsend CA; Clavulanic acid is only a modestly effective antibiotic against bacterial infections in humans, but a potent inhibitor/inactivator of beta-lactamase enzymes that confer bacterial resistance . The biosynthetic pathway to clavulanic acid is considerably more complex than that to the structurally related penicillins and cephalosporins and has revealed several interesting reactions.

Antimicrob Agents Chemother, 2002 Nov, 46(11), 3627 - 9
TEM-103/IRT-28 beta-lactamase, a new TEM variant produced by Escherichia coli BM4511; Alonso R et al.; Clinical isolate Escherichia coli BM4511 was resistant to broad-spectrum penicillins in the presence or in the absence of beta-lactamase inhibitors but remained susceptible to cephalosporins . Resistance was due to production of a new TEM-type beta-lactamase, designated TEM-103/IRT-28, characterized by the Arg(275)Leu substitution and encoded by the ca . 62-kb pIP845 conjugative plasmid of the IncI1 incompatibility group.

Antimicrob Agents Chemother, 2002 Nov, 46(11), 3568 - 73
Resistance to beta-lactamase inhibitor protein does not parallel resistance to clavulanic acid in TEM beta-lactamase mutants; Schroeder WA et al.; In order to compare patterns of resistance to inhibition by clavulanic acid with patterns of resistance to inhibition by a beta-lactamase inhibitor protein (BLIP), R164S, R244S, and R164S/R244S mutant forms of TEM beta-lactamase were prepared by site-directed mutagenesis . When kinetic parameters were determined for these mutant and wild-type forms of TEM, the single mutants showed properties that were similar to those in the literature but the double mutant showed properties that were very different . The R164S/R244S double mutant form of TEM retained its resistance to inhibition by clavulanic acid (characteristic of the R244S mutation) but lost all its ability to hydrolyze ceftazidime (characteristic of the R164S mutation) . While these characteristics are contrary to those previously reported for an R164S/R244S double mutant, this discrepancy resulted from the use of a defective mutant in the earlier study . Both the single and double mutant forms of TEM remained highly sensitive when tested for inhibition by BLIP, showing only slightly increased resistance compared to that of the wild type; this pattern of resistance is quite different from the pattern of clavulanic acid resistance . The slight increases in resistance to inhibition by BLIP seen in the mutants may have been related to the fact that all of the mutations effected changes in the net charge on the TEM protein that could impede interactions with BLIP.

Plasmid, 2002 Sep, 48(2), 160 - 3
Construction of two pGEM-7Zf(+) phagemid T-tail vectors using AhdI-restriction endonuclease sites for direct cloning of PCR products; Jeung JU et al.; For applications such as sequencing, transfection, and in vitro transcription, PCR products have to be subcloned into plasmids . Many strategies are used for cloning, blunt-end ligation or the incorporation of restriction endonuclease sites into PCR primers for appropriate vectors . However, the most convenient and direct method is T/A cloning . In this study, we developed two of the pGEM-7Zf(+) phagemid T-tail vectors using AhdI-restriction endonuclease sites, and these T vectors have all the features of pGEM-7Zf(+): f1 ori, T7, and SP6 RNA polymerase promoters, the alpha-peptide coding region of beta-galactosidase for X-gal blue/white color selection, the beta-lactamase gene for recombinant colony selection, and binding sites for pUC/M13 forward and reverse sequencing primers . These AhdI-containing phagemid vectors, pGEM-NJ105 and pGEM-NJ107, are useful for the easy and inexpensive preparation of T vectors and direct cloning of PCR products.

Proc Natl Acad Sci U S A, 2002 Oct 15, 99(21), 13843 - 8 Epub 2002 Oct 04.
A dominant block to HIV-1 replication at reverse transcription in simian cells; Munk C et al.; Although nonhuman primates are genetically close to humans, their T cells do not support productive replication of HIV-1 . In contrast, HIV-1 replicates in activated human CD4(+) T cells, monocytes, and metabolically active human cells of a variety of cell types become permissive for HIV-1 replication when transduced to express CD4 and CCR5 or CXCR4 . The molecular basis of this species restriction to HIV-1 replication was investigated by using African green monkey and rhesus macaque cell lines that were stably transduced to express human CD4 and CCR5 . The cells supported replication of cognate viruses {simian immunodeficiency virus from African green monkeys (SIV-AGM) and macaques (SIVmac239)} but did not support replication of an R5-tropic cytopathic HIV-1 . A beta-lactamase-based HIV-1 entry assay was used to show that the virus efficiently entered the nonhuman primate cells . Provirus formation was reduced 50-fold compared with similarly infected human cells . Real-time PCR quantitation demonstrated that reverse transcription failed to initiate efficiently in the simian cells . The block to reverse transcription was overridden at multiplicity of infection >1 or by preincubation of the nonhuman primate cells with virus, a feature reminiscent of the Friend virus resistance gene-1 (FV-1), restriction to murine leukemia virus replication in mouse cells . Heterokaryon analysis in which human and simian cells were fused demonstrated that the block was dominant . These findings suggested that the primate cells contain a dominant inhibitor that prevents HIV-1 reverse transcription.

Farmaco, 2002 Aug, 57(8), 663 - 9
Synthesis and biological evaluation of 6-bromo-6-substituted penicillanic acid derivatives as beta-lactamase inhibitors; Bedini A et al.; The synthesis of a selected set of 6-bromopenicillanic acid derivatives with an additional C6 substituent is reported . All these substances were tested as inhibitors of class A and C beta-lactamase enzymes derived from Escherichia coli (TEM-1) and E . cloacae (P99) . As 6-(1-hydroxyethyl) derivatives 4c and 6c were found to be weak beta-lactamase inhibitors, they were further investigated in combination with amoxicillin against a series of beta-lactamase-producing bacterial strains . Some structure-activity relationships are discussed.

Nat Biotechnol, 2002 Nov, 20(11), 1151 - 4 Epub 2002 Sep 30.
A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes; Cavrois M et al.; As an early event in the viral life cycle, the entry of enveloped viruses into target cells has received considerable attention . Viral fusion to cellular targets has been studied principally with fusion assays in which cells engineered to express the viral envelope are cultured with the target cells . These assays yield valuable information but do not fully recapitulate all of the variables governing the fusion of actual virions to their cellular targets . The virion membrane and the plasma membrane, for example, differ strikingly in their lipid and protein compositions . Two virion-based fusion assays have been described . One is based on the redistribution of a self-quenching fluorophore, whereas the second depends on photosensitized activation of a hydrophobic probe by a fluorescent lipid loaded into the target membrane . These assays are complex and have not been adapted to study fusion in complex cell populations . We have developed a simple, rapid assay allowing the detection of HIV-1 virion fusion to biologically relevant target cells, including primary CD4(+) T lymphocytes . It is based on the incorporation of beta-lactamase-Vpr chimeric proteins (BlaM-Vpr) into HIV-1 virions and their subsequent delivery into the cytoplasm of target cells as a result of virion fusion . This transfer is then detected by enzymatic cleavage of the CCF2 dye, a fluorescent substrate of beta-lactamase (BlaM), loaded in the target cells . BlaM cleaves the beta-lactam ring in CCF2, changing its fluorescence emission spectrum from green (520 nm) to blue (447 nm) and thereby allowing fusion to be detected by fluorescence microscopy, flow cytometry, or UV photometry.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2002 Sep, 94(3), 301 - 4
Effects of 0.2% chlorhexidine gluconate and amoxicillin plus clavulanic acid on the prevention of alveolar osteitis following mandibular third molar extractions; Delilbasi C et al.; OBJECTIVE: The purpose of this study was to evaluate the use of a 0.2% chlorhexidine gluconate and amoxicillin plus clavulanic acid combination as a prophylactic therapy for the prevention of alveolar osteitis after mandibular third molar extractions and to investigate adverse reactions to chlorhexidine . STUDY DESIGN: This randomized, placebo-controlled, parallel group study was conducted in a group of 177 subjects, from which 3 groups were formed . The first group (n = 62) received 0.2% chlorhexidine gluconate, the second group (n = 56) received a 0.2% chlorhexidine gluconate and amoxicillin plus clavulanic acid combination, and the third group (n = 59) received 0.09% sterile saline solution . All patients were recalled for the diagnosis of alveolar osteitis on the third and seventh postoperative days . RESULTS: When patients in the antibiotic group were compared with those in the other 2 groups, a significant reduction in alveolar osteitis was noted (P <.05) . An alteration in taste, the bad taste of the solution, and staining of dentures and oral tissues were the major complaints about chlorhexidine . CONCLUSION: It would be more beneficial to use chlorhexidine solution with a beta-lactamase inhibitor-containing antibiotic to enhance its effectiveness for the prevention of alveolar osteitis.

Hua Xi Yi Ke Da Xue Xue Bao, 1999 Sep, 30(3), 245 - 8
{Sub-cloning and preliminary sequence analysis of the gene encoding of a cefoperazone hydrolyzing beta-lactamase isolated from Escherichia coli}; Bao Y et al.; Escherichia coli HX88108, which is resistant to cefoperazone(CPZ), was isolated from a severely infected patient . We studied genetical basis of beta-lactamase produced in E . coli HX88108 by pFL25, one of the recombinant plasmid of pFC . Largescale pFL25 plasmid was extracted, purified, and cleaved with restriction endonuclease EcoR I, Sal I, Pvu I, then subcloned into vector pUC19 as 1.9 kb, 0.9 kb, 0.65 kb fragments respectively . Recomminant plasmids were selected by alpha-complementation and determined by restriction endonuclease analysis . DNA sequencing was performed by the dideoxy polymerase chain termination method . Partial nucleotide sequence(1-78 nucleotide position) of the gene was found to be highly homologous (97%) with the gene coding for TEM-52 extended spectrum beta-lactamase of K . pneumonise, suggesting that the beta-lactamase coded by the cefoperazone resistant gene might be derived from TEM-type beta-lactamase.

J Comput Chem, 2002 Oct, 23(13), 1281 - 96
Binding of D- and L-captopril inhibitors to metallo-beta-lactamase studied by polarizable molecular mechanics and quantum mechanics; Antony J et al.; The bacterial Zn2+ metallo-beta-lactamase from B . fragilis is a zinc-enzyme with two potential metal ion binding sites . It cleaves the lactam ring of antibiotics, thus contributing to the acquired resistance of bacteria against antibiotics . The present study bears on the binuclear form of the enzyme . We compare several possible binding modes of captopril, a mercaptocarboxamide inhibitor of several zinc-metalloenzymes . Two diastereoisomers of captopril were considered, with either a D- or an L-proline residue . We have used the polarizable molecular mechanics procedure SIBFA (Sum of Interactions Between Fragments ab initio computed) . Two beta-lactamase models were considered, encompassing 104 and 188 residues, respectively . The energy balances included the inter and intramolecular interaction energies as well as the contribution from solvation computed using a continuum reaction field procedure . The thiolate ion of the inhibitor is binding to both metal ions, expelling the bridging solvent molecule from the uncomplexed enzyme . Different competing binding modes of captopril were considered, either where the inhibitor binds in a monodentate mode to the zinc cations only with its thiolate ion, or in bidentate modes involving additional zinc binding by its carboxylate or ketone carbonyl groups . The additional coordination by the inhibitor's carboxylate or carbonyl group always occurs at the zinc ion, which is bound by a histidine, a cysteine, and an aspartate side chain . For both diastereomers, the energy balances favor monodentate binding of captopril via S- . The preference over bidentate binding is small . The interaction energies were recomputed in model sites restricted to captopril, the Zn2+ cations, and their coordinating end side chains from beta-lactamase (98 atoms) . The interaction energies and their ranking among competing arrangements were consistent with those computed by ab initio HF and DFT procedures .

Biotechniques, 2002 Aug, 33(2), 326 - 8, 330
Normalization of transfection efficiency using the beta-lactamase gene of the pGL3 luciferase vector in primary anterior pituitary cells; Kang SW et al.; The beta-galactosidase reporter gene is commonly used as a control for transfection efficiency in the promoter reporter assay system . While investigating vasoactive intestinal peptide response elements in the promoter of the prolactin gene, we found that primary pituitary cells from turkey hens highly expressed endogenous beta-galactosidase . Therefore, we developed a new protocol for determining transfection efficiency using the beta-lactamase gene, which is present on many expression vectors . Transcript levels of beta-lactamase were measured by RT-PCR after transfection of different amounts of the pGL3-basic and pGL3-control vectors . A high correlation was observed between the amount of plasmid transfected and beta-lactamase mRNA levels . Although no eukaryotic promoter was present, there was apparently leaky expression of the beta-lactamase gene . Expression of beta-lactamase was independent of expression from the simian virus 40 or turkey prolactin promoters cloned upstream of the luciferase gene.

Antimicrob Agents Chemother, 2002 Sep, 46(9), 3045 - 9
Beta-lactamases of Kluyvera ascorbata, probable progenitors of some plasmid-encoded CTX-M types; Humeniuk C et al.; Kluyvera ascorbata produces a beta-lactamase that results in an atypical susceptibility pattern, including low-level resistance to penicillins, cephalothin, and cefuroxime, but this resistance is reversed by clavulanate . Ten nucleotide sequences of the corresponding gene, bla(KLUA), were obtained and were found to have minor variations (96 to 100%) . Otherwise, bla(KLUA) was found to be similar (95 to 100%) to some plasmid-encoded CTX-M-type beta-lactamases . Finally, mobilization of bla(KLUA) on a plasmid was found to be mediated probably by a genetic mobile element like ISEcp1.

Nucleic Acids Res . 2002 Aug 15;30(16):e84.
Novel ceftazidime-resistance beta-lactamases generated by a codon-based mutagenesis method and selection; Gaytan P et al.; Four known and nine new ceftazidime-resistance beta-lactamases were generated by a novel, contaminating codon-based mutagenesis approach . In this method, wild-type codons are spiked with a set of mutant codons during oligonucleotide synthesis, generating random combinatorial libraries of primers that contain few codon replacements per variant . Mutant codons are assembled by tandem addition of a diluted mixture of five Fmoc-dimer amidites to the growing oligo and a mixture of four DMTr-monomer amidites to generate 20 trinucleotides that encode a set of 18 amino acids . Wild-type codons are assembled with conventional chemistry and the whole process takes place in only one synthesis column, making its automation feasible . The random and binomial behavior of this approach was tested in the polylinker region of plasmid pUC19 by the synthesis of three oligonucleotide libraries mutagenized at different rates and cloned as mutagenic cassettes . Additionally, the method was biologically assessed by mutating six contiguous codons that encode amino acids 237-243 (ABL numbering) of the TEM(pUC19) beta-lactamase, which is functionally equivalent to the clinically important TEM-1 beta-lactamase . The best ceftazidime-recognizing variant was a triple mutant, R164H:E240K: R241A, displaying a 333-fold higher resistance than the wild-type enzyme.

J Am Chem Soc, 2002 Aug 14, 124(32), 9396 - 403
In vitro selection for catalytic activity with ribosome display; Amstutz P et al.; We report what is, to our knowledge, the first in vitro selection for catalytic activity based on catalytic turnover by using ribosome display, a method which does not involve living cells at any step . RTEM-beta-lactamase was functionally displayed on ribosomes as a complex with its encoding mRNA . We designed and synthesized a mechanism-based inhibitor of beta-lactamase, biotinylated ampicillin sulfone, appropriate for selection of catalytic activity of the ribosome-displayed beta-lactamase . This derivative of ampicillin inactivated beta-lactamase in a specific and irreversible manner . Under appropriate selection conditions, active RTEM-beta-lactamase was enriched relative to an inactive point mutant over 100-fold per ribosome display selection cycle . Selection for binding, carried out with beta-lactamase inhibitory protein (BLIP), gave results similar to selection with the suicide inhibitor, indicating that ribosome display is similarly efficient in catalytic activity and affinity selections . In the future, the capacity to select directly for enzymatic activity using an entirely in vitro process may allow for a significant increase in the explorable sequence space relative to existing strategies.

J Mol Biol, 2002 Aug 9, 321(2), 285 - 96
Structural bases of stability-function tradeoffs in enzymes; Beadle BM et al.; The structures of enzymes reflect two tendencies that appear opposed . On one hand, they fold into compact, stable structures; on the other hand, they bind a ligand and catalyze a reaction . To be stable, enzymes fold to maximize favorable interactions, forming a tightly packed hydrophobic core, exposing hydrophilic groups, and optimizing intramolecular hydrogen-bonding . To be functional, enzymes carve out an active site for ligand binding, exposing hydrophobic surface area, clustering like charges, and providing unfulfilled hydrogen bond donors and acceptors . Using AmpC beta-lactamase, an enzyme that is well-characterized structurally and mechanistically, the relationship between enzyme stability and function was investigated by substituting key active-site residues and measuring the changes in stability and activity . Substitutions of catalytic residues Ser64, Lys67, Tyr150, Asn152, and Lys315 decrease the activity of the enzyme by 10(3)-10(5)-fold compared to wild-type . Concomitantly, many of these substitutions increase the stability of the enzyme significantly, by up to 4.7kcal/mol . To determine the structural origins of stabilization, the crystal structures of four mutant enzymes were determined to between 1.90A and 1.50A resolution . These structures revealed several mechanisms by which stability was increased, including mimicry of the substrate by the substituted residue (S64D), relief of steric strain (S64G), relief of electrostatic strain (K67Q), and improved polar complementarity (N152H) . These results suggest that the preorganization of functionality characteristic of active sites has come at a considerable cost to enzyme stability . In proteins of unknown function, the presence of such destabilized regions may indicate the presence of a binding site.

Arch Biochem Biophys, 2002 Aug 1, 404(1), 147 - 57
The plastid translocon component TOC36 exhibits an affinity for the bacterial protein translocation process; Gordon B et al.; The 44-kDa envelope polypeptides are active components of the plastid translocon, but their role in plastid protein import remains elusive . One form from Brassica napus (bnToc36B) was previously observed to exert a significant overall effect on bacterial protein translocation, but the nature of the influence requires further characterization . The experimental strategies employed in this study thus focus specifically on the nature of the bnToc36B-bacterial Sec translocon relationship to gain an understanding of Toc36's function . BnToc36B's presence in bacteria created a number of effects related to the protein transport process that together point to functional interactions with the bacterial Sec translocon . These effects are (1) reduced sensitivity to azide impairment as measured by a higher recovery rate from azide treatment, (2) reduced sensitivity to suboptimal temperatures manifesting as sustained levels of protein synthesis and translocation, (3) sustained levels of growth and beta-lactamase transport in high ampicillin concentrations, and (4) evidence for a physical affinity for the bacterial translocon . A reduction in overall SecA levels and a more stable SecA profile, when subjected to azide treatment, was observed in bnToc36B-containing bacteria . The implications of the bacterial data are discussed.

J Biol Chem, 2002 Oct 18, 277(42), 39713 - 21 Epub 2002 Jul 16.
Spontaneous calcium oscillations control c-fos transcription via the serum response element in neuroendocrine cells; Maturana A et al.; In excitable cells the localization of Ca2+ signals plays a central role in the cellular response, especially in the control of gene transcription . To study the effect of localized Ca2+ signals on the transcriptional activation of the c-fos oncogene, we stably expressed various c-fos beta-lactamase reporter constructs in pituitary AtT20 cells . A significant, but heterogenous expression of c-fos beta-lactamase was observed in unstimulated cells, and a further increase was observed using KCl depolarization, epidermal growth factor (EGF), pituitary adenylate cyclase-activating polypeptide (PACAP), and serum . The KCl response was almost abolished by a nuclear Ca2+ clamp, indicating that a rise in nuclear Ca2+ is required . In contrast, the basal expression was not affected by the nuclear Ca2+ clamp, but it was strongly reduced by nifedipine, a specific antagonist of l-type Ca2+ channels . Spontaneous Ca2+ oscillations, blocked by nifedipine, were observed in the cytosol but did not propagate to the nucleus, suggesting that a rise in cytosolic Ca2+ is sufficient for basal c-fos expression . Inactivation of the c-fos promoter cAMP/Ca2+ response element (CRE) had no effect on basal or stimulated expression, whereas inactivation of the serum response element (SRE) had the same marked inhibitory effect as nifedipine . These experiments suggest that in AtT20 cells spontaneous Ca2+ oscillations maintain a basal c-fos transcription through the serum response element . Further induction of c-fos expression by depolarization requires a nuclear Ca2+ increase.

Structure (Camb), 2002 Jul, 10(7), 1013 - 23
Structure-based discovery of a novel, noncovalent inhibitor of AmpC beta-lactamase; Powers RA et al.; beta-lactamases are the most widespread resistance mechanisms to beta-lactam antibiotics, and there is a pressing need for novel, non-beta-lactam drugs . A database of over 200,000 compounds was docked to the active site of AmpC beta-lactamase to identify potential inhibitors . Fifty-six compounds were tested, and three had K(i) values of 650 microM or better . The best of these, 3-{(4-chloroanilino)sulfonyl}thiophene-2-carboxylic acid, was a competitive noncovalent inhibitor (K(i) = 26 microM), which also reversed resistance to beta-lactams in bacteria expressing AmpC . The structure of AmpC in complex with this compound was determined by X-ray crystallography to 1.94 A and reveals that the inhibitor interacts with key active-site residues in sites targeted in the docking calculation . Indeed, the experimentally determined conformation of the inhibitor closely resembles the prediction . The structure of the enzyme-inhibitor complex presents an opportunity to improve binding affinity in a novel series of inhibitors discovered by structure-based methods.

Eur J Pharm Sci, 2002 Jul, 16(1-2), 45 - 51
Evaluation of the role of intestinal and liver metabolism in the conversion of two different ester prodrugs of sanfetrinem to the parent drug in vitro and in vivo using different rat tissues and a surgically prepared rat model; Braggio S et al.; To improve oral absorption of sanfetrinem, a broad-spectrum, beta-lactamase-stable antibiotic, two different ester prodrugs have been selected . Both prodrugs proved to be readily hydrolyzed after absorption before reaching the systemic circulation . The objective of this study was to evaluate the role of intestinal and liver metabolism in the conversion of the two prodrugs into the active compound . In vitro experiments were performed in different rat tissues involved in the absorption process . Moreover data obtained with in vitro experiments have been integrated with data obtained in vivo using a surgically prepared rat model which allows for the measurement of the amount of intact prodrug that overcomes the intestinal mucosa and its presence in the portal vein . Both prodrugs proved to be readily cleaved by jejunum and liver microsomes . The rates of ester hydrolysis with these two tissues were 10- to 30-fold higher than those calculated in intestinal juice at pH 7.4 and about 100-fold higher than in buffer at pH 5.5 . These data suggest that both the intestinal wall and liver could play an important role in the conversion of the two prodrugs in active parent compound . In the in vivo experiment, relative to sanfetrinem levels, very low concentrations of intact esters were measured in the portal vein blood, indicating that the two prodrugs are nearly completely hydrolyzed to the active drug by the intestinal wall . In conclusion this study demonstrated that the intestinal epithelium plays a major role in the conversion of the two prodrugs into sanfetrinem . The liver, despite its high esterase activity seems to be only marginally involved.

J Med Chem, 2002 Jul 18, 45(15), 3222 - 34
Structure-based approach for binding site identification on AmpC beta-lactamase; Powers RA et al.; Beta-lactamases are the most widespread resistance mechanism to beta-lactam antibiotics and are an increasing menace to public health . Several beta-lactamase structures have been determined, making this enzyme an attractive target for structure-based drug design . To facilitate inhibitor design for the class C beta-lactamase AmpC, binding site "hot spots" on the enzyme were identified using experimental and computational approaches . Experimentally, X-ray crystal structures of AmpC in complexes with four boronic acid inhibitors and a higher resolution (1.72 A) native apo structure were determined . Along with previously determined structures of AmpC in complexes with five other boronic acid inhibitors and four beta-lactams, consensus binding sites were identified . Computationally, the programs GRID, MCSS, and X-SITE were used to predict potential binding site hot spots on AmpC . Several consensus binding sites were identified from the crystal structures . An amide recognition site was identified by the interaction between the carbonyl oxygen in the R1 side chain of beta-lactams and the atom Ndelta2 of the conserved Asn152 . Surprisingly, this site also recognizes the aryl rings of arylboronic acids, appearing to form quadrupole-dipole interactions with Asn152 . The highly conserved "oxyanion" hole defines a site that recognizes both carbonyl and hydroxyl groups . A hydroxyl binding site was identified by the O2 hydroxyl in the boronic acids, which hydrogen bonds with Tyr150 and a conserved water . A hydrophobic site is formed by Leu119 and Leu293 . A carboxylate binding site was identified by the ubiquitous C3(4) carboxylate of the beta-lactams, which interacts with Asn346 and Arg349 . Four water sites were identified by ordered waters observed in most of the structures; these waters form extensive hydrogen-bonding networks with AmpC and occasionally the ligand . Predictions by the computational programs showed some correlation with the experimentally observed binding sites . Several sites were not predicted, but novel binding sites were suggested . Taken together, a map of binding site hot spots found on AmpC, along with information on the functionality recognized at each site, was constructed . This map may be useful for structure-based inhibitor design against AmpC.

J Pept Res, 2002 Aug, 60(2), 75 - 80
Beta-turn formation by a six-residue linear peptide in solution; Gao F et al.; A model peptide AAGDYY-NH2 (B1), which is found to adopt a beta-turn conformation in the TEM-1 beta-lactamase inhibitor protein (BLIP) in the TEM-1/BLIP co-crystal, was synthesized to elucidate the mechanism of its beta-turn formation and stability . Its structural preferences in solution were comprehensively characterized using CD, FT-IR and 1H NMR spectroscopy, respectively . The set of observed diagnostic NOEs, the restrained molecular dynamics simulation, CD and FT-IR spectroscopy confirmed the formation of a beta-turn in solution by the model peptide . The dihedral angles {(phi3, phi3) (phi4, phi4)} of {(-52 degrees, -32 degrees ) (-38 degrees, -44 degrees )} of Gly-Asp fragment in the model peptide are consistent with those of a type III beta-turn . In a conclusion, the conformational preference of the linear hexapeptide B1 in solution was determined, and it would provide a simple template to study the mechanism of beta-turn formation and stability.

Biochim Biophys Acta, 2002 Aug 19, 1564(1), 47 - 56
Stability of the lactose permease in detergent solutions; Engel CK et al.; Protein stability, as measured by irreversible protein aggregation, is one of the central difficulties in the handling of detergent-solubilized membrane proteins . We present a quantitative analysis of the stability of the Escherichia coli lactose (lac) permease and a series of lac permease fusion proteins containing an insertion of cytochrome(b562), T4 lysozyme or beta-lactamase in the central hydrophilic loop of the permease . The stability of the proteins was evaluated under a variety of storage conditions by both a qualitative SDS-PAGE assay and by a quantitative hplc assay . Long-chain maltoside detergents were more effective at maintaining purified protein in solution than detergents with smaller head groups and/or shorter alkyl tails . A full factorial experiment established that the proteins were insensitive to sodium chloride concentrations, but greatly stabilized by glycerol, low temperature and the combination of glycerol and low temperature . The accurate quantitation of the protein by absorbance spectroscopy required exclusion of all contact with clarified polypropylene or polyvinyl chloride (PVC) materials . Although some of the fusion proteins were more prone to aggregation than the wild-type permease, the stability of a fusion protein containing a cytochrome(b562) insertion was indistinguishable from that of native lac permease.

Biochim Biophys Acta, 2002 Aug 19, 1564(1), 38 - 46
Insertion of carrier proteins into hydrophilic loops of the Escherichia coli lactose permease; Engel CK et al.; We describe the design and characterization of a set of fusion proteins of the Escherichia coli lactose (lac) permease in which a set of five different soluble "carrier" proteins (cytochrome(b562), flavodoxin, T4 lysozyme, beta-lactamase and 70 kDa heat shock ATPase domain) were systematically inserted into selected loop positions of the transporter . The design goal was to increase the exposed hydrophilic surface area of the permease, while minimizing the internal flexibility of the resulting fusion proteins in order to improve the crystallization properties of the membrane protein . Fusion proteins with insertions into the central hydrophilic loop of the lac permease were active in transport lactose, although only the fusion proteins with E . coli cytochrome(b562), E . coli flavodoxin or T4 lysozyme were expressed at near wild-type lac permease levels . Eight other loop positions were tested with these three carriers, leading to the identification of additional fusion proteins that were active and well-expressed . By combining the results from the single carrier insertions, we have expressed functional "double fusion" proteins containing cytochrome(b562) domains inserted in two different loop positions.

Microbiology, 2002 Jun, 148(Pt 6), 1757 - 65
The Escherichia coli small heat-shock proteins IbpA and IbpB prevent the aggregation of endogenous proteins denatured in vivo during extreme heat shock; Kuczynska-Wisnik D et al.; The roles of the Escherichia coli IbpA and IbpB chaperones in protection of heat-denatured proteins against irreversible aggregation in vivo were investigated . Overproduction of IbpA and IbpB resulted in stabilization of the denatured and reversibly aggregated proteins (the S fraction), which could be isolated from E . coli cells by sucrose gradient centrifugation . This finding is in agreement with the present model of the small heat-shock proteins' function, based mainly on in vitro studies . Deletion of the ibpAB operon resulted in almost twofold increase in protein aggregation and in inactivation of an enzyme (fructose-1,6-biphosphate aldolase) in cells incubated at 50 degrees C for 4 h, decreased efficiency of the removal of protein aggregates formed during prolonged incubation at 50 degrees C and affected cell viability at this temperature . IbpA/B proteins were not needed for removal of protein aggregates or for the enzyme protection/renaturation in cells heat shocked at 50 degrees C for 15 min . These results show that the IbpA/B proteins are required upon an extreme, long-term heat shock . Overproduction of IbpA but not IbpB caused an increase of the level of beta-lactamase precursor, which was localized in the S fraction, together with the IbpA protein, which suggests that the unfolded precursor binds to IbpA but not to IbpB . Although in the wild-type cells both E . coli small heat-shock proteins are known to localize in the S fraction, only 2% of total IbpB co-localized with the aggregated proteins in the absence of IbpA, while in the absence of IbpB, the majority of IbpA was present in the aggregates fraction.

FEMS Microbiol Lett, 2002 May 21, 211(1), 13 - 6
Substitution of Met-69 by Ala or Gly in TEM-1 beta-lactamase confer an increased susceptibility to clavulanic acid and other inhibitors; Madec S et al.; In some inhibitor-resistant TEM-derived beta-lactamases, Met-69 is substituted by Leu, Ile or Val . Residue 69 is located in a region of strong structural constraints, at the beginning of H2 alpha-helix, and in the vicinity of B3 and B4 beta-strands . Analysis of the three-dimensional structure of TEM-1 beta-lactamase suggests that alteration of the substrate-binding site can be produced by changes of the size of residue 69 side chain . Met-69 was substituted by alanine or glycine in TEM-Bs beta-lactamase (a TEM-1-related enzyme) using site-directed mutagenesis . The minimum inhibitory concentrations of the mutants compared with the wild-type revealed an increased susceptibility to beta-lactamase inhibitor-beta-lactam combinations and to first-generation cephalosporins . Comparing the Met69Ala and Met69Gly beta-lactamases with TEM-Bs, K(m) constants of the mutants showed an increased affinity for most beta-lactams but the kcat for most substrates did not change substantially . Mutants also demonstrated lower IC50 for the three inhibitors (clavulanic acid, tazobactam and sulbactam) . The two substitutions of the residue 69 by alanine and glycine had a noticeable effect on K(m) values of TEM-Bs beta-lactamase, and on affinity for beta-lactamase inhibitors.

Acta Crystallogr C, 2002 Jun, 58(Pt 6), o367 - 9 Epub 2002 May 31.
Precursor of a beta-lactamase inhibitor: allyl (4S,8S,9R)-10-{(E)-ethylidene}-4-methoxy-11-oxo-1-azatricyclo{7.2.0.0(3,8)}undec-2-ene-2-carboxylate; Leban I et al.; The molecular structure of the title tricyclic compound, C(17)H(21)NO(4), which is the immediate precursor of a potent synthetic inhibitor {Lek157: sodium (8S,9R)-10-{(E)-ethylidene}-4-methoxy-11-oxo-1-azatricyclo{7.2.0.0(3,8)}undec-2-ene-2-carboxylate} with remarkable potency, provides experimental evidence for the previously modelled relative position of the fused cyclohexyl ring and the carbonyl group of the beta-lactam ring, which takes part in the formation of the initial tetrahedral acyl-enzyme complex . In this hydrophobic molecule, the overall geometry is influenced by C{bond}H...O intramolecular hydrogen bonds {3.046 (4) and 3.538 (6) A, with corresponding normalized H.O distances of 2.30 and 2.46 A}, whereas the molecules are interconnected through intermolecular C{bond}H...O hydrogen bonds {3.335 (4)-3.575 (5) A}.

Phytochemistry, 2002 Jun, 60(3), 295 - 9
Inhibition of alpha-glucosidase by oleanolic acid and its synthetic derivatives; Ali MS et al.; Oleanolic acid (1) and five synthetic derivatives (2-6) were tested spectrophotometrically for inhibition of urease, beta-lactamase, acetyl cholinesterase and alpha-glucosidase . All products showed a positive response only against alpha-glucosidase but not against the other enzymes; IC(50) calculations showed that the dihydroxy-olide derivative (4) was the most potent among all tested samples.

Protein Sci, 2002 Jun, 11(6), 1506 - 18
TEM-1 beta-lactamase as a scaffold for protein recognition and assay; Legendre D et al.; A large number of different proteins or protein domains have been investigated as possible scaffolds to engineer antibody-like molecules . We have previously shown that the TEM-1 beta-lactamase can accommodate insertions of random sequences in two loops surrounding its active site without compromising its activity . From the libraries that were generated, active enzymes binding with high affinities to monoclonal antibodies raised against prostate-specific antigen, a protein unrelated to beta-lactamase, could be isolated . Antibody binding was shown to affect markedly the enzyme activity . As a consequence, these enzymes have the potential to be used as signaling molecules in direct or competitive homogeneous immunoassay . Preliminary results showed that beta-lactamase clones binding to streptavidin could also be isolated, indicating that some enzymes in the libraries have the ability to recognize proteins other than antibodies . In this paper, we show that, in addition to beta-lactamases binding to streptavidin, beta-lactamase clones binding to horse spleen ferritin and beta-galactosidase could be isolated . Affinity maturation of a clone binding to ferritin allowed obtaining beta-lactamases with affinities comprised between 10 and 20 nM (Kd) for the protein . Contrary to what was observed for beta-lactamases issued from selections on antibodies, enzyme complexation induced only a modest effect on enzyme activity, in the three cases studied . This kind of enzyme could prove useful in replacement of enzyme-conjugated antibodies in enzyme-linked immunosorbant assays (ELISA) or in other applications that use antibodies conjugated to an enzyme.

Biochem J, 2002 Sep 1, 366(Pt 2), 423 - 34
Oligomeric structure of proclavaminic acid amidino hydrolase: evolution of a hydrolytic enzyme in clavulanic acid biosynthesis; Elkins JM et al.; During biosynthesis of the clinically used beta-lactamase inhibitor clavulanic acid, one of the three steps catalysed by clavaminic acid synthase is separated from the other two by a step catalysed by proclavaminic acid amidino hydrolase (PAH), in which the guanidino group of an intermediate is hydrolysed to give proclavaminic acid and urea . PAH shows considerable sequence homology with the primary metabolic arginases, which hydrolyse arginine to ornithine and urea, but does not accept arginine as a substrate . Like other members of the bacterial sub-family of arginases, PAH is hexameric in solution and requires Mn2+ ions for activity . Other metal ions, including Co2+, can substitute for Mn2+ . Two new substrates for PAH were identified, N-acetyl-(L)-arginine and (3R)-hydroxy-N-acetyl-(L)-arginine . Crystal structures of PAH from Streptomyces clavuligerus (at 1.75 A and 2.45 A resolution, where 1 A=0.1 nm) imply how it binds beta-lactams rather than the amino acid substrate of the arginases from which it evolved . The structures also suggest how PAH selects for a particular alcohol intermediate in the clavam biosynthesis pathway . As observed for the arginases, each PAH monomer consists of a core of beta-strands surrounded by alpha-helices, and its active site contains a di-Mn2+ centre with a bridging water molecule responsible for hydrolytic attack on to the guanidino group of the substrate . Comparison of structures obtained under different conditions reveals different conformations of a flexible loop, which must move to allow substrate binding.

Int J Clin Pract Suppl, 2002 Mar, (125), 2 - 9; discussion 37-9
The emergence of beta-lactamase resistance in respiratory pathogens; Gur D; Bacteria have evolved a variety of mechanisms to express resistance to beta-lactam antibiotics . beta-Lactamase-induced hydrolysis of the beta-lactam ring is the principal and most important mediator of clinically significant resistance . Almost 200 beta-lactamases have now been identified, of which class 1 chromosomal beta-lactamases, class 2b plasmid-mediated beta-lactamases (especially TEM-1) and class 2be extended-spectrum beta-lactamases (ESBLs) are among the most important in respiratory pathogens . The combination of enzymatic and non-enzymatic resistance mechanisms has led to a steady rise in the prevalence of resistance to beta-lactams among isolates of the major respiratory pathogens, and, in turn, increasing rates of treatment failure, increased mortality, and prolonged morbidity . The combination of a beta-lactam antibiotic with a beta-lactamase inhibitor such as sulbactam, which protects the antibiotic from beta-lactamase destruction and so restores its activity, provides an innovative solution to this problem.

Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 6749 - 54
Identification of four candidate cGMP targets in Dictyostelium; Goldberg JM et al.; In Dictyostelium, a transient increase in intracellular cGMP is important for cytoskeletal rearrangements during chemotaxis . There must be cGMP-binding proteins in Dictyostelium that regulate key cytoskeletal components after treatment with chemoattractants, but to date, no such proteins have been identified . Using a bioinformatics approach, we have found four candidate cGMP-binding proteins (GbpA-D) . GbpA and -B have two tandem cGMP-binding sites downstream of a metallo beta-lactamase domain, a superfamily that includes cAMP phosphodiesterases . GbpC contains the following nine domains (in order): leucine-rich repeats, Ras, MEK kinase, Ras guanine nucleotide exchange factor N-terminal (RasGEF-N), DEP, RasGEF, cGMP-binding, GRAM, and a second cGMP-binding domain . GbpD is related to GbpC, but is much shorter; it begins with the RasGEF-N domain, and lacks the DEP domain . Disruption of the gbpC gene results in loss of all high-affinity cGMP-binding activity present in the soluble cellular fraction . GbpC mRNA levels increase dramatically 8 h after starvation is initiated . GbpA, -B, and -D mRNA levels show less dramatic changes, with gbpA mRNA levels highest 4 h into starvation, gbpB mRNA levels highest in vegetative cells, and gbpD levels highest at 8 h . The identification of these genes is the first step in a molecular approach to studying downstream effects of cGMP signaling in Dictyostelium.

Structure (Camb), 2002 Mar, 10(3), 413 - 24
Structural milestones in the reaction pathway of an amide hydrolase: substrate, acyl, and product complexes of cephalothin with AmpC beta-lactamase; Beadle BM et al.; Beta-lactamases hydrolyze beta-lactam antibiotics and are the leading cause of bacterial resistance to these drugs . Although beta-lactamases have been extensively studied, structures of the substrate-enzyme and product-enzyme complexes have proven elusive . Here, the structure of a mutant AmpC in complex with the beta-lactam cephalothin in its substrate and product forms was determined by X-ray crystallography to 1.53 A resolution . The acyl-enzyme intermediate between AmpC and cephalothin was determined to 2.06 A resolution . The ligand undergoes a dramatic conformational change as the reaction progresses, with the characteristic six-membered dihydrothiazine ring of cephalothin rotating by 109 degrees . These structures correspond to all three intermediates along the reaction path and provide insight into substrate recognition, catalysis, and product expulsion.

Ear Nose Throat J, 2002 Apr, 81(4), 234 - 6, 238-40, 242 passim
Bilateral Lemierre's syndrome: a case report and literature review; Moore BA et al.; Lemierre's syndrome is characterized by thrombosis of the internal jugular vein that develops following an oropharyngeal infection . Sepsis and septic metastases frequently ensue and affect the lungs, the musculoskeletal system, and occasionally the liver . Most cases are caused by infection with Fusobacterium necrophorum . This infection responds to antibiotic therapy with beta-lactamase-resistant compounds that exert good anaerobic coverage . Anticoagulation and surgical intervention can be helpful in advanced cases . Fewer than 160 cases of classic Lemierre's syndrome have been described; approximately one-third of these reported cases have occurred since 1988 . We describe a new case of Lemierre's syndrome that occurred in an otherwise healthy 27-year-old man . Thrombosis of both internal jugular veins extended through the subclavian system and into both upper extremities . The patient was treated with intravenous antibiotics and heparin during 14 days of hospitalization . He was discharged on oral clindamycin and warfarin sodium, and after 6 months he was able to return to full activity . To our knowledge, this is the first reported case of Lemierre's syndrome in which internal jugular vein thrombosis occurred bilaterally . By reporting this previously undescribed manifestation of Lemierre's syndrome, we hope to increase practitioner awareness of this disease entity.

Bioorg Med Chem Lett, 2002 Mar 25, 12(6), 971 - 5
Design, synthesis and bioactivity evaluation of tribactam beta lactamase inhibitors; Copar A et al.; Known carbapenem compounds with inhibitory effect towards beta-lactamase enzymes are formed from bicyclical beta lactam structural scaffolds . On the basis of results from theoretical computational methods and molecular modelling we have designed and developed a synthetic route towards novel, biologically active tricyclic derivatives of carbapenems.

J Med Chem, 2002 Apr 11, 45(8), 1712 - 22
A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening; McGovern SL et al.; High-throughput and virtual screening are widely used to discover novel leads for drug design . On examination, many screening hits appear non-drug-like: they act noncompetitively, show little relationship between structure and activity, and have poor selectivity . Attempts to develop these peculiar molecules into viable leads are often futile, and much time can be wasted on the characterization of these "phony" hits . Despite their common occurrence, the mechanism of action of these promiscuous molecules remains unknown . To investigate this problem, 45 diverse screening hits were studied . Fifteen of these were previously reported as inhibitors of various receptors, including beta-lactamase, malarial protease, dihydrofolate reductase, HIV Tar RNA, thymidylate synthase, kinesin, insulin receptor, tyrosine kinases, farnesyltransferase, gyrase, prions, triosephosphate isomerase, nitric oxide synthase, phosphoinositide 3-kinase, and integrase; 30 were from an in-house screening library of a major pharmaceutical company . In addition to their original targets, 35 of these 45 compounds were shown to inhibit several unrelated model enzymes . These 35 screening hits included compounds, such as fullerenes, dyes, and quercetin, that have repeatedly shown activity against diverse targets . When tested against the model enzymes, the compounds showed time-dependent but reversible inhibition that was dramatically attenuated by albumin, guanidinium, or urea . Surprisingly, increasing the concentration of the model enzymes 10-fold largely eliminated inhibition, despite a 1000-fold excess of inhibitor; a well-behaved competitive inhibitor did not show this behavior . One model to explain these observations was that the active form of the promiscuous inhibitors was an aggregate of many individual molecules . To test this hypothesis, light scattering and electron microscopy experiments were performed . The nonspecific inhibitors were observed to form particles of 30-400 nm diameter by both techniques . In control experiments, a well-behaved competitive inhibitor and an inactive dye-like molecule were not observed to form aggregates . Consistent with the hypothesis that the aggregates are the inhibitory species, the particle size and IC(50) values of the promiscuous inhibitors varied monotonically with ionic strength; a competitive inhibitor was unaffected by changes in ionic strength . Unexpectedly, aggregate formation appears to explain the activity of many nonspecific inhibitors and may account for the activity of many promiscuous screening hits . Molecules acting via this mechanism may be widespread in drug discovery screening databases . Recognition of these compounds may improve screening results in many areas of pharmaceutical interest.

J Clin Microbiol, 2002 Apr, 40(4), 1546 - 8
Production of BRO beta-lactamases and resistance to complement in European Moraxella catarrhalis isolates; Schmitz FJ et al.; Of the 419 Moraxella catarrhalis isolates collected during the 1997-1999 European SENTRY surveillance study, 385 (92%) were beta-lactamase positive . Twenty-two (5.7%) produced BRO-2 beta-lactamase . Twenty-one new mutations were found in the putative promoter region of the bro genes . Nineteen percent of all isolates tested were complement sensitive . Resistance to beta-lactams is not linked to the phylogenetic lineages associated with susceptibility to complement.

Proc Natl Acad Sci U S A, 2002 Mar 19, 99(6), 3469 - 74
Protein-protein interactions monitored in mammalian cells via complementation of beta -lactamase enzyme fragments; Wehrman T et al.; We have defined inactive alpha and omega fragments of beta-lactamase that can complement to form a functional enzyme in both bacteria and mammalian cells, serving as a readout for the interaction of proteins fused to the fragments . Critical to this advance was the identification of a tripeptide, Asn-Gly-Arg, which when juxtaposed at the carboxyl terminus of the alpha fragment increased complemented enzyme activity by up to 4 orders of magnitude . beta-Lactamase is well suited to monitoring constitutive and inducible protein interactions because it is small (29 kDa), monomeric, and assayable with a fluorescent cell-permeable substrate . The negligible background, the magnitude of induced signal caused by enzymatic amplification, and detection of signal within minutes are unparalleled in mammalian protein interaction detection systems published to date.

Genetics, 2002 Mar, 160(3), 823 - 32
Predicting evolutionary potential: in vitro evolution accurately reproduces natural evolution of the tem beta-lactamase; Barlow M et al.; To evaluate the validity of our in vitro evolution method as a model for natural evolutionary processes, the TEM-1 beta-lactamase gene was evolved in vitro and was selected for increased resistance to cefotaxime, cefuroxime, ceftazadime, and aztreonam, i.e., the "extended-spectrum" phenotype . The amino acid substitutions recovered in 10 independent in vitro evolvants were compared with the amino acid substitutions in the naturally occurring extended-spectrum TEM alleles . Of the nine substitutions that have arisen multiple times in naturally occurring extended-spectrum TEM alleles, seven were recovered multiple times in vitro . We take this result as evidence that our in vitro evolution technique accurately mimics natural evolution and can therefore be used to predict the results of natural evolutionary processes . Additionally, our results predict that a phenotype not yet observed among TEM beta-lactamases in nature-resistance to cefepime-is likely to arise in nature.

Antimicrob Agents Chemother, 2002 Apr, 46(4), 966 - 70
Molecular and biochemical characterization of Ambler class A extended-spectrum beta-lactamase CGA-1 from Chryseobacterium gleum; Bellais S et al.; Antibiotic susceptibility testing by disk diffusion of a Chryseobacterium gleum isolate, strain CIP 103039, showed a typical synergy image between clavulanic acid and expanded-spectrum cephalosporins . Shotgun cloning gave a recombinant plasmid in Escherichia coli that produced a beta-lactamase, CGA-1, with a pI value of 8.9 that conferred resistance to most penicillins (except ureidopenicillins) and narrow-spectrum cephalosporins and an intermediate susceptibility to expanded-spectrum cephalosporins and aztreonam . The CGA-1 amino acid sequence shared only 60% amino acid identity with CME-1 and CME-2 from Chryseobacterium meningosepticum, the most closely related beta-lactamases . CGA-1 was very likely chromosome encoded . It is a novel member of the PER subgroup of Ambler class A beta-lactamases (Bush functional group 2be).

Proc Natl Acad Sci U S A, 2002 Mar 19, 99(6), 3651 - 6 Epub 2002 Mar 12.
Design of an HIV-1 lentiviral-based gene-trap vector to detect developmentally regulated genes in mammalian cells; Lai Z et al.; The recent development of HIV-1 lentiviral vectors is especially useful for gene transfer because they achieve efficient integration into nondividing cell genomes and successful long-term expression of the transgene . These attributes make the vector useful for gene delivery, mutagenesis, and other applications in mammalian systems . Here we describe two HIV-1-based lentiviral vector derivatives, pZR-1 and pZR-2, that can be used in gene-trap experiments in mammalian cells in vitro and in vivo . Each lentiviral gene-trap vector contains a reporter gene, either beta-lactamase or enhanced green fluorescent protein (EGFP), that is inserted into the U3 region of the 3' long terminal repeat . Both of the trap vectors readily integrate into the host genome by using a convenient infection technique . Appropriate insertion of the vector into genes causes EGFP or beta-lactamase expression . This technique should facilitate the rapid enrichment and cloning of the trapped cells and provides an opportunity to select subpopulations of trapped cells based on the subcellular localization of reporter genes . Our findings suggest that the reporter gene is driven by an upstream, cell-specific promoter during cell culture and cell differentiation, which further supports the usefulness of lentivirus-based gene-trap vectors . Lentiviral gene-trap vectors appear to offer a wealth of possibilities for the study of cell differentiation and lineage commitment, as well as for the discovery of new genes.

J Am Chem Soc, 2002 Mar 20, 124(11), 2461 - 5
High-resolution X-ray structure of an acyl-enzyme species for the class D OXA-10 beta-lactamase; Maveyraud L et al.; Beta-lactamases are resistance enzymes for beta-lactam antibiotics . These enzymes hydrolyze the beta-lactam moieties of these antibiotics, rendering them inactive . Of the four classes of known beta-lactamases, the enzymes of class D are the least understood . We report herein the high-resolution (1.9 A) crystal structure of the class D OXA-10 beta-lactamase inhibited by a penicillanate derivative . The structure provides evidence that the carboxylated Lys-70 (a carbamate) is intimately involved in the mechanism of the enzyme.

Proteins, 2002 Apr 1, 47(1), 86 - 96
Noncovalent interaction energies in covalent complexes: TEM-1 beta-lactamase and beta-lactams; Wang X et al.; The class A beta-lactamase TEM-1 is a key bacterial resistance enzyme against beta-lactam antibiotics, but little is known about the energetic bases for complementarity between TEM-1 and its inhibitors . Most inhibitors form a covalent adduct with the catalytic Ser70, making the measurement of equilibrium constants, and hence interaction energies, technically difficult . This study evaluates noncovalent interactions within covalent complexes by examining the differential stability of TEM-1 and its inhibitor adducts . The thermal denaturation of TEM-1 follows a two-state, reversible model with a melting temperature (T(m)) of 51.6C and a van't Hoff enthalpy of unfolding (DeltaH(VH)) of 146.2 kcal/mol at pH 7.0 . The stability of the enzyme changes on forming an inhibitor adduct . As expected, some inhibitors stabilize TEM-1; transition-state analogues increase the T(m) by up to 3.7C (1.7 kcal/mol) . Surprisingly, all beta-lactam covalent acyl--enzyme complexes tested destabilize TEM-1 significantly relative to the apo-enzyme . For instance, the clinically used inhibitor clavulanic acid and the beta-lactamase-resistant beta-lactams moxalactam and imipenem destabilize TEM-1 by over 2.6C (1.2 kcal/mol) in their covalent adducts . Based on the structure of the TEM-1/imipenem complex (Maveyraud et al., J Am Chem Soc 1998;120:9748--52), destabilization by moxalactam and imipenem is thought to be caused by a steric clash between the side-chain of Asn132 and the 6(7)-alpha group of these beta-lactams . To test this hypothesis, the mutant enzyme N132A was made . In contrast with wild-type, the covalent complexes between N132A and both imipenem and moxalactam stabilize the enzyme, consistent with the hypothesis . To investigate the structural bases of this dramatic change in stability, the structure of N132A/imipenem was determined by X-ray crystallography . In the complex with N132A, imipenem adopts a very different conformation from that observed in the wild-type complex, and the putative destabilizing interaction with residue 132 is relieved . Studies of several enzymes suggest that beta-lactams, and covalent inhibitors in general, can have either net favorable or net unfavorable noncovalent interaction energies within the covalent complex . In the case of TEM-1, such unfavorable interactions convert substrate analogues into very effective inhibitors .

Clin Microbiol Infect, 1997 Feb, 3 Suppl 4, S2 - S9
beta-Lactamases: quality and resistance; Medeiros AA; beta-Lactamase-mediated resistance to beta-lactam antibiotics is a feature of great clinical significance . beta-Lactamases are a diverse group of bacterial enzymes that vary in their abilities to hydrolyze beta-lactam antibiotics . beta-Lactamases possess an active site containing either a serine moiety or a zinc atom; serine beta-lactamases are currently of greater clinical prevalence . This review considers the molecular classification of beta-lactamases, the structure of the serine beta-lactamase active site and the mechanisms by which beta-lactamase production may become derepressed . The spread of beta-lactamases in the clinical setting and some important structural mutations that have extended the hydrolysis profiles of serine beta-lactamases are also discussed.

J Am Chem Soc, 2002 Feb 27, 124(8), 1809 - 16
Role of protein flexibility in enzymatic catalysis: quantum mechanical-molecular mechanical study of the deacylation reaction in class A beta-lactamases; Castillo R et al.; We present a theoretical study of a mechanism for the hydrolysis of the acyl-enzyme complex formed by a class A beta-lactamase (TEM1) and an antibiotic (penicillanate), as a part of the process of antibiotic's inactivation by this type of enzymes . In the presented mechanism the carboxylate group of a particular residue (Glu166) activates a water molecule, accepting one of its protons, and afterward transfers this proton directly to the acylated serine residue (Ser70) . In our study we employed a quantum mechanics (AM1)-molecular mechanics partition scheme (QM/MM) where all the atoms of the system were allowed to relax . For this purpose we used the GRACE procedure in which part of the system is used to define the Hessian matrix while the rest is relaxed at each step of the stationary structures search . By use of this computational scheme, the hydrolysis of the acyl-enzyme is described as a three-step process: The first step corresponds to the proton transfer from the hydrolytic water molecule to the carboxylate group of Glu166 and the subsequent formation of a tetrahedral adduct as a consequence of the attack of this activated water molecule to the carbonyl carbon atom of the beta-lactam . In the second step, the acyl-enzyme bond is broken, obtaining a negatively charged Ser70 . In the last step this residue is protonated by means of a direct proton transfer from Glu166 . The large mobility of Glu166, a residue that is placed in a Ohms-loop, is essential to facilitate this mechanism . The geometry of the acyl-enzyme complex shows a large distance between Glu166 and Ser70 and thus, if protein coordinates were kept frozen during the reaction path, it would be difficult to get a direct proton transfer between these two residues . This computational study shows how a flexible treatment suggests the feasibility of a mechanism that could have been discounted on the basis of crystallographic positions.

Antimicrob Agents Chemother, 2002 Mar, 46(3), 646 - 53
Mutant TEM beta-lactamase producing resistance to ceftazidime, ampicillins, and beta-lactamase inhibitors; Vakulenko S et al.; A derivative of the TEM-1 beta-lactamase producing clinically significant levels of resistance to ceftazidime and beta-lactamase inhibitors in the presence of penicillins was generated following five rounds of DNA shuffling and selection . This complex mutant enzyme contained three amino acid substitutions including those of residues 104 and 276 that are known to produce extended-spectrum resistance and, correspondingly, resistance to beta-lactamase inhibitors . Although the Glu104Lys substitution by itself produced low levels of ceftazidime resistance, additional amino acid replacements in the enzyme with the triple mutation resulted in further enhancement of resistance to ceftazidime . Kinetic studies of the purified beta-lactamase enzyme with the triple mutation indicated enhancement of the catalytic efficiency for turnover (kcat/Km) of ceftazidime . The increases in the Ki values of both clavulanic acid and tazobactam for the enzyme with the triple mutation were consistent with the observed bacterial resistance to the reversibility of beta-lactam resistance with these inhibitors.

Bioorg Med Chem Lett, 2002 Feb 25, 12(4), 597 - 9
Enzymatic synthesis of monocyclic beta-lactams; Sleeman MC et al.; An Mg2+ and ATP dependent beta-lactam synthetase (BLS) catalyses formation of a beta-lactam ring during the biosynthesis of clavulanic acid, an important beta-lactamase inhibitor . An epimeric mixture of a 2-methylated derivative of the natural BLS substrate N2-(2-carboxyethyl)-L-arginine was synthesised and found to be a substrate for the enzyme . The epimeric products were characterised by 1H NMR and mass spectrometric analyses . The results suggest that a modified version of BLS might be used to catalyse the preparation of intermediates useful for the synthesis of beta-lactam antibiotics.

Chembiochem, 2001 Jun 1, 2(6), 445 - 55
The outstanding biological stability of beta- and gamma-peptides toward proteolytic enzymes: an in vitro investigation with fifteen peptidases; Frackenpohl J et al.; A series of 36 linear and cyclic beta- and gamma-peptides consisting of as few as two, and as many as 15 residues, was offered as substrates to 15 commercially available proteases of bacterial, fungal, and eukaryotic origin, including a beta-lactamase and amidases, as well as most vigorous, nonspecific proteases, such as the 20S proteasome from human erythrocytes . For comparison, an alpha-eicosapeptide and standard substrates of the proteolytic enzymes were included in the investigation . Under conditions of complete cleavage of the alpha-peptide within 15 min the beta- and gamma-peptides were stable for at least 48 h . Inhibition studies with seven beta- and gamma-peptides and alpha-chymotrypsin show that the residual enzyme activity toward succinyl-Ala-Ala-Pro-Phe-p-nitroanilide is unchanged within experimental error after incubation for 15 min with the peptide analogues . Thus, beta- and gamma-peptides with proteinogenic side chains, that is, consisting of the singly or doubly homologated natural alpha-amino acids (one or two CH(2) groups inserted in the backbone of each residue), are completely stable to common proteases, without inhibiting their normal activity (as demonstrated for alpha-chymotrypsin) . This proteolytic stability of peptides built of homologated amino acids is a prerequisite for their potential use as drugs.

J Antimicrob Chemother, 2002 Feb, 49(2), 387 - 9
Differential regulation of L1 and L2 beta-lactamase expression in Stenotrophomonas maltophilia; Avison MB et al.; It has been reported that Stenotrophomonas maltophilia L1 and L2 beta-lactamase expression is coordinated . We have isolated S . maltophilia mutants where (i) L1 is constitutively hyper-expressed and L2 is inducible; (ii) L2 is hyper-expressed and L1 is inducible; and (iii) L1 and L2 are constitutively hyper-expressed . The frequency of isolating type 1 and 2 mutants is c . 10(-7), indicating that promoter mutations are probably not involved and providing strong evidence that L1 and L2 expression is not, after all, coordinated . The frequency of isolating type 3 mutants is c . 10(-9), however, implying that there is a significant overlap between the regulatory mechanisms.

Nucleic Acids Res, 2002 Feb 1, 30(3), 803 - 9
In vitro transcription of a torsionally constrained template; Bentin T et al.; RNA polymerase (RNAP) and the DNA template must rotate relative to each other during transcription elongation . In the cell, however, the components of the transcription apparatus may be subject to rotary constraints . For instance, the DNA is divided into topological domains that are delineated by rotary locked boundaries . Furthermore, RNAPs may be located in factories or attached to matrix sites limiting or prohibiting rotation . Indeed, the nascent RNA alone has been implicated in rotary constraining RNAP . Here we have investigated the consequences of rotary constraints during transcription of torsionally constrained DNA by free RNAP . We asked whether or not a newly synthesized RNA chain would limit transcription elongation . For this purpose we developed a method to immobilize covalently closed circular DNA to streptavidin-coated beads via a peptide nucleic acid (PNA)-biotin conjugate in principle mimicking a SAR/MAR attachment . We used this construct as a torsionally constrained template for transcription of the beta-lactamase gene by Escherichia coli RNAP and found that RNA synthesis displays similar characteristics in terms of rate of elongation whether or not the template is torsionally constrained . We conclude that transcription of a natural bacterial gene may proceed with high efficiency despite the fact that newly synthesized RNA is entangled around the template in the narrow confines of torsionally constrained supercoiled DNA.

Genome Biol . 2001;2(12):RESEARCH0051 . Epub 2001 Nov 13.
Quoderat demonstrandum? The mystery of experimental validation of apparently erroneous computational analyses of protein sequences; Iyer LM et al.; BACKGROUND: Computational predictions are critical for directing the experimental study of protein functions . Therefore it is paradoxical when an apparently erroneous computational prediction seems to be supported by experiment . RESULTS: We analyzed six cases where application of novel or conventional computational methods for protein sequence and structure analysis led to non-trivial predictions that were subsequently supported by direct experiments . We show that, on all six occasions, the original prediction was unjustified, and in at least three cases, an alternative, well-supported computational prediction, incompatible with the original one, could be derived . The most unusual cases involved the identification of an archaeal cysteinyl-tRNA synthetase, a dihydropteroate synthase and a thymidylate synthase, for which experimental verifications of apparently erroneous computational predictions were reported . Using sequence-profile analysis, multiple alignment and secondary-structure prediction, we have identified the unique archaeal 'cysteinyl-tRNA synthetase' as a homolog of extracellular polygalactosaminidases, and the 'dihydropteroate synthase' as a member of the beta-lactamase-like superfamily of metal-dependent hydrolases . CONCLUSIONS: In each of the analyzed cases, the original computational predictions could be refuted and, in some instances, alternative strongly supported predictions were obtained . The nature of the experimental evidence that appears to support these predictions remains an open question . Some of these experiments might signify discovery of extremely unusual forms of the respective enzymes, whereas the results of others could be due to artifacts.

Biochemistry, 2002 Jan 22, 41(3), 797 - 808
Structure of spin-labeled methylmethanethiolsulfonate in solution and bound to TEM-1 beta-lactamase determined by electron nuclear double resonance spectroscopy; Mustafi D et al.; Site-directed spin-labeling of proteins whereby the spin-label methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)methanethiolsulfonate (SLMTS) is reacted with the -SH groups of cysteinyl residues incorporated into a protein by mutagenesis has been successfully applied to investigate secondary structure and conformational transitions of proteins . In these studies, it is expected that the spin-label moiety adopts different conformations dependent on its local environment . To determine the conformation of SLMTS in solution reacted with L-cysteine (SLMTCys) and bound in the active site of the Glu240Cys mutant of TEM-1 beta-lactamase, we have synthesized SLMTS both of natural abundance isotope composition and in site-specifically deuterated forms for electron nuclear double resonance (ENDOR) studies . ENDOR-determined electron-proton distances from the unpaired electron of the nitroxyl group of the spin-label to the methylene and methyl protons of SLMTS showed three conformations of the oxypyrrolinyl ring with respect to rotation around the S-S bond dependent on the solvent dielectric constant . For SLMTCys, two conformations of the molecule were compatible with the ENDOR-determined electron-nucleus distances to the side-chain methylene protons and to H(alpha) and H(beta1,2) of cysteine . To determine SLMTS conformation reacted with the Glu240Cys mutant of TEM-1 beta-lactamase, enzyme was overexpressed in both ordinary and perdeuterated minimal medium . Resonance features of H(alpha) and H(beta1,2) of the Cys240 residue of the mutant and of the side-chain methylene protons within the spin-label moiety yielded electron-proton distances that sterically accommodated the two conformations of free SLMTCys in solution.

Mikrobiol Z, 2001 Sep-Oct, 63(5), 49 - 58
{Construction of the shuttle vector pSTS1 and development of the method of its introduction into the cells of the cyanbacterium Plectonema boryanum CALU 465}; Syrchin SA et al.; It has been shown that cells of trichoma cyanobacterium Plectonema boryanum G o m . strain CALU 465 do not possess natural resistance to antibiotics ampicyllin, chloramphenicol, streptomycin and canamycin . A shuttle vector pSTS1 has been constructed on the basis of cyanobacterial plasmid pSM1 and vector pBR 322 . Conditions have been developed and conjugative transfer of pSTS1 to the cells of cyanobacterium P . boryanum has been performed . As the result of expression of beta-lactamase gene pSTS1 the resistance of cells-conjugantes to ampicillin increased by three orders.

Biochem Biophys Res Commun, 2002 Jan 11, 290(1), 191 - 6
Insertion of dibasic residues directs a constitutive protein to the regulated secretory pathway; Feliciangeli S et al.; The mechanisms for sorting proteins to the regulated secretory pathway (RSP) remains poorly understood . We recently reported that dibasic sequences that are cleaved by pro-protein convertases (PCs) in pro-neurotensin also acted as sorting signal for the precursor . Here we addressed two questions regarding the role of dibasics as sorting signal: (i) Are dibasics sufficient to direct proteins to the RSP? (ii) Do they sort proteins by virtue of their interaction with PCs? The first question was studied by inserting dibasics in beta-lactamase, a constitutively secreted protein and comparing the regulated secretion of beta-lactamase to that of its mutant in transfected endocrine cells . The second question was investigated by comparing the regulated release of pro-neurotensin in PC12 cells that are devoid of PCs to that in PC1- and PC2-transfected PC12 cells . The data show that the mutant beta-lactamase was indeed targeted in part to the RSP and that pro-neurotensin was sorted to the RSP without the assistance of the PCs, thus indicating that dibasics can act as sorting signal by themselves independently of their interaction with PCs . (c)2002 Elsevier Science.

Eur J Med Res, 2001 Dec 17, 6(12), 535 - 42
Hepatotoxic reactions induced by beta-lactamase inhibitors; Berg P et al.; Since amoxicillin/clavulanate was first introduced in the UK in 1981, beta-lactamase inhibitors are used increasingly worldwide . Two more drugs of this class are currently available, sulbactam and tazobactam . Meanwhile, adverse drug reactions associated with amoxicillin/clavulanate occurring late after the end of therapy have been repeatedly published . In many cases, a cholestatic hepatitis was diagnosed that was most likely caused by the clavulanic acid component of the combination . Symptoms were mostly mild and reversible, whereas a number of cases showing a protracted, even fatal course of the disease have been documented . This article summarizes and analyzes all relevant studies and case reports dealing with hepatotoxicity caused by beta-lactamase inhibitors . The description of a typical case from our own patient population illustrates the clinical challenge associated with this adverse drug reaction.

FEMS Microbiol Lett, 2001 Nov 13, 204(2), 281 - 5
Secretion of the Escherichia coli K-12 SheA hemolysin is independent of its cytolytic activity; del Castillo FJ et al.; The Escherichia coli K-12 sheA gene encodes a pore-forming hemolysin that is secreted to the medium by a hitherto unidentified mechanism . To study SheA secretion, we constructed fusions between SheA and the mature form of the periplasmic enzyme beta-lactamase, and performed site-directed mutagenesis on these constructs . The SheA-Bla and Bla-SheA hybrid proteins displayed hemolytic activity and were efficiently exported to the extracellular medium . Our results with mutant hybrid proteins show that secretion of SheA is independent of its cytolytic activity, that secretion is paralleled by a transient leakage of periplasmic contents to the extracellular medium, and that deletion of the 11 C-terminal residues of SheA has no effect on its secretion and cytolytic activity.

Proc Natl Acad Sci U S A, 2001 Dec 4, 98(25), 14280 - 5 Epub 2001 Nov 27.
Critical involvement of a carbamylated lysine in catalytic function of class D beta-lactamases; Golemi D et al.; beta-Lactamases are the resistance enzymes for beta-lactam antibiotics, of which four classes are known . beta-lactamases hydrolyze the beta-lactam moieties of these antibiotics, rendering them inactive . It is shown herein that the class D OXA-10 beta-lactamase depends critically on an unusual carbamylated lysine as the basic residue for both the enzyme acylation and deacylation steps of catalysis . The formation of carbamylated lysine is reversible . Evidence is presented that this enzyme is dimeric and carbamylated in living bacteria . High-resolution x-ray structures for the native enzyme were determined at pH values of 6.0, 6.5, 7.5, and 8.5 . Two dimers are present per asymmetric unit . One monomer in each dimer was carbamylated at pH 6.0, whereas all four monomers were fully carbamylated at pH 8.5 . At the intermediate pH values, one monomer of each dimer was carbamylated, and the other showed a mixture of carbamylated and non-carbamylated lysines . It would appear that, as the pH increased for the sample, additional lysines were "titrated" by carbamylation . A handful of carbamylated lysines are known from protein crystallographic data, all of which have been attributed roles in structural stabilization (mostly as metal ligands) of the proteins . This paper reports a previously unrecognized role for a noncoordinated carbamylate lysine as a basic residue involved in mechanistic reactions of an enzyme, which indicates another means for expansion of the catalytic capabilities of the amino acids in nature beyond the 20 common amino acids in development of biological catalysts.

Acta Crystallogr D Biol Crystallogr, 2001 Dec, 57(Pt 12), 1912 - 4 Epub 2001 Nov 21.
Crystallization and preliminary X-ray study of OXA-1, a class D beta-lactamase; Sun T et al.; The Escherichia coli OXA-1 oxacillinase, a beta-lactamase which provides resistance to beta-lactam antibiotics (penicillins and cephalosporins), has been crystallized . A member of the class D family of serine beta-lactamases, OXA-1 is especially active against the penicillins oxacillin and cloxacillin and is now found in 10% of E . coli clinical isolates . Crystals grown from PEG 8000 at pH 7.5 diffract to 1.5 A resolution at 100 K and have space group P1 (Z = 2), with unit-cell parameters a = 36.0, b = 51.6, c = 72.9 A, alpha = 70.2, beta = 84.1, gamma = 81.5 degrees.

Protein Sci, 2001 Dec, 10(12), 2556 - 65
Identification of residues critical for metallo-beta-lactamase function by codon randomization and selection; Materon IC et al.; IMP-1 beta-lactamase is a zinc metallo-enzyme encoded by the transferable bla(IMP-1) gene, which confers resistance to virtually all beta-lactam antibiotics including carbapenems . To understand how IMP-1 recognizes and hydrolyzes beta-lactam antibiotics it is important to determine which amino acid residues are critical for catalysis and which residues control substrate specificity . We randomized 27 individual codons in the bla(IMP-1) gene to create libraries that contain all possible amino acid substitutions at residue positions in and near the active site of IMP-1 . Mutants from the random libraries were selected for the ability to confer ampicillin resistance to Escherichia coli . Of the positions randomized, >50% do not tolerate amino acid substitutions, suggesting they are essential for IMP-1 function . The remaining positions tolerate amino acid substitutions and may influence the substrate specificity of the enzyme . Interestingly, kinetic studies for one of the functional mutants, Asn233Ala, indicate that an alanine substitution at this position significantly increases catalytic efficiency as compared with the wild-type enzyme.

Antimicrob Agents Chemother, 2001 Dec, 45(12), 3651 - 3
Evolution of TEM-related extended-spectrum beta-lactamases in Korea; Pai H et al.; TEM-52, differing from TEM-1 by having the substitutions Glu-104-->Lys, Met-182-->Thr, and Gly-238-->Ser, has previously been described as the most prevalent extended-spectrum beta-lactamase (ESBL) in Korea . In a further survey, we discovered the ESBLs TEM-15, which is like TEM-52 but lacks the substitution at residue 182, and TEM-88, which is like TEM-52 with an additional Gly-196-->Asp substitution . TEM-88 retained the activity of TEM-52 against moxalactam . Otherwise, the kinetic properties of the three ESBLs failed to show an advantage to this evolution.

Med Sci Monit, 2001 Nov-Dec, 7(6), 1230 - 5
Role of reactive oxygen species (ROS) in patients with erythema migrans, an early manifestation of Lyme borreliosis; Pancewicz SA et al.; BACKGROUND: Lyme borreliosis is a tick-transmitted, chronic, zoogenous disease caused by Borrelia burgdorferi spirochete . The clinical picture of Lyme disease is characterized by the variety of tissue and organ involvement and differing severity of symptoms . One of the pathogenic symptoms of early Lyme disease is a skin lesion called erythema migrans . MATERIAL AND METHODS: The purpose of our research was to estimate the parameters of the antioxidant system and the concentration of lipid peroxidation products in the plasma of patients with erythema migrans (EM) . The parameters measured included the activity levels of superoxide dismutase (SOD) according to Sykes, gluthatione reductase (GSSG-R) according to Mize and Langdon, glutathione peroxidase (GSH-Px) according to Paglia and Valentine; the concentrations of malondialdehyde (MDA) were examined by means of a Bioxytech LPO-586 kit . The total sulphydryl groups (-SH) according to Ellman and reduced glutathione (GSH) were measured using a Bioxytech GSH-400 test in plasma samples collected from 20 patients with EM aged from 19 to 50, taken before (examination 1) and after (examination 2) therapy with amoxycycline . The control group consisted of 8 healthy people . RESULTS: The results of our examinations prove that beta-lactamase antibiotic therapy brings non-enzymatic antioxidant parameters to control values, though the treatment causes no change in enzymatic antioxidant parameters, resulting in the further activation of free radicals . CONCLUSIONS: In patients with Erythema migrans, the decreased capability to reduce lipid superoxidants leads to maintaining a high concentration of membrane lipid peroxidation products.

Int J Antimicrob Agents, 2001 Sep, 18(3), 199 - 209
Role of sultamicillin and ampicillin/sulbactam in the treatment of upper and lower bacterial respiratory tract infections; Lode H; The emergence of beta-lactamase-mediated resistance to beta-lactam antibiotics among key respiratory tract pathogens has threatened the usefulness of the beta-lactam agents familiar to physicians as being clinically effective and well tolerated . This article reassesses the clinical usefulness of ampicillin when administered in combination with the beta-lactamase inhibitor sulbactam, either intravenously or orally (as the mutual prodrug sultamicillin), in the treatment of upper and lower respiratory tract infections . Numerous clinical studies and several meta-analyses indicate that ampicillin/sulbactam and sultamicillin are clinically effective and well tolerated in both adults and children, in agreement with published North American and European guidelines.

J Biol Chem, 2001 Dec 21, 276(51), 48356 - 61 Epub 2001 Oct 16.
C-terminal periplasmic domain of Escherichia coli quinoprotein glucose dehydrogenase transfers electrons to ubiquinone; Elias M et al.; Membrane-bound quinoprotein glucose dehydrogenase (GDH) in Escherichia coli donates electrons directly to ubiquinone during the oxidation of d-glucose as a substrate, and these electrons are subsequently transferred to ubiquinol oxidase in the respiratory chain . To determine whether the specific ubiquinone-reacting site of GDH resides in the N-terminal transmembrane domain or in the large C-terminal periplasmic catalytic domain (cGDH), we constructed a fusion protein between the signal sequence of beta-lactamase and cGDH . This truncated GDH was found to complement a GDH gene-disrupted strain in vivo . The signal sequence of the fused protein was shown to be cleaved off, and the remaining cGDH was shown to be recovered in the membrane fraction, suggesting that cGDH has a membrane-interacting site that is responsible for binding to membrane, like peripheral proteins . Kinetic analysis and reconstitution experiments revealed that cGDH has ubiquinone reductase activity nearly equivalent to that of the wild-type GDH . Thus, it is likely that the C-terminal periplasmic domain of GDH possesses a ubiquinone-reacting site and transfers electrons directly to ubiquinone.

J Biol Chem, 2001 Dec 14, 276(50), 46759 - 64 Epub 2001 Oct 15.
In vivo mutagenesis by Escherichia coli DNA polymerase I . Ile(709) in motif A functions in base selection; Shinkai A et al.; The fidelity of DNA replication by Escherichia coli DNA polymerase I (pol I) was assessed in vivo using a reporter plasmid bearing a ColE1-type origin and an ochre codon in the beta-lactamase gene . We screened 53 single mutants within the region Val(700)-Arg(712) in the polymerase active-site motif A . Only replacement of Ile(709) yielded mutator polymerases, with substitution of Met, Asn, Phe, or Ala increasing the beta-lactamase reversion frequency 5-23-fold . Steady-state kinetic analysis of the I709F polymerase revealed reductions in apparent K(m) values for both insertion of non-complementary nucleotides and extension of mispaired primer termini . Abolishment of the 3'-5' exonuclease activity of wild-type pol I increased mutation frequency 4-fold, whereas the combination of I709F and lack of the 3'-5' exonuclease yielded a 400-fold increase . We conclude that accurate discrimination of the incoming nucleotide at the polymerase domain is more critical than exonucleolytic proofreading for the fidelity of pol I in vivo . Surprisingly, the I709F polymerase enhanced mutagenesis in chromosomal DNA, although the increase was 10-fold less than in plasmid DNA . Our findings indicate the feasibility of obtaining desired mutations by replicating a target gene at a specific locus in a plasmid under continuous selection pressure.

Antimicrob Agents Chemother, 2001 Nov, 45(11), 3253 - 5
Susceptibilities of Eikenella corrodens, Prevotella intermedia, and Prevotella nigrescens clinical isolates to amoxicillin and tetracycline; Luong N et al.; The AB Biodisk Etest showed that 106 (100%) and 98 (92%) isolates of Eikenella corrodens were susceptible to amoxicillin and tetracycline, respectively . Twenty-three (68%) Prevotella intermedia isolates and 14 (67%) Prevotella nigrescens isolates were susceptible to amoxicillin . Seventy-nine percent of the P . intermedia isolates and 67% of the P . nigrescens isolates were susceptible to tetracycline . A higher percentage of beta-lactamase-producing isolates of P . intermedia and P . nigrescens were identified with selective agar containing amoxicillin than with nonselective agar.

Chem Biol, 2001 Sep, 8(9), 883 - 90
A general approach for the generation of orthogonal tRNAs; Wang L et al.; BACKGROUND: The addition of new amino acids to the genetic code of Escherichia coli requires an orthogonal suppressor tRNA that is uniquely acylated with a desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase . A tRNA(Tyr)(CUA)-tyrosyl-tRNA synthetase pair imported from Methanococcus jannaschii can be used to generate such a pair . In vivo selections have been developed for selecting mutant suppressor tRNAs with enhanced orthogonality, which can be used to site-specifically incorporate unnatural amino acids into proteins in E . coli . RESULTS: A library of amber suppressor tRNAs derived from M . jannaschii tRNA(Tyr) was generated . tRNA(Tyr)(CUA)s that are substrates for endogenous E . coli aminoacyl-tRNA synthetases were deleted from the pool by a negative selection based on suppression of amber nonsense mutations in the barnase gene . The remaining tRNA(Tyr)(CUA)s were then selected for their ability to suppress amber nonsense codons in the beta-lactamase gene in the presence of the cognate M . jannaschii tyrosyl-tRNA synthetase (TyrRS) . Four mutant suppressor tRNAs were selected that are poorer substrates for E . coli synthetases than M . jannaschii tRNA(Tyr)(CUA), but still can be charged efficiently by M . jannaschii TyrRS . CONCLUSIONS: The mutant suppressor tRNA(Tyr)(CUA) together with the M . jannaschii TyrRS is an excellent orthogonal tRNA-synthetase pair for the in vivo incorporation of unnatural amino acids into proteins . This general approach may be expanded to generate additional orthogonal tRNA-synthetase pairs as well as probe the interactions between tRNAs and their cognate synthetases.

Antimicrob Agents Chemother, 2001 Oct, 45(10), 2947 - 8
Sequences of the NPS-1 and TLE-1 beta-lactamase genes; Pai H et al.; The NPS-1 and TLE-1 beta-lactamase genes were cloned and sequenced . NPS-1 differed from LCR-1 beta-lactamase in 8 of 260 amino acids . TLE-1 differed from TEM-1 by a single Asp(115)-->Gly substitution and has been renamed TEM-90.

Antimicrob Agents Chemother, 2001 Oct, 45(10), 2807 - 12
Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae; Conrath KE et al.; Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors . The immunization of dromedaries with the TEM-1 and BcII beta-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those beta-lactamases . Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified . All inhibitory VHHs were tight-binding inhibitors . The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay . Addition of the VHHs to the TEM-1 beta-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria . This innovative strategy could generate multiple potent inhibitors for all types of beta-lactamases.

Protein Eng, 2001 Jul, 14(7), 487 - 92
Protein minimization by random fragmentation and selection; Rudgers GW et al.; Protein-protein interactions are involved in most biological processes and are important targets for drug design . Over the past decade, there has been increased interest in the design of small molecules that mimic functional epitopes of protein inhibitors . BLIP is a 165 amino acid protein that is a potent inhibitor of TEM-1 beta-lactamase (K(i) = 0.1 nM) . To aid in the development of new inhibitors of beta-lactamase, the gene encoding BLIP was randomly fragmented and DNA segments encoding peptides that retain the ability to bind TEM-1 beta-lactamase were isolated using phage display . The selected peptides revealed a common, overlapping region that includes BLIP residues C30-D49 . Synthesis and binding analysis of the C30-D49 peptide indicate that this peptide inhibits TEM-1 beta-lactamase . Therefore, a peptide derivative of BLIP that has been reduced in size by 88% compared with wild-type BLIP retains the ability to bind and inhibit beta-lactamase.

J Bacteriol, 2001 Sep, 183(18), 5230 - 8
Absence of the outer membrane phospholipase A suppresses the temperature-sensitive phenotype of Escherichia coli degP mutants and induces the Cpx and sigma(E) extracytoplasmic stress responses; Langen GR et al.; DegP is a periplasmic protease that is a member of both the sigma(E) and Cpx extracytoplasmic stress regulons of Escherichia coli and is essential for viability at temperatures above 42 degrees C . {U-(14)C}acetate labeling experiments demonstrated that phospholipids were degraded in degP mutants at elevated temperatures . In addition, chloramphenicol acetyltransferase, beta-lactamase, and beta-galactosidase assays as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that large amounts of cellular proteins are released from degP cells at the nonpermissive temperature . A mutation in pldA, which encodes outer membrane phospholipase A (OMPLA), was found to rescue degP cells from the temperature-sensitive phenotype . pldA degP mutants had a normal plating efficiency at 42 degrees C, displayed increased viability at 44 degrees C, showed no degradation of phospholipids, and released far lower amounts of cellular protein to culture supernatants . degP and pldA degP mutants containing chromosomal lacZ fusions to Cpx and sigma(E) regulon promoters indicated that both regulons were activated in the pldA mutants . The overexpression of the envelope lipoprotein, NlpE, which induces the Cpx regulon, was also found to suppress the temperature-sensitive phenotype of degP mutants but did not prevent the degradation of phospholipids . These results suggest that the absence of OMPLA corrects the degP temperature-sensitive phenotype by inducing the Cpx and sigma(E) regulons rather than by inactivating the phospholipase per se.

FEBS Lett, 2001 Aug 10, 503(1), 1 - 6
Expansion of the zinc metallo-hydrolase family of the beta-lactamase fold; Daiyasu H et al.; Recently, the zinc metallo-hydrolase family of the beta-lactamase fold has grown quite rapidly, accompanied by the accumulation of sequence and structure data . The variety of the biological functions of the family is higher than expected . In addition, the members often have mosaic structures with additional domains . The family includes class B beta-lactamase, glyoxalase II, arylsulfatase, flavoprotein, cyclase/dehydrase, an mRNA 3'-processing protein, a DNA cross-link repair enzyme, a DNA uptake-related protein, an alkylphosphonate uptake-related protein, CMP-N-acetylneuraminate hydroxylase, the romA gene product, alkylsulfatase, and insecticide hydrolases . In this minireview, the functional and structural varieties of the growing protein family are described.

Drug Resist Updat, 2000 Apr, 3(2), 109 - 125
b-Lactamase inhibitors; Page MG; The use of beta-lactamase inhibitors in combination with a beta-lactamase-susceptible antibiotic is a useful strategy to rescue otherwise good antibiotics from failure . However, recent years have seen a rise in the numbers of beta-lactamases that are insensitive to the available beta-lactamase inhibitors . This review summarizes of the mechanisms of action of the principal types of inhibitors and the ways in which beta-lactamase are thought to develop resistance towards them . Ten general classes of inhibitors are reviewed, especially those of therapeutic importance (clavulanic acid, penam sulfones and carbapenems) .

J Biol Chem, 2001 Sep 14, 276(37), 34553 - 9 Epub 2001 Jul 10.
Inhibition of translocation of beta -lactamase into the yeast endoplasmic reticulum by covalently bound benzylpenicillin; Paunola E et al.; We found recently that beta-lactamase folds in the yeast cytosol to a native-like, catalytically active, and trypsin-resistant conformation, and is thereafter translocated into the ER and secreted to the medium . Previously, it was thought that pre-folded proteins cannot be translocated . Here we have studied in living yeast cells whether beta-lactamase, a tight globule in authentic form, must be unfolded for ER translocation . A beta-lactamase mutant (E166A) binds irreversibly benzylpenicillin via Ser(70) in the active site . We fused E166A to the C terminus of a yeast-derived polypeptide having a post-translational signal peptide . In the presence of benzylpenicillin, the E166A fusion protein was not translocated into the endoplasmic reticulum, whereas translocation of the unmutated variant was not affected . The benzylpenicillin-bound protein adhered to the endoplasmic reticulum membrane, where it prevented translocation of BiP, carboxypeptidase Y, and secretory proteins . Although the 321-amino acid-long N-terminal fusion partner adopts no regular secondary structure and should have no constraints for pore penetration, the benzylpenicillin-bound protein remained fully exposed to the cytosol, maintaining its signal peptide . Our data suggest that the beta-lactamase portion must unfold for translocation, that the unfolding machinery is cytosolic, and that unfolding of the remote C-terminal beta-lactamase is required for initiation of pore penetration.

FEMS Microbiol Lett, 2001 Jul 10, 201(1), 37 - 40
Substitution of Thr for Ala-237 in TEM-17, TEM-12 and TEM-26: alterations in beta-lactam resistance conferred on Escherichia coli; Giakkoupi P et al.; Non-naturally occurring mutants of TEM-17 (E104K), TEM-12 (R164S) and TEM-26 (E104K:R164S) extended-spectrum (ES) beta-lactamases bearing threonine at position 237 were constructed by site-specific mutagenesis and expressed under isogenic conditions in Escherichia coli . Quantification of beta-lactamase activities and immunoblotting indicated that Ala-237-->Thr did not significantly affect expression levels of these ES enzymes . Minimum inhibitory concentrations of beta-lactam antibiotics showed that the presence of threonine at position 237 exerted a dominant effect increasing the enzymes' preference for various early generation cephalosporins over penicillins . Activity against broad-spectrum oxyimino-beta-lactams was also changed . The effect of Ala-237-->Thr on the activity against ceftazidime, aztreonam, cefepime and cefpirome of all three ES TEM enzymes was detrimental . Introduction of Thr-237 improved activity against cefotaxime and ceftriaxone in TEM-12 and TEM-26, but not in TEM-17.

Biochemistry, 2001 Jul 10, 40(27), 7992 - 9
Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site; Trehan I et al.; Beta-lactamases hydrolyze beta-lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechanism to these drugs and pose a growing threat to public health . beta-Lactams that contain a bulky 6(7)alpha substituent, such as imipenem and moxalactam, actually inhibit serine beta-lactamases and are widely used for this reason . Although mutant serine beta-lactamases have arisen that hydrolyze beta-lactamase resistant beta-lactams (e.g., ceftazidime) or avoid mechanism-based inhibitors (e.g., clavulanate), mutant serine beta-lactamases have not yet arisen in the clinic with imipenemase or moxalactamase activity . Structural and thermodynamic studies suggest that the 6(7)alpha substituents of these inhibitors form destabilizing contacts within the covalent adduct with the conserved Asn152 in class C beta-lactamases (Asn132 in class A beta-lactamases) . This unfavorable interaction may be crucial to inhibition . To test this destabilization hypothesis, we replaced Asn152 with Ala in the class C beta-lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam . Consistent with the hypothesis, the Asn152 --> Ala substitution relieved 0.44 and 1.10 kcal/mol of strain introduced by imipenem and moxalactam, respectively, relative to the wild-type complexes . However, the kinetic efficiency of AmpC N152A was reduced by 6300-fold relative to that of the wild-type enzyme . To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determined to a resolution of 1.83 A . Moxalactam in the mutant complex is significantly displaced from its orientation in the wild-type complex; however, moxalactam does not adopt an orientation that would restore competence for hydrolysis . Although Asn152 forces beta-lactams with 6(7)alpha substituents out of a catalytically competent configuration, making them inhibitors, the residue is essential for orienting beta-lactam substrates and cannot simply be replaced with a much smaller residue to restore catalytic activity . Designing beta-lactam inhibitors that interact unfavorably with this conserved residue when in the covalent adduct merits further investigation.

FEMS Microbiol Lett, 2001 Jun 25, 200(2), 157 - 61
Restriction fragment length dimorphism-PCR method for the detection of extended-spectrum beta-lactamases unrelated to TEM- and SHV-types; Lee SH et al.; The diagnostic ability of the restriction fragment length dimorphism-polymerase chain reaction (RFLD-PCR) method was evaluated . Seven primer pairs, newly designed from 44 beta-lactamase genes encoding extended-spectrum beta-lactamases not related to TEM- and SHV-types, were used to differentiate OXA-2, FOX-3, CMY-3, IMP-1, and IMI-1 beta-lactamases . The RFLD-PCR was carried out successfully, and these genes were differentiated by the sizes of their PCR products and by the difference in restriction fragment length when each amplicon was digested with a unique restriction enzyme . This discriminatory detection of the genes was confirmed by sequencing the PCR products.

J Am Vet Med Assoc, 2001 Jun 15, 218(12), 1893 - 6
Penicillins and beta-lactamase inhibitor combinations; Mealey KL; beta-Lactamase production by bacteria continues to be one of the main mechanisms of bacterial resistance to beta-lactam antibiotics, and it seems likely to remain so . beta-Lactamase inhibitors provide 1 strategy to overcome this mechanism of bacterial resistance . Although 3 beta-lactamase inhibitor/antibiotic combinations are currently available, only 1 is approved for veterinary use . Because the beta-lactamase inhibitor must be present concurrently with the antibiotic for synergistic activity, it is important to consider the pharmacokinetic profile of these drugs in combination . These combinations were developed and optimized for human patients, so it is unlikely that they would achieve the ideal plasma and tissue concentrations and ratios in veterinary patients . Indeed, several differences in pharmacokinetic variables of beta-lactam antibiotic/beta-lactamase inhibitor agents have been described in dogs, compared with people . Such pharmacokinetic differences should be considered when interpreting in vitro susceptibility results in veterinary species, because these tests use ratios of drug that were established for humans . The beta-lactamase inhibitors represent a successful example of targeted drug development . However, the currently available inhibitors are active primarily against class-A beta-lactamases . Because the frequency with which class-C beta-lactamases are recognized is rapidly increasing in human isolates, and because beta-lactamase enzymes continue to evolve, new beta-lactamase inhibitors will need to be developed to target these enzymes.

Biochim Biophys Acta, 2001 Jun 11, 1547(2), 196 - 205
Inactivation of CMY-2 beta-lactamase by tazobactam: initial mass spectroscopic characterization; Bonomo RA et al.; The CMY-2 beta-lactamase, a plasmid determined class C cephalosporinase, was shown to be susceptible to inhibition by tazobactam (K(i)=40 microM) . The reaction product(s) of CMY-2 beta-lactamase with the beta-lactamase inhibitor tazobactam were analyzed by electrospray ionization/mass spectrometry (ESI/MS) to characterize the prominent intermediates of the inactivation pathway . The ESI/MS determined mass of CMY-2 beta-lactamase was 39851+/-3 Da . After inactivating CMY-2 beta-lactamase with excess tazobactam, a single species, M(r)=39931+/-3.0, was detected . Comparison of the peptide maps from tryptic digestion of the native enzyme and the inactivated beta-lactamase followed by LC/MS identified two 22 amino acid peptides containing the active site Ser64 modified by a fragment of tazobactam . These two peptides were increased in mass by 70 and 88 Da, respectively . UV difference spectra following inactivation revealed the presence of a new species with a 302 nm lambda(max) . Based upon the increase in molecular mass of the tazobactam inactivated CMY-2 beta-lactamase, we propose that during the inactivation of this beta-lactamase by tazobactam an imine is formed . Tautomerization forms the spectrally observed enamine . Hydrolysis generates the covalently attached malonyl semialdehyde, its hydrate, or an enol . This work provides information on the mass of a stable enzyme intermediate of a class C beta-lactamase inactivated by tazobactam and, for the first time, unequivocal evidence that a cross-linked species is not required for apparent inactivation.

Antimicrob Agents Chemother, 2001 Jul, 45(7), 2110 - 4
Discrimination of SHV beta-lactamase genes by restriction site insertion-PCR; Chanawong A et al.; Restriction site insertion-PCR (RSI-PCR) is a simple, rapid technique for detection of point mutations . This technique exploits primers with one to three base mismatches near the 3' end to modulate a restriction site . We have developed this technique to identify described mutations of the bla(SHV) genes for differentiation of SHV variants that cannot be distinguished easily by other techniques . To validate this method, eight standard strains were used, each producing a different SHV beta-lactamase: SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, SHV-6, SHV-8, and SHV-18 . Mismatch primers were designed to detect mutations affecting amino acids at positions 8 (SspI), 179 (HinfI), 205 (PstI), 238 (Gly-->Ala) (BsrI), and 240 (NruI) of bla(SHV) genes . All amplimers of the bla(SHV) genes used in this study yielded the predicted restriction endonuclease digestion products . In addition, this study also makes theoretical identification of bla(SHV-6), bla(SHV-8), and 12 novel bla(SHV) variants using the PCR-restriction fragment length polymorphism (RFLP) technique possible . By using a combination of PCR-RFLP and RSI-PCR techniques, up to 27 SHV variants can now be distinguished rapidly and reliably . These simple techniques are readily applied to epidemiological studies of the SHV beta-lactamases and may be extended to the characterisation of other resistance determinants.

FEMS Microbiol Lett, 1998 Jan 15, 158(2), 191 - 4
A beta-lactamase belonging to group 2e from oral clinical isolates of Prevotella intermedia; Valle G et al.; A beta-lactamase in oral clinical isolates of Prevotella intermedia that hydrolyzed cefuroxime and cephalothin with rates of 600 and 53.3 respectively, relative to that for cephaloridine (100), was characterized as a 2e-cephalosporinase . Inhibition was observed by clavulanic acid (IC50 0.72 microM), tazobactam (IC50 0.21 microM) and sulbactam (IC50 0.07 microM) and was not inhibited by cloxacillin, EDTA, NaCl or p-CMB . The pI and pH optima were 4.7 and 5.4, respectively.

Clin Infect Dis, 2001 Jul 1, 33(1), 126 - 8 Epub 2001 May 23.
Control of an outbreak of infection due to extended-spectrum beta-lactamase--producing Escherichia coli in a liver transplantation unit; Paterson DL et al.; We report an outbreak of infection due to genotypically identical extended-spectrum beta-lactamase--producing Escherichia coli among patients in a liver transplantation unit . Control of the outbreak was achieved by a combination of contact isolation, feedback on hand hygiene, and gut decontamination with an orally administered fluoroquinolone . These interventions led to abrupt curtailment of the outbreak.

Bioorg Med Chem, 2001 May, 9(5), 1175 - 83
The synthesis and evaluation of benzofuranones as beta-lactamase substrates; Adediran SA et al.; 6- and 7-Carboxy-3-phenylacetamido-3H-1-benzofuran-2-one have been synthesized as potential beta-lactamase substrates and/or inhibitors . These compounds were prepared by lactonization of the corresponding, appropriately substituted phenylglycines . The latter compounds were prepared by either the Strecker or the Bucherer-Berg method . The benzofuran-2-ones were less stable in aqueous solution than the analogous acyclic phenaceturate esters but comparably stable to analogous benzopyran-2-ones . They differed from the latter compounds however in that the C-3 hydrogen of the furan-2-ones, adjacent to the lactone carbonyl group, was distinctly acidic; 7-carboxy-3-phenylacetamido-3H-1-benzofuran-2-one exists largely as an enolate at pH 7.5 . The furan-2-ones were beta-lactamase substrates with reactivity very similar to the analogous acyclic phenaceturates . They were not, however, DD-peptidase inhibitors and are thus unlikely to have antibiotic activity . The structural basis for these observations is discussed.

Bioorg Med Chem Lett, 2001 May 7, 11(9), 1161 - 4
Reaction of Lys-Tyr-Lys triad mimics with benzylpenicillin: insight into the role of Tyr150 in class C beta-lactamase; Kato-Toma Y et al.; Small and simple molecules mimicking a Lys-Tyr-Lys triad and some 'mutant' derivatives were designed and synthesized . These-compounds react with benzylpenicillin in water (75mM phosphate buffer, pH 7), apparently through general base assistance by the phenolic moiety . Class C beta-lactamase has a Lys-Tyr-Lys triad in its active site, and our finding gives some insight into the role of this triad in the enzymatic beta-lactam hydrolysis mechanism.

Bioorg Chem, 2000 Dec, 28(6), 338 - 56
The Oxyanion Hole in Serine beta-Lactamase Catalysis: Interactions of Thiono Substrates with the Active Site; Curley K et al.; Both functional and structural studies of serine beta-lactamases indicate the existence of an oxyanion hole at the active site with an important role in catalysis . The functional presence of the oxyanion hole is demonstrated by the previous observation that thiono-beta-lactams are very poor substrates of beta-lactamases (B . P . Murphy, and R . F . Pratt, 1988, Biochem . J . 256, 669-672) and in the present paper by the inability of these enzymes to catalyze hydrolysis of a thiono analog of a depsipeptide substrate . This thiono effect was first noted and interpreted in regard to classical serine hydrolases although the chemical basis for it has not been firmly established either in those enzymes or in beta-lactamases . In this paper a computational approach to a further understanding of the effect has been taken . The results for a class C beta-lactamase show that the deacylation tetrahedral intermediate interacted more strongly with the enzyme with an O(-) placed in the oxyanion hole than an S(-) . On the other hand, the converse was true for acylation tetrahedral intermediate species, a result distinctly not in accord with experiment . These results indicate that the thiono effect does not arise from unfavorable interactions between enzyme and thiono substrate at the tetrahedral intermediate stage but must be purely kinetic in nature, i.e., arise in a transitional species at an early stage of the acylation reaction . The same conclusion as to the origin of the thiono effect was also indicated by a less extensive series of calculations on a class A beta-lactamase and on chymotrypsin .

J Antimicrob Chemother, 2001 May, 47(5), 547 - 54
Amino acid substitutions causing inhibitor resistance in TEM beta-lactamases compromise the extended-spectrum phenotype in SHV extended-spectrum beta-lactamases; Randegger CC et al.; Three amino acid substitutions, Met-69-->Ile, Arg-244-->Ser and/or Asn-276-->Asp, mediate inhibitor resistance (IR) in TEM beta-lactamases . They were introduced in all possible combinations at homologous positions into either SHV-1 or the respective extended-spectrum beta-lactamases (ESBLs), SHV-2 or SHV-5 . Susceptibility testing of the resulting set of seven variants of each parental strain, all in an isogenic background, was performed . The phenotypes of the constructions revealed that most substitutions resulted in reduced resistance to most tested single beta-lactam formulations . This decrease over-compensated for the expected increase in inhibitor resistance, so that most mutants showed no rise in resistance to inhibitor/beta-lactam combinations, although increases of MICs from one- to 43-fold compared with the respective parental strains were also measured . Combination of several IR-determining substitutions impaired both phenotypes in the carrier strains even more . None of the 14 mutants derived from the ESBLs, SHV-2 and SHV-5, showed a clinically relevant combined ESBL-IR phenotype . These findings indicate that the SHV beta-lactamase does not benefit proportionally from simultaneous substitution of residues relevant for the ESBL and the IR phenotype.

Biochemistry, 2001 Feb 27, 40(8), 2397 - 409
ENDOR structural characterization of a catalytically competent acylenzyme reaction intermediate of wild-type TEM-1 beta-lactamase confirms glutamate-166 as the base catalyst; Mustafi D et al.; The catalytically competent active-site structure of a true acylenzyme reaction intermediate of TEM-1 beta-lactamase formed with the kinetically specific spin-labeled substrate 6-N-(2,2,5,5-tetramethyl-1-oxypyrrolinyl-3-carboxyl)-penicillanic acid isolated under cryoenzymologic conditions has been determined by angle-selected electron nuclear double resonance (ENDOR) spectroscopy . Cryoenzymologic experiments with use of the chromophoric substrate 6-N-{3-(2-furanyl)-propen-2-oyl}-penicillanic acid showed that the acylenzyme reaction intermediate could be stabilized in the -35 to -75 degrees C range with a half-life suitably long to allow freeze-quenching of the reaction species for ENDOR studies while a noncovalent Michaelis complex could be optically identified at temperatures only below -70 degrees C . The wild-type, Glu166Asn, Glu240Cys, and Met272Cys mutant forms of the mature enzyme were overexpressed in perdeuterated minimal medium to allow detection and assignment of proton resonances specific for the substrate and chemically modified amino acid residues in the active site . From analysis of the dependence of the ENDOR spectra on the setting of the static laboratory magnetic field H0, the dipolar contributions to the principal hyperfine coupling components were estimated to calculate the separations between the unpaired electron of the nitroxyl group and isotopically identified nuclei . These electron-nucleus distances were applied as constraints to assign the conformation of the substrate in the active site and of amino acid side chains by molecular modeling . Of special interest was that the ENDOR spectra revealed a water molecule sequestered in the active site of the acylenzyme of the wild-type protein that was not detected in the deacylation impaired Glu166Asn mutant . On the basis of the X-ray structure of the enzyme, the ENDOR distance constraints placed this water molecule within hydrogen-bonding distance to the carboxylate side chain of glutamate-166 as if it were poised for nucleophilic attack of the scissile ester bond . The ENDOR results provide experimental evidence of glutamate-166 in its functional role as the general base catalyst in the wild-type enzyme for hydrolytic breakdown of the acylenzyme reaction intermediate of TEM-1 beta-lactamase.

Biochemistry, 2001 Feb 13, 40(6), 1861 - 6
Inhibition of the SHV-1 beta-lactamase by sulfones: crystallographic observation of two reaction intermediates with tazobactam; Kuzin AP et al.; Two species resulting from the reaction of the SHV-1 class A beta-lactamase with the sulfone inhibitor tazobactam have been trapped at 100 K and mapped by X-ray crystallography at 2.0 A resolution . An acyclic form of tazobactam is covalently bonded to the catalytic Ser70 side chain, and a second species, a five-atom vinyl carboxylic acid fragment of tazobactam, is bonded to Ser130 . It is proposed that the electron density map of the crystal is a composite picture of two complexes, each with only a single bound species . It is estimated that the two complexes exist in the crystal in approximately equal populations . Results are discussed in relation to the mechanism-based inhibition of class A beta-lactamases by the similar inhibitors sulbactam and clavulanic acid.

Bioorg Med Chem Lett, 2001 Apr 23, 11(8), 997 - 1000
Allyl and propargyl substituted penam sulfones as versatile intermediates toward the syntheses of new beta-lactamase inhibitors; Sandanayaka VP et al.; Several alkenyl derivatives were prepared using allyl penam sulfone as the key intermediate . Isomers of these derivatives having beta configuration at C-6 showed potent activity against CcrA enzyme . A new method was developed to prepare propargyl penam sulfone . The majority of the triazoles prepared by this route exhibited good activity against all three representative enzymes used for the inhibition assay.

Microbiology, 2001 May, 147(Pt 5), 1253 - 8
Isolation of strong expression signals of Mycobacterium tuberculosis; Triccas JA et al.; The natural fluorescence of the Aequoria victoria green fluorescent protein was exploited to isolate strong expression signals of Mycobacterium tuberculosis . Mycobacterium bovis bacille Calmette-Guerin harbouring M . tuberculosis fragments driving high levels of gfp expression were isolated by fluorescence-activated cell sorting (FACS) . DNA sequencing and subsequent comparison with the M . tuberculosis genome sequence revealed that a total of nine postulated promoters had been identified . The majority of the promoters displayed activity that was greater than or equal to the Mycobacterium fortuitum beta-lactamase promoter, one of the strongest mycobacterial promoters characterized to date . Two of the promoters corresponded to proteins predicted to be involved in calcium and magnesium utilization, the importance of such functions for cell physiology suggesting why these two genes are controlled by strong transcription signals . The seven other promoters corresponded to genes encoding proteins of unknown function . Promoter activity was maintained after prolonged incubation within macrophages, implying that these promoters could be used to drive sustained foreign gene expression in vivo . The strength of these expression signals identified could be employed for the overexpression of foreign genes in mycobacteria to aid protein purification and vaccine vector development . Furthermore, this study demonstrated that FACS provides a sensitive and efficient technique to measure and select strong mycobacterial expression signals.

Biotechnol Prog, 2001 Mar-Apr, 17(2), 336 - 47
A quantitative approach to characterizing cell lysis caused by mechanical agitation of Streptomyces clavuligerus; Roubos JA et al.; Streptomyces clavuligerus is a commercially important actinomycete that is used to produce clavulanic acid, a beta-lactamase inhibitor . Observations during 10 batch cultivations with S . clavuligerus on defined media have led to the finding that the organism is very sensitive to shear when grown in batch cultures with increasing stirrer speed . The stirrer speed was increased to keep the dissolved oxygen level above 50% air saturation . A quantitative approach based on the calculation of elemental balances and a simple mathematical model is proposed to characterize the biomass lysis . Finally, a linear relation between biomass yield and observed specific growth rate is determined . Results show that cell lysis occurs at a high degradation rate, e.g., mu(max) = 0.16 h(-1) and k(d) = 0.07 h(-1), when the gassed power input increases above 1.1, 1.7, or 2.0 kW/m(3), respectively, depending on the medium composition . The overall biomass yield on substrate is dramatically reduced in all experiments (>30%).

Nippon Rinsho, 2001 Apr, 59(4), 771 - 6
{Fundamental and clinical studies on beta-lactamase inhibitors}; Niki Y; Cluvulanic acid and sulbactam have been widely used in Japan as beta-lactamase inhibitors, and we will soon have tazobactam, the third beta-lactamase inhibitor . It will be available in combination with piperacillin(1:4), and expected excellent clinical efficacy in various infection, caused by class A, class D and class C beta-lactamase producing bacteria, including ESBLs producing gram negatives . The clinical usefulness of tazobactam/piperacillin might be exceeded those of drugs which combined with cluvulanic acid and sulbactam . However, it dose not inhibit class B beta-lactamase and large amount of class C beta-lactamase . There are some reports of newer beta-lactamase inhibitors, which show much more strong activities against class C or even against class B beta-lactamase in fundamental studies . Continuous studies will be needed to these agent for the future clinical use.

Nippon Rinsho, 2001 Apr, 59(4), 694 - 700
{Extended-spectrum beta-lactamases (ESBLs) producing bacteria}; Hirakata Y; TEM- or SHV-type extended-spectrum beta-lactamases(ESBLs) are of clinical concern in Europe and the United States, whereas bacterial strains producing such types of ESBLs had not been reported in Japan for many years . Toho-1, a different type class A ESBL, has been reported in 1995, in which any prototypical enzyme has not been identified so far . At present Toho-1 is the major ESBL in Japan, however, SHV5 alpha has been reported in 1998, followed by TEM-26, SHV-2, and SHV12 . More recently, SHV-24, a novel SHV-derived ESBL has also been found . Since Toho-1-type ESBL, AmpC-type beta-lactamase, and class B metallo-beta-lactamase have been widely found in Japan, a novel detection system for ESBLs suitable for this country should be developed.

Nucleic Acids Res, 2001 Apr 15, 29(8), 1672 - 82
Development of an inducible pol III transcription system essentially requiring a mutated form of the TATA-binding protein; Meissner W et al.; We attempted to devise a transcription system in which a particular DNA sequence of interest could be inducibly expressed under the control of a modified polymerase III (pol III) promoter . Its activation requires a mutated transcription factor not contained endogenously in human cells . We constructed such a promoter by fusing elements of the beta-lactamase gene of Escherichia coli, containing a modified TATA-box and a pol III terminator, to the initiation region of the human U6 gene . This construct functionally resembles a 5'-regulated pol III gene and its transcribed segment can be exchanged for an arbitrary sequence . Its transcription in vitro by pol III requires the same factors as the U6 gene with the major exception that the modified TATA-box of this construct only interacts with a TATA-binding protein (TBP) mutant (TBP-DR2) but not with TBP wild-type (TBPwt) . Its transcription therefore requires TBP-DR2 exclusively instead of TBPWT: In order to render the system inducible, we fused the gene coding for TBP-DR2 to a tetracycline control element and stably transfected this new construct into HeLa cells . Induction of such a stable and viable clone with tetracycline resulted in the expression of functional TBP-DR2 . This system may conceptually be used in the future to inducibly express an arbitrary DNA sequence in vivo under the control of the above mentioned promoter.

FEMS Microbiol Lett, 2001 Apr 1, 197(1), 85 - 9
Characterization of the active-site residues asparagine 167 and lysine 161 of the IMP-1 metallo beta-lactamase; Haruta S et al.; The roles of lysine at position 161 and asparagine at position 167 in IMP-1 metallo beta-lactamase were studied by site-directed mutagenesis . These residues are highly conserved in metallo beta-lactamases and are thought to be present in the active-site cavity . Mutant enzymes with alanine or aspartic acid at position 167 showed almost the same properties as the wild-type enzyme . Kinetic parameters for the mutant enzymes differing at position 161 indicated that the positive charge of lysine 161 is required for electrostatic interaction with the carboxyl moiety of the substrate, i.e . C-3 of penicillins or C-4 of cephalosporins.

Mol Microbiol, 2001 Mar, 39(6), 1623 - 37
The Caulobacter crescentus flagellar gene, fliX, encodes a novel trans-acting factor that couples flagellar assembly to transcription; Muir RE et al.; The first flagellar assembly checkpoint of Caulobacter crescentus couples assembly of the early class II components of the basal body complex to the expression of class III and IV genes, which encode extracytoplasmic structures of the flagellum . The transcription of class III/IV flagellar genes is activated by the response regulator factor, FlbD . Gain of function mutations in flbD, termed bfa, can bypass the transcriptional requirement for the assembly of class II flagellar structures . Here we show that the class II flagellar gene fliX encodes a trans-acting factor that couples flagellar assembly to FlbD-dependent transcription . We show that the overexpression of fliX can suppress class III/IV gene expression in both wild-type and flbD-bfa cells . Introduction of a bfa allele of flbD into cells possessing a deletion in fliX restores motility indicating that FliX is not a structural component of the flagellum, but rather a trans-acting factor . Furthermore, extragenic motile suppressors which arise in DeltafliX cells map to the flbD locus . These results indicate that FlbD functions downstream of FliX in activating class III/IV transcription . beta-Lactamase fusions to FliX and analysis of cellular fractions demonstrate that FliX is a cytosolic protein that demonstrates some peripheral association with the cytoplasmic membrane . In addition, we have isolated a mutant allele of fliX that exhibits a bfa-like phenotype, restoring flbD-dependent class III/IV transcription in strains that contain mutations in class II flagellar structural genes . Taken together, these results indicated both a positive and negative regulatory function for FliX in coupling the assembly of class II basal body components to gene expression.

FASEB J, 2001 Mar, 15(3), 815 - 22
Overpassing an aberrant V(kappa) gene to sequence an anti-idiotypic abzyme with (beta)-lactamase-like activity that could have a linkage with autoimmune diseases; Debat H et al.; A monoclonal antibody 9G4H9 that exhibits a beta-lactamase-like activity was previously obtained in accordance with the idiotypic network theory . This abzyme presents the most catalytic efficiency in amidase activity described in literature (kcat = 0.9 min-1) . Some reports have demonstrated that functionality as complex as catalysis may be mimicked in this way . Comparison of the catalytic properties of both enzyme and abzyme previously allowed us to obtain better knowledge about 9G4H9 abzymatic machinery . In attempt to characterize this abzyme, the variable regions of kappa and heavy chain were cloned . We present a 'universal' method to clone the correct Vkappa gene to bypass aberrant Vkappa (abVkappa) produced by MOPC-21-derived hybridomas . Sequences obtained are compared in the GenBank database . The VH and Vkappa genes present some important sequence homology with autoantibodies suggesting a direct relationship between catalytic anti-idiotypic antibody and autoimmunity.

Antimicrob Agents Chemother, 2001 Apr, 45(4), 1254 - 62
Biochemical characterization of the FEZ-1 metallo-beta-lactamase of Legionella gormanii ATCC 33297T produced in Escherichia coli; Mercuri PS et al.; The bla(FEZ-1) gene coding for the metallo-beta-lactamase of Legionella (Fluoribacter) gormanii ATCC 33297T was overexpressed via a T7 expression system in Escherichia coli BL21(DE3)(pLysS) . The product was purified to homogeneity in two steps with a yield of 53% . The FEZ-1 metallo-beta-lactamase exhibited a broad-spectrum activity profile, with a preference for cephalosporins such as cephalothin, cefuroxime, and cefotaxime . Monobactams were not hydrolyzed . The beta-lactamase was inhibited by metal chelators . FEZ-1 is a monomeric enzyme with a molecular mass of 29,440 Da which possesses two zinc-binding sites . Its zinc content did not vary in the pH range of 5 to 9, but the presence of zinc ions modified the catalytic efficiency of the enzyme . A model of the FEZ-1 three-dimensional structure was built.

Protein Expr Purif, 2001 Mar, 21(2), 323 - 32
Escherichia coli FtsH (HflB) degrades a membrane-associated TolAI-II-beta-lactamase fusion protein under highly denaturing conditions; Cooper KW et al.; TolAI--II--beta-lactamase, a fusion protein consisting of the inner membrane and transperiplasmic domains of TolA followed by TEM--beta-lactamase associated with the inner membrane but remained confined to the cytoplasm when expressed at high level in Escherichia coli . Although the fusion protein was resistant to proteolysis in vivo, it was hydrolyzed during preparative SDS-polyacrylamide electrophoresis and when insoluble cellular fractions unfolded with 5 M urea were subjected to microdialysis . Inhibitor profiling studies revealed that both a metallo- and serine protease were involved in TolAI--II--beta-lactamase degradation under denaturing conditions . The in vitro degradation rates of the fusion protein were not affected when insoluble fractions were harvested from a strain lacking protease IV, but were significantly reduced when microdialysis experiments were conducted with material isolated from an isogenic ftsH1 mutant . Adenine nucleotides were not required for degradation, and ATP supplementation did not accelerate the apparent rate of TolAI--II--beta-lactamase hydrolysis under denaturing conditions . Our results indicate that the metalloprotease active site of FtsH remains functional in the presence of 3--5 M urea and suggest that the ATPase and proteolytic activities of FtsH can be uncoupled if the substrate is sufficiently unstructured . Thus, a key role of the FtsH AAA module appears to be the net unfolding of bound substrates so that they can be efficiently engaged by the protease active site .

FEBS Lett, 2001 Jan 26, 489(1), 25 - 8
A suicide-substrate mechanism for hydrolysis of beta-lactams by an anti-idiotypic catalytic antibody; Lefevre S et al.; The catalytic mechanism of an anti-idiotypic antibody, 9G4H9, displaying a beta-lactamase activity was investigated . Kinetics experiments suggest that some penicillinic derivatives behave both as substrates and inactivators . Biochemical and immunological experiments strongly indicate that ampicillin may be regarded as a suicide substrate for hydrolysis by 9G4H9 . The anti-idiotypic network appears as a way to create enzyme mimics with modified catalytic activities.

Lijec Vjesn, 2000 Sep-Oct, 122(9-10), 217 - 21
{Characteristics of SHV-2 beta-lactamase from 2 hospitals in Zagreb}; Bedenic B et al.; The aim of the investigation was to determine the properties of SHV-2 type of beta-lactamase from two hospitals in Zagreb . The production of SHV-2 beta-lactamase was determined in 22 clinical isolates of K . pneumoniae by isoelectric focusing, PCR and PCR/Nhe test . The organisms were collected from the Sestre Milosrdnice Hospital and Dubrava Hospital in Zagreb during 1994-1995 . Nine out of 22 strains transferred resistance to E . coli recipient . In spite of the fact that SHV-2 beta-lactamase is a cefotaximase, only 18% of the strains were resistant to cefotaxime with standard inoculum size but the percentage of resistant strains rose to 39% when the inoculum size was increased to 10(7) CFU/ml . The frequency of transfer was lower when more stringent conditions were applied (higher concentration of cefotaxime in the medium) but the transconjugants obtained with such conditions, displayed higher level of resistance to third generation cephalosporins . The genes responsible for resistance to aminoglycosides were usually transferred on the same plasmid with the genes encoding ESBLs . All strains produced an enzyme with the isoelectric point of 7.6, yielded an amplicon with SHV specific primers and had positive PCR/Nhe test.

J Recept Signal Transduct Res, 2000 Nov, 20(4), 189 - 210
A fluorescent reporter assay for the detection of ligands acting through Gi protein-coupled receptors; Xing H et al.; Accompanying the advances in basic biology of G protein-coupled receptors (GPCRs) is the practical need among biopharmaceutical companies for sensitive assays to assess GPCR function, particularly formats that are compatible with high-throughput drug screening . Here we describe a novel cell-based assay format for the high-throughput detection of ligands for Gi protein-coupled receptors . Two Gi-GPCRs, mu-opioid receptor (mu-OPR) and 5-hydroxytryptamine receptor la (5HT1aR) are employed as model receptor targets . The key feature of this assay system is the isolation of stable, clonal Chinese hamster ovary (CHO) cell lines that carry three separate expression plasmids: (1) a chimeric Gq/i5 protein (which re-directs a negative Gi-type signal to a positive Gq-type response), (2) a given Gi-GPCR, and (3) a beta-lactamase (beta1a) reporter gene responsive to Gi-GPCR signaling . Cell-based assays built using this format show appropriate rank order of potency among a reference set of receptor agonist and antagonist compounds . Such assays are also robust, reliable, and can be used for industrial-scale applications such as high-throughput screening for drug leads.

Antimicrob Agents Chemother, 2001 Mar, 45(3), 878 - 82
Molecular and biochemical analysis of AST-1, a class A beta-lactamase from Nocardia asteroides sensu stricto; Poirel L et al.; A beta-lactamase gene was cloned from a Nocardia asteroides sensu stricto clinical isolate . A recombinant plasmid, pAST-1, expressed the beta-lactamase AST-1 in Escherichia coli JM109 . Its pI was 4.8, and its relative molecular mass was 31 kDa . E . coli JM109(pAST-1) was resistant to penicillins and narrow-spectrum cephalosporins . The beta-lactamase AST-1 had a restricted hydrolytic activity spectrum . Its activity was partially inhibited by clavulanic acid but not by sulbactam and tazobactam . AST-1 is an Ambler class A beta-lactamase sharing 65% amino acid identity with beta-lactamase FAR-1, the most closely related enzyme.

Diagn Microbiol Infect Dis, 2001 Jan, 39(1), 65 - 7
Rapid discrimination between BRO beta-lactamases from clinical isolates of Moraxella catarrhalis using restriction endonuclease analysis; du Plessis M; An important feature of Moraxella catarrhalis is the production of beta-lactamases, which causes resistance to the penicillins . Restriction enzyme analysis was able to distinguish between the bro-1 and bro-2 beta-lactamase-encoding genes from 89 clinical isolates of M . catarrhalis . This is a rapid, simple and cost effective method of characterizing these genes.

Prog Neurobiol, 2001 Apr, 63(6), 673 - 86
Using reporter genes to label selected neuronal populations in transgenic mice for gene promoter, anatomical, and physiological studies; Spergel DJ et al.; This review summarizes recent work on the use of reporter genes to label selected neuronal populations in transgenic mice, with particular emphasis on gonadotropin-releasing hormone (GnRH) neurons . Reporter genes discussed are the lacZ, green fluorescent protein (GFP), luc, and bla genes, which encode the reporter proteins beta-galactosidase, GFP, luciferase, and beta-lactamase, respectively . Targeted transgenic expression of these reporter proteins is obtained by fusing the corresponding reporter gene, with or without a subcellular localization signal, to a cell type- or brain region-specific gene promoter . Mice carrying GnRH promoter-driven reporter genes have proven useful for revealing the promoter elements required for cell type-specific expression of GnRH, the full anatomical profile of the GnRH neuronal network, and its electrophysiological activity, suggesting that similar approaches will assist in elucidating the properties of other neuronal populations as well.

Infect Immun, 2001 Feb, 69(2), 869 - 74
Induction of neutralizing antibodies against diphtheria toxin by priming with recombinant Mycobacterium bovis BCG expressing CRM(197), a mutant diphtheria toxin; Miyaji EN et al.; BCG, the attenuated strain of Mycobacterium bovis, has been widely used as a vaccine against tuberculosis and is thus an important candidate as a live carrier for multiple antigens . With the aim of developing a recombinant BCG (rBCG) vaccine against diphtheria, pertussis, and tetanus (DPT), we analyzed the potential of CRM(197), a mutated nontoxic derivative of diphtheria toxin, as the recombinant antigen for a BCG-based vaccine against diphtheria . Expression of CRM(197) in rBCG was achieved using Escherichia coli-mycobacterium shuttle vectors under the control of pBlaF*, an upregulated beta-lactamase promoter from Mycobacterium fortuitum . Immunization of mice with rBCG-CRM(197) elicited an anti-diphtheria toxoid antibody response, but the sera of immunized mice were not able to neutralize diphtheria toxin (DTx) activity . On the other hand, a subimmunizing dose of the conventional diphtheria-tetanus vaccine, administered in order to mimic an infection, showed that rBCG-CRM(197) was able to prime the induction of a humoral response within shorter periods . Interestingly, the antibodies produced showed neutralizing activity only when the vaccines had been given as a mixture in combination with rBCG expressing tetanus toxin fragment C (FC), suggesting an adjuvant effect of rBCG-FC on the immune response induced by rBCG-CRM(197) . Isotype analysis of the anti-diphtheria toxoid antibodies induced by the combined vaccines, but not rBCG-CRM(197) alone, showed an immunoglobulin G1-dominant profile, as did the conventional vaccine . Our results show that rBCG expressing CRM(197) can elicit a neutralizing humoral response and encourage further studies on the development of a DPT vaccine with rBCG.

FEMS Microbiol Lett, 2001 Jan 1, 194(1), 13 - 7
Topological analysis of DctQ, the small integral membrane protein of the C4-dicarboxylate TRAP transporter of Rhodobacter capsulatus; Wyborn NR et al.; Tripartite ATP-independent periplasmic ('TRAP') transporters are a novel group of bacterial and archaeal secondary solute uptake systems which possess a periplasmic binding protein, but which are unrelated to ATP-binding cassette (ABC) systems . In addition to the binding protein, TRAP transporters contain two integral membrane proteins or domains, one of which is 40-50 kDa with 12 predicted transmembrane (TM) helices, thought to be the solute import protein, while the other is 20-30 kDa and of unknown function . Using a series of plasmid-encoded beta-lactamase fusions, we have determined the topology of DctQ, the smaller integral membrane protein from the high-affinity C4-dicarboxylate transporter of Rhodobacter capsulatus, which to date is the most extensively characterised TRAP transporter . DctQ was predicted by several topology prediction programmes to have four TM helices with the N- and C-termini located in the cytoplasm . The levels of ampicillin resistance conferred by the fusions when expressed in Escherichia coli were found to correlate with this predicted topology . The data have provided a topological model which can be used to test hypotheses concerning the function of the different regions of DctQ and which can be applied to other members of the DctQ family.

J Biol Inorg Chem, 2000 Dec, 5(6), 774 - 83
High-throughput synthesis and screening of platinum drug candidates; Ziegler CJ et al.; Platinum drugs play an important role in the treatment of cancer, but there is room for improvement . Here we present a new platinum drug-discovery strategy to identify compounds having efficacy equivalent to that of cisplatin with the expectation that some may increase the spectrum of treatable tumors and/or reduce dose-limiting toxicity . Platinum drug candidates were generated through the use of automated synthesis, taking advantage either of the trans effect or by using silver chloride precipitation to activate the starting materials . Reaction products were screened for activity in a high-throughput transcription assay and the most promising candidates characterized . Over 3,600 reaction products were screened for their ability to inhibit transcription of beta-lactamase in the BlaM HeLa cell line by monitoring cleavage of a lactam ring linking the two halves of a fluorescent resonance energy transfer (FRET) dye, CCF2/AM . From this screen, three reactions produced good candidates, and four species were identified among these reaction products . Three of the compounds, cis-{(isopropylamine)2PtCl2}, cis-{(cyclobutylamine)2PtCl2}, and cis-{ammine(cyclobutylamine)PtCl2}, have been previously determined to be active cisplatin analogs . The fourth compound, cis-{ammine(2-amino-3-picoline)PtCl2}, represents a new kind of antitumor drug candidate similar to ZD0473, a recently reported analog . The discovery of these compounds represents an important proof of principle that platinum anticancer drug candidates can be rapidly prepared and screened in this manner.

Virology, 2000 Dec 20, 278(2), 570 - 7
Neutralization of human papillomavirus (HPV) pseudovirions: a novel and efficient approach to detect and characterize HPV neutralizing antibodies; Yeager MD et al.; The development of vaccines against human papillomaviruses (HPVs) has long been hampered by the inability to grow HPVs in tissue culture and the lack of an efficient neutralization assay . To date, less than 10% of more than 100 different HPV types can be grown in athymic and "SCID" mouse xenograft systems or raft culture systems . Recently, the in vitro generation of HPV pseudovirions and their use in neutralization assays were demonstrated . The major shortcomings of the current approaches to HPV neutralization are the lack of HPV virions for most types for the xenograft methods and the time-consuming and inefficient generation of infective pseudovirions for the latter methods, which precludes their use in large-scale HPV clinical trials or epidemiological studies . We describe here a novel and efficient approach to generating pseudovirions in which HPV virus-like particles (VLPs) are coupled to the beta-lactamase gene as a reporter . We show that it is not necessary to encapsidate the reporter gene constructs into the pseudovirions . Using sera from human volunteers immunized with HPV-11 VLPs expressed in yeast, we demonstrate that our novel neutralization assay compares favorably with the athymic mouse neutralization assay . Furthermore, our assay was used to define neutralizing monoclonal antibodies to HPV-6, which were previously unknown .

Eur J Clin Microbiol Infect Dis, 2000 Oct, 19(10), 759 - 64
Genetic diversity among strains of Moraxella catarrhalis cultured from the nasopharynx of young and healthy Brazilian, Angolan and Dutch children; Wolf B et al.; The present study describes the carriage patterns and genetic variability of Moraxella catarrhalis strains isolated from children living in different countries . Moraxella catarrhalis is genetically heterogeneous, but little is known about its geographic distribution and phenotypic and genetic diversity in warm-climate countries . A collection of 99 isolates from 30 Brazilian, 19 Angolan and 50 Dutch healthy children, all less than 5 years of age, was investigated for phenotypic and genotypic relatedness . The isolates from the three countries were similar where biochemical reactivity was concerned: 89 strains were beta-lactamase-producing and 87 were complement-resistant as determined by phenotype . There was no geographical difference in the prevalence of beta-lactamase-producing isolates, but the carriage rate of complement-resistant strains was significantly higher in Dutch than in Angolan children (P=0.004) . Complement resistance of 66 randomly selected strains was genetically confirmed in a Southern hybridization assay by a novel DNA probe that is specific for complement-resistant strains and that demonstrated a sensitivity of 97% and a specificity of 100% . PCR amplification based on the probe sequence had a sensitivity of 98% and a specificity of 57% when compared to the outcome of a conventional culture spot test . PCR restriction fragment length polymorphism analysis of the MU 46 locus and pulsed-field gel electrophoresis of SpeI DNA macrorestriction fragments revealed genetic heterogeneity of strains from within and between the three countries, and no geographical clustering could be established . In conclusion, similar phenotypic characteristics but genotypic heterogeneity was found among Moraxella catarrhalis strains colonizing children in three different continents.

J Biol Chem, 2001 Mar 2, 276(9), 6140 - 50 Epub 2000 Dec 04.
The role of dibasic residues in prohormone sorting to the regulated secretory pathway . A study with proneurotensin; Feliciangeli S et al.; The mechanisms by which prohormone precursors are sorted to the regulated secretory pathway in neuroendocrine cells remain poorly understood . Here, we investigated the presence of sorting signal(s) in proneurotensin/neuromedin N . The precursor sequence starts with a long N-terminal domain followed by a Lys-Arg-(neuromedin N)-Lys-Arg-(neurotensin)-Lys-Arg- sequence and a short C-terminal tail . An additional Arg-Arg dibasic is contained within the neurotensin sequence . Mutated precursors were expressed in endocrine insulinoma cells and analyzed for their regulated secretion . Deletion mutants revealed that the N-terminal domain and the Lys-Arg-(C-terminal tail) sequence were not critical for precursor sorting to secretory granules . In contrast, the Lys-Arg-(neuromedin N)-Lys-Arg-(neurotensin) sequence contained essential sorting information . Point mutation of all three dibasic sites within this sequence abolished regulated secretion . However, keeping intact any one of the three dibasic sequences was sufficient to maintain regulated secretion . Finally, fusing the dibasic-containing C-terminal domain of the precursor to the C terminus of beta-lactamase, a bacterial enzyme that is constitutively secreted when expressed in neuroendocrine cells, resulted in efficient sorting of the fusion protein to secretory granules in insulinoma cells . We conclude that dibasic motifs within the neuropeptide domain of proneurotensin/neuromedin N constitute a necessary and sufficient signal for sorting proteins to the regulated secretory pathway.

J Antimicrob Chemother, 2000 Dec, 46(6), 879 - 84
A TEM-2beta-lactamase encoded on an active Tn1-like transposon in the genome of a clinical isolate of Stenotrophomonas maltophilia; Avison MB et al.; A constitutively expressed beta-lactamase gene from a clinical isolate of Stenotrophomonas maltophilia, J675Ia, has been cloned . Its DNA sequence is almost identical to that of bla(TEM2) (one nucleotide change) and the expressed enzyme is a Bush type 2a penicillinase with an amino acid sequence identical to that of TEM-2 . The bla(TEM) gene was present within a novel Tn1/Tn3-type transposon in the genome of isolate J675Ia and the transposon was able to mobilize bla(TEM) on to the broad host-range conjugative plasmid, R388 . When transferred to an Escherichia coli recipient, R388::Tn conferred high-level ampicillin resistance . This represents the first identification of a TEM beta-lactamase in S . maltophilia and the first evidence that this important clinical pathogen is able to act as a reservoir for mobile beta-lactamase genes in the hospital environment.

Braz J Infect Dis, 1998 Jun, 2(3), 128 - 134
Frequency, Type and Associated Diseases of Bacteria and Virus in The Oropharynx of Children Born to Human Immunodeficiency Virus-Infected Mothers; Palacio R et al.; HIV-infected children are more likely than other children to develop pneumonia, which in these children is often recurrent or persistent . The main reservoir of the major pathogens is the nasopharynx, but to date no data has been published on the frequency and biologic characteristics of S.pneumoniae, H.influenzae and respiratory viruses found in the upper respiratory tract of children born to human immunodeficiency virus-infected mothers . To document these aspects, 105 children was monitored by pharyngeal swab (PS) and nasopahryngeal aspirates (NPA) who attended an outpatient clinic for HIV-infection evaluation . Bacterial identification was performed by standard procedures . Serotype, biotype and beta-lactamase production was investigated in H.influenzae isolates . S.pneumoniae serotypes were recognized by "quellung" and the susceptibility to 4 antibiotics was assessed . Respiratory syncytial viruses, parainfluenza, influenza A and B, and adenoviruses were diagnosed by indirect immunofluorescence and/or viral isolation in cell cultures . Twenty-nine children were identified as infected by HIV as a result of maternal-child transmission . Seventy children born to HIV-positive mothers but who were not HIV-infected served as controls . Of 269 PS, 110 110 S . pneumoniae and 92 H.influenzae were identified . Also 31 viruses were detected in 188 NPA . After stratifying by age no differences were observed in the frequency of bacterial colonization or in the presence of viruses in the upper respiratory tract of the two groups . Some biologic characteristics of the agents were noteworthy such as the frequency of colonization by S.pneumoniae serotype 14, the predominance of H.influenzae biotype I and the high frequency of viruses in NPA of asymptomatic children . Of note, although colonization frequencies were similar, children presenting with acute respiratory illness (ARI) were more likely to have bacteria isolated if they also had HIV-infection than if they were HIV-negative . It is concluded that HIV-infection in infants as a result of maternal virus transmission have a similar frequency of bacteria and virus colonization of their respiratory tract, but a higher frequency of ARI and perhaps a higher frequency of types of bacteria with special characteristics.

Nat Biotechnol, 2000 Dec, 18(12), 1298 - 302
A ubiquitin-based tagging system for controlled modulation of protein stability; Stack JH et al.; Many biotechnology applications depend on the expression of exogenous proteins in a predictable and controllable manner . A key determinant of the intracellular concentration of a given protein is its stability or "half-life." We have developed a versatile and reliable system for producing short half-life forms of proteins expressed in mammalian cells . The system consists of a series of destabilization domains composed of varying numbers of a mutant form of ubiquitin (UbG76V) that cannot be cleaved by ubiquitin hydrolases . We show that increasing the number of UbG76V moieties within the destabilization domain results in a graded decrease in protein half-life and steady-state levels when fused to heterologous reporter proteins as well as cellular proteins . Cells expressing a destabilized beta-lactamase reporter act as a robust, high-throughput screening (HTS)-compatible assay for proteasome activity within cells.

Appl Microbiol Biotechnol, 2000 Oct, 54(4), 467 - 75
Clavulanic acid, a beta-lactamase inhibitor: biosynthesis and molecular genetics; Liras P et al.; Clavulanic acid is a secondary metabolite produced by Streptomyces clavuligerus . It possesses a clavam structure and a characteristic 3R,5R stereochemistry essential for action as a beta-lactamase inhibitory molecule . It is produced from glyceraldehyde-3-phosphate and arginine in an eight step biosynthetic pathway . The pathway is carried out by unusual enzymes, such as (1) the enzyme condensing both precursors, N2-(2-carboxyethyl)-arginine (CEA) synthetase, (2) the beta-lactam synthetase cyclizing CEA and (3) the clavaminate synthetase, a well-characterized multifunctional enzyme . Genes for biosynthesis of clavulanic acid and other clavams have been cloned and characterized . They offer new possibilities for modification of the pathway and for obtaining new molecules with a clavam structure . The state of the regulatory proteins controlling clavulanic acid biosynthesis, as well as the relationship between the biosynthetic pathway of clavulanic acid and other clavams, is discussed.

Expert Opin Investig Drugs, 2000 Feb, 9(2), 247 - 61
beta-Lactamase epidemiology and the utility of established and novel beta-lactamase inhibitors; Payne DJ et al.; beta-Lactamase inhibitor:beta-lactam combinations remain one of the most successful strategies for the treatment of bacterial infections . Over the last 20 years the number and diversity of serine and metallo active site beta-lactamases has increased dramatically . This review highlights some of the new additions to the beta-lactamase arena and discusses how the commercially available beta-lactamase inhibitors are keeping pace with the changing epidemiology of beta-lactamases . In addition, we survey the progress with the design of novel inhibitors of serine and metallo-beta-lactamases . Focus is given to the recent advances in the design of metallo-beta-lactamase inhibitors as these enzymes pose a serious emerging threat to the use of all beta-lactam based therapies.

Bioorg Med Chem, 2000 Sep, 8(9), 2317 - 35
Studies on anti-Helicobacter pylori agents . Part 2: new cephem derivatives; Yoshida Y et al.; The synthesis and optimization of the anti-Helicobacter pylori activity of a novel series of cephem derivatives are described . Introduction of thio-heterocyclic groups containing N- and S-atoms to the 3-position and phenyl or thienyl acetamido groups to the 7-position of the cephem nucleus dramatically improved the activity . From this series of derivatives, compound 13i was found to have extremely potent in vitro anti-H . pylori activity, superior therapeutic efficacy compared to AMPC and CAM, no cross-resistance between CAM or MNZ and low potential for causing diarrhea due to instability to beta-lactamase.

Bioorg Med Chem Lett, 2000 Oct 2, 10(19), 2179 - 82
The synthesis and SAR of rhodanines as novel class C beta-lactamase inhibitors; Grant EB et al.; Beta-lactam antibiotics such as the cephalosporins and penicillins have diminished clinical effectiveness due to the hydrolytic activity of diverse beta-lactamases, especially those in molecular classes A and C . A structure activity relationship (SAR) study of a high-throughput screening lead resulted in the discovery of a potent and selective non-beta-lactam inhibitor of class C beta-lactamases.

Org Lett, 2000 Oct 5, 2(20), 3087 - 90
Dipolar cycloaddition of novel 6-(nitrileoxidomethyl) penam sulfone: an efficient route to a new class of beta-lactamase inhibitors; Sandanayaka VP et al.; 6-(Nitrileoxidomethyl) penam sulfone intermediate was prepared in a few steps starting from commercially available (+)-6-aminopenicillanic acid . This intermediate underwent smooth 1, 3-dipolar cycloaddition reactions with various alkenes and alkynes to give cycloadducts in moderate to good yields . By this new method, several potent beta-lactamase inhibitors were synthesized . The regio- and stereoselectivity outcomes of the cycloaddition process are also discussed.

Diagn Microbiol Infect Dis, 2000 Aug, 37(4), 293 - 5
Laboratory detection of extended-spectrum beta-lactamases (ESBLs)--the need for a reliable, reproducible method; Essack SY; A reason for the under-recognition of extended-spectrum beta-lactamase (ESBL)-mediated resistance has been the lack of a convenient and sensitive method for recognising ESBL-producing strains . This paper reviews the relative merits and demerits of various methods used.

Biochem Biophys Res Commun, 2000 Aug 28, 275(2), 360 - 4
Molecular mechanisms for antibody-mediated modulation of peptide-displaying enzyme sensors; Ferrer-Miralles N et al.; The generation of molecular sensors based on peptide-displaying enzymes for the detection of antibodies or antigens represents an innovative field of protein engineering . The knowledge of the underlying molecular mechanisms of enzymatic modulation in such sensors would be of great importance for the rational construction and improvement of responsiveness of new peptide-enzyme molecules . Here we analyze the enzymatic characteristics of three different kinds of sensors based in engineered beta-galactosidase, alkaline phosphatase and beta-lactamase, to explore a common activation basis . We describe two different categories of enzyme sensors . In one of them, including only some modified beta-lactamases, the enzymatic activity is inhibited upon ligand binding and it seems to be caused by the steric coverage of the active site by the bound antibody . In a second group, embracing members of the three studied enzymes, the ability to be modulated upon effector binding depends on the ratio between the k(cat) of the engineered enzyme and the k(cat) of the intact enzyme . This proves a common mechanism for enzymatic modulation of enzyme biosensors that is probably caused by conformational effects induced by the bound antibody on the enzyme .

Antimicrob Agents Chemother, 2000 Sep, 44(9), 2549 - 53
Molecular characterization of FOX-4, a new AmpC-type plasmid-mediated beta-lactamase from an Escherichia coli strain isolated in Spain; Bou G et al.; A clinical strain of Escherichia coli (Ec GCE) displayed resistance to cefoxitin, cefotetan, cefotaxime, and ceftazidime . Susceptibility was not restored by the addition of clavulanic acid . Two beta-lactamases with apparent pIs of 5.4 and 6.4 were identified; the beta-lactamase with a pI of 6.4 was transferred by conjugation and associated with a 40-kb plasmid . Analysis of the nucleotide sequence showed a new ampC beta-lactamase gene that is closely related to those encoding the FOX-3, FOX-2, and FOX-1 beta-lactamases but whose product has four novel amino acid mutations, at positions 11 (M-->T), 43 (A-->E), 233 (V-->A), and 280 (Y-->H) . This first cephamycinase from Spain was named FOX-4.

Antimicrob Agents Chemother, 2000 Sep, 44(9), 2534 - 6
Activities of imipenem and cephalosporins against clonally related strains of Escherichia coli hyperproducing chromosomal beta-lactamase and showing altered porin profiles; Martinez-Martinez L et al.; Forty clonally related clinical isolates of Escherichia coli from hospitalized patients were resistant to cefoxitin (MICs, >256 microg/ml) and ceftazidime (MICs, 32 to 256 microg/ml) and were intermediate or resistant to cefotaxime (MICs, 16 to 128 microg/ml) but susceptible to both cefepime (MICs, 0.5 to 2 microg/ml) and imipenem (MICs, 0.125 to 0.25 microg/ml) . Resistance to beta-lactams was related to high-level production of AmpC beta-lactamase and loss of OmpF porin.

Pathol Biol (Paris), 2000 Jun, 48(5), 478 - 84
{Bacterial growth delay in E . coli: demonstration and evaluation of the beta-lactamase post-inhibitor effect on 6 strains with different resistance phenotypes}; Murbach V et al.; Six E . coli, whose phenotypes of resistance were different, were tested in vitro in order to evaluate a regrowth delay, the post beta-lactamases inhibitor effect (PLIE) . This PLIE was investigated after a brief incubation in contact with clavulanic acid (CA) alone or associated with amoxicillin (AMX) . After removal of the drugs used during the pre-exposure phase, the bacteria were incubated with AMX at different concentrations . The PLIE was shown not to be in association with any other regrowth delay (post-antibiotic effect or effect inherent to the technical procedures used) . A PLIE was evaluated on the five intermediary or high-level beta-lactamases-producing strains . Generally, the duration of the PLIE was prolonged after the CA alone pre-exposure phase and could reach values up to 22 hours . The concentrations of AMX added in cultures previously exposed to sufficient CA concentrations were related to an extended PLIE.

Infect Immun, 2000 Sep, 68(9), 4877 - 83
Recombinant Mycobacterium bovis BCG expressing pertussis toxin subunit S1 induces protection against an intracerebral challenge with live Bordetella pertussis in mice; Nascimento IP et al.; The recent development of acellular pertussis vaccines has been a significant improvement in the conventional whole-cell diphtheria-pertussis-tetanus toxoid vaccines, but high production costs will limit its widespread use in developing countries . Since Mycobacterium bovis BCG vaccination against tuberculosis is used in most developing countries, a recombinant BCG-pertussis vaccine could be a more viable alternative . We have constructed recombinant BCG (rBCG) strains expressing the genetically detoxified S1 subunit of pertussis toxin 9K/129G (S1PT) in fusion with either the beta-lactamase signal sequence or the whole beta-lactamase protein, under control of the upregulated M . fortuitum beta-lactamase promoter, pBlaF* . Expression levels were higher in the fusion with the whole beta-lactamase protein, and both were localized to the mycobacterial cell wall . The expression vectors were relatively stable in vivo, since at two months 85% of the BCG recovered from the spleens of vaccinated mice maintained kanamycin resistance . Spleen cells from rBCG-S1PT-vaccinated mice showed elevated gamma interferon (IFN-gamma) and low interleukin-4 (IL-4) production, as well as increased proliferation, upon pertussis toxin (PT) stimulation, characterizing a strong antigen-specific Th1-dominant cellular response . The rBCG-S1PT strains induced a low humoral response against PT after 2 months . Mice immunized with rBCG-S1PT strains displayed high-level protection against an intracerebral challenge with live Bordetella pertussis, which correlated with the induction of a PT-specific cellular immune response, reinforcing the importance of cell-mediated immunity in the protection against B . pertussis infection . Our results suggest that rBCG-expressing pertussis antigens could constitute an effective, low-cost combined vaccine against tuberculosis and pertussis.

In Vitro Cell Dev Biol Anim, 2000 May, 36(5), 293 - 8
Efficient translocation and processing with Xenopus egg extracts of proteins synthesized in rabbit reticulocyte lysate; Zhou X et al.; Cell-free translation/translocation systems are broadly applied to examine gene expression and characterize the structure-function relationship of gene products . We present the characterization of Xenopus egg extract (XEE) translocation and processing of proteins synthesized in rabbit reticulocyte lysate . The XEE was prepared from eggs laid by adult female frogs that received serial injections of gonadotropins . The eggs were then dejellied in 2% L-cysteine-HCl and the cytoplasm extracted by centrifugation at 10,000 rpm for 15 min . The in vitro translocation and processing of XEE was examined with a cell-free translation system containing reticulocyte lysate, and appropriate messenger ribonucleic acid (RNA) or complementary deoxyribonucleic acid plasmids with RNA polymerase . Cell-free production of the following proteins were used to assess posttranslational modifications: Escherichia coli beta-lactamase for signal sequence cleavage, Saccharomyces cerevisiae alpha-mating factor for translocation and N-linked glycosylation, the soluble protein luciferase for functional activity, and the membrane-bound human insulin receptor for translation efficiency . All translation products were identified by {35S}-methionine labeling, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography . The results demonstrate that (1) XEE produces near-complete signal sequence and N-glycosylation processing of proteins synthesized in reticulocyte lysate, (2) XEE contains endoplasmic reticulum-equivalent microsomes, which allows for protein translocation and protease protection, (3) the addition of XEE in the translation reaction does not affect synthesis and chemiluminescence activity of luciferase, (4) XEE is efficient in processing the nascent 160-kDa human insulin receptor precursor, a transmembrane protein, and (5) as compared to canine pancreatic microsomes, XEE translocation efficiency is minimally decreased with the addition of dimethylsulfoxide . These results are the first description of the combined use of XEE with reticulocyte lysate and clearly demonstrate a higher efficiency of translocation and processing compared to canine pancreatic microsomes . This method of cell-free translation and processing allows for more extensive in vitro examination of posttranslational modifications of secretory and membrane-bound proteins.

Mol Microbiol, 2000 Jul, 37(2), 364 - 70
'Intergenic' blr gene in Escherichia coli encodes a 41-residue membrane protein affecting intrinsic susceptibility to certain inhibitors of peptidoglycan synthesis; Wong RS et al.; In the annotation of genomic sequences, small open reading frames (ORFs) are often neglected, particularly if they have no homology to other ORFs or proteins . A mini-TnphoA insertion in a 602 bp 'intergenic' region of the Escherichia coli chromosome at genomic nucleotide 1702674 gave rise to a membrane-bound PhoA fusion protein and a two- to fourfold increase in the intrinsic susceptibility to a wide spectrum of beta-lactam antibiotics without affecting beta-lactamase activity or susceptibility to tetracycline, chloramphenicol, gentamicin or quinolones . Susceptibility was also increased to cycloserine and bacitracin, but not to fosfomycin or valinomycin; these drugs, like beta-lactams, inhibit peptidoglycan synthesis, although by different mechanisms . A clone bearing only 358 bp of this 'blr' region restored resistance to the parental level . Two amber mutations in the clone prevented such restoration and were counteracted by an amber suppressor, proving that the active species is a protein . The Blr protein has 41 amino acids, with a single predicted transmembrane helix, but no clear homology to any other protein . A transcriptional start exists 39 bp upstream from the translational start . The membrane location of Blr suggests that it may be part of an efflux pump or involved in murein metabolism . The results indicate that genes for other very small functional proteins may lie within 'intergenic' regions.

Biochem Biophys Res Commun, 2000 Aug 11, 274(3), 732 - 5
Inhibition of serine amidohydrolases by complexes of vanadate with hydroxamic acids; Bell JH et al.; Serine beta-lactamases are inhibited by phosphonate monoester monoanions . These compounds phosphonylate the active site serine hydroxyl group to form inert, covalent complexes . Since spontaneous hydrolysis of these phosphonates is generally quite slow, the beta-lactamase active site must have considerable affinity for the (presumably) pentacoordinated phosphonyl transfer transition state . Structural analogs of such a transition state might well therefore be effective and novel beta-lactamase inhibitors . Complexes of vanadate with hydroxamic acids may be able to achieve such a structure . Indeed, mixtures of these two components, but neither one alone, were found to inhibit a typical class C beta-lactamase . A Job plot of the inhibition by vanadate/benzohydroxamic acid mixtures indicated that the inhibitor was a 1:1 complex for which an inhibition constant of 4.2 microM could be calculated . A bacterial DD-peptidase, structurally similar to the beta-lactamase, was also inhibited (K(i) = 22 microM) by this complex . A similar rationale would suggest that other serine hydrolases might also be inhibited by these mixtures . In fact, chymotrypsin was inhibited by a complex of vanadate with benzohydroxamic acid (K(i) = 10 microM) and elastase by a complex with acetohydroxamic acid (K(i) = 90 microM) .

Biochemistry, 2000 Jul 25, 39(29), 8666 - 73
Site-directed mutagenesis and biochemical analysis of the endogenous ligands in the ferrous active site of clavaminate synthase . The His-3 variant of the 2-His-1-carboxylate model; Khaleeli N et al.; The facial 2-His-1-carboxylate (Asp/Glu) motif has emerged as the structural paradigm for metal binding in the alpha-ketoglutarate (alpha-KG)-dependent nonheme iron oxygenases . Clavaminate synthase (CS2) is an unusual member of this enzyme family that mediates three different, nonsequential reactions during the biosynthesis of the beta-lactamase inhibitor clavulanic acid . In this study, covalent modification of CS2 by the affinity label N-bromoacetyl-L-arginine near His297, which is within the HRV signature of a His-2 motif, suggested this histidine could play a role in metal coordination . However, site-specific mutagenesis of eight His residues to Gln identified His145 and His280, but not His297, as involved in iron binding . Weak homology of His145 and its flanking sequence and the presence of Glu147 fitting the canonical acidic residue of the His-Xaa-Asp/Glu signature are consistent with His145 being a coordinating ligand (His-1) . His280 and its flanking sequence, which give poor alignments to most other members of this enzyme family, are similar among a subset of these enzymes and notably to CarC, an apparent oxygenase involved in carbapenem biosynthesis . The separation of His145 and His280 is more than twice that seen in the current 2-His-1-carboxylate model and may define an alternative iron binding motif, which we propose as His-3 . These ligand assignments, based on kinetic measurements of both oxidative cyclization/desaturation and hydroxylation assays, establish that no histidine ligand switching occurs during the catalytic cycle . These results are confirmed in a recent X-ray crystal structure of CS1, a highly similar isozyme of CS2 (81% identical) . Tyr299, Tyr300 in CS2 modified by N-bromoacetyl-L-arginine, is hydrogen bonded to Glu146 (Glu147 in CS2) in this structure and well-positioned for reaction with the affinity label.

Antimicrob Agents Chemother, 2000 Aug, 44(8), 2182 - 4
Selection of naturally occurring extended-spectrum TEM beta-lactamase variants by fluctuating beta-lactam pressure; Blazquez J et al.; Despite the large number of in vitro mutations that increase resistance to extended-spectrum cephalosporins in TEM-type beta-lactamases, only a small number occur in naturally occurring enzymes . In nature, and particularly in the hospital, bacteria that contain beta-lactamases encounter simultaneous or consecutive selective pressure with different beta-lactam molecules . All variants obtained by submitting an Escherichia coli strain that contains a bla(TEM-1) gene to fluctuating challenge with both ceftazidime and amoxicillin contained only mutations previously detected in naturally occurring beta-lactamases . Nevertheless, some variants obtained by ceftazidime challenge alone contained mutations never detected in naturally occurring TEM beta-lactamases, suggesting that extended-spectrum TEM variants in hospital isolates result from fluctuating selective pressure with several beta-lactams rather than selection with a single antibiotic.

Bioorg Med Chem Lett, 2000 Jun 19, 10(12), 1389 - 91
Synthesis and biological activities of an alpha-methyl and a beta-methyl carbapenem and the corresponding unsubstituted compound; Pfaendler HR et al.; The carbapenem potassium salts 4, 7 and 8 were prepared . Their rates of beta-lactam hydrolysis and their biological activities, particularly their beta-lactamase inhibiting properties, were examined and explained on the basis of their different substitution and pyramidality.

Nat Struct Biol, 2000 Jul, 7(7), 537 - 41
Rational design of faster associating and tighter binding protein complexes; Selzer T et al.; A protein design strategy was developed to specifically enhance the rate of association (k(on)) between a pair of proteins without affecting the rate of dissociation (k(off)) . The method is based on increasing the electrostatic attraction between the proteins by incorporating charged residues in the vicinity of the binding interface . The contribution of mutations towards the rate of association was calculated using a newly developed computer algorithm, which predicted accurately the rate of association of mutant protein complexes relative to the wild type . Using this design strategy, the rate of association and the affinity between TEM1 beta-lactamase and its protein inhibitor BLIP was enhanced 250-fold, while the dissociation rate constant was unchanged . The results emphasize that long range electrostatic forces specifically alter k(on), but do not effect k(off) . The design strategy presented here is applicable for increasing rates of association and affinities of protein complexes in general.

EMBO J, 1983, 2(8), 1275 - 9
The ultimate localization of an outer membrane protein of Escherichia coli K-12 is not determined by the signal sequence; Tommassen J et al.; To study the role of the signal sequences in the biogenesis of outer membrane proteins, we have constructed two hybrid genes: a phoE-ompF hybrid gene, which encodes the signal sequence of outer membrane PhoE protein and the structural sequence of outer membrane OmpF protein, and a bla-phoE hybrid gene which encodes the signal sequence as well as 158 amino acids of the structural sequence of the periplasmic enzyme beta-lactamase and the complete structural sequence of PhoE protein . The products of these genes are normally transported to and assembled into the outer membrane These results show: (i) that signal sequences of exported proteins are export signals which function independently of the structural sequence, and (ii) that the information which determines the ultimate location of an outer membrane protein is located in the structural sequence of this protein, and not in the signal sequence.

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