Appl Environ Microbiol, 1989 Aug, 55(8), 2056 - 60 Cloning and expression in Escherichia coli of the Azospirillum brasilense Sp7 gene encoding ampicillin resistance; Verreth C et al.; The Azospirillum brasilense ATCC 29145 gene coding for beta-lactamase was cloned in Escherichia coli . The gene was expressed in E . coli from its own promoter as a 30-kilodalton protein, conferring resistance to high levels of beta-lactam antibiotics . The DNA sequence containing the beta-lactamase gene was found to be highly amplified in the Azospirillum genome, scattered in the chromosomal as well as in the plasmidic DNA. Nucleic Acids Res, 1989 Jul 25, 17(14), 5537 - 45 Transcription regulation in vitro by an E . coli promoter containing a DNA cruciform in the '-35' region; Horwitz MS; A promoter with the potential to adopt a 50 basepair (bp) cruciform spanning from -19 to -69 has been constructed in the plasmid pBR322 tetracycline resistance gene (tet) by forming an inverted repeat from '-35' sequences . Compared to a control promoter, the sequence of this cruciform promoter differs only by a 22 bp insertion between -48 and -69, upstream from the usual location of promoter sequences . The cruciform is extruded in a supercoil-dependent manner, and transcription from this promoter in vitro by RNA polymerase decreases as the negative supercoil density of the plasmid DNA increases . In contrast, transcription from the control promoter increases with negative supercoiling . Thus, DNA secondary structure in the '-35' region can affect promoter-polymerase interaction . The tet promoter cruciform also influences expression of the pBR322 beta-lactamase gene (bla) . This apparently results when extrusion of the cruciform reduces the superhelicity of the plasmid molecule to a level that is below the optimum for expression from the bla promoter, illustrating one mechanism for how DNA secondary structure may effect action-at-a-distance . Transcription from both promoters in vivo does not differ from controls, suggesting that this cruciform is not generated to a significant extent intracellularly, most probably as a result of the slow kinetics of extrusion. Biochemistry, 1989 Jul 11, 28(14), 5703 - 7 Mutants generated by the insertion of random oligonucleotides into the active site of the beta-lactamase gene; Dube DK et al.; We have remodeled the gene coding for beta-lactamase by replacing DNA at the active site with random nucleotide sequences . The oligonucleotide replacement (Phe66XXXSer70XXLys73) preserves the codon for the active serine-70 but also contains 15 base pairs of chemically synthesized random sequences that code for 2.5 x 10(6) amino acid substitutions . From a population of Escherichia coli infected with plasmids containing these random inserts, we have selected seven new active-site mutants that render E . coli resistant to carbenicillin and a series of related analogues . Each of the new mutants contains multiple nucleotide substitutions that code for different amino acids surrounding serine-70 . Each of the mutants exhibits a temperature-sensitive beta-lactamase activity . This technique offers the possibility of constructing alternative active sites in enzymes on the basis of biological selection for functional variants. Yale J Biol Med, 1989 Jul-Aug, 62(4), 379 - 92 Oncogene expression in vivo by ovarian adenocarcinomas and mixed-mullerian tumors; Kacinski BM et al.; Six-micron paraffin sections of paraformaldehyde-fixed specimens of 24 ovarian benign and neoplastic specimens were assayed for tumor cell-specific oncogene expression by a sensitive, quantitative in situ hybridization technique with probes for 17 oncogenes, beta-actin, and E . coli beta-lactamase . In the benign, borderline, and invasive adenocarcinomas, multiple oncogenes, including neu, fes, fms, Ha-ras, trk, c-myc, fos, and PDGF-A chains, were expressed at significant levels relative to a housekeeping gene (beta-actin) . In the mixed-Mullerian tumors, a rather different pattern of oncogene expression was observed, characterized primarily by expression of sis (PDGF-B chain) . For the adenocarcinomas, statistical analysis demonstrated that expression of several genes (fms, neu, PDGF-A) was closely linked to others (c-fos, c-myc) known to have important roles in the control of cell proliferation, but only one gene, fms, correlated very strongly with clinicopathologic features (high FIGO histologic grade and high FIGO clinical stage) predictive of aggressive clinical behavior and poor outcome . The authors discuss the role that tumor epithelial cell expression of the fms gene product might play in the auto- and paracrine control of growth and dissemination of ovarian adenocarcinomas. Arch Biochem Biophys, 1989 Jul, 272(1), 52 - 68 The Michaelis constants of a nonchromogenic substrate may be determined using a chromogenic substrate; Blake RC 2nd et al.; A general method is presented for the determination of the KM and Vmax for a nonchromogenic substrate in an experimental system where a chromogenic and a nonchromogenic substrate compete for the active site of a single enzyme . Entire progress curves of absorbance versus time for the transformation of the chromogenic substrate must be obtained in the absence and presence of the nonchromgenic substrate . Two quantities may then be extracted from the entire kinetic curves: the value of a delta Area, the area bounded by the kinetic traces of absorbance versus time in the presence and absence of the nonchromogenic substrate; and the value of delta t(5%), the time required to transform 5% of the chromogenic substrate in the presence of the nonchromogenic substrate minus the corresponding time required in its absence . The values of KM and Vmax for the nonchromogenic substrate may be obtained from the dependencies of delta Area and delta t(5%) upon the concentration of the nonchromogenic substrate . The ability of this procedure to yield the correct values of KM and Vmax was demonstrated using beta-lactamase, beta-galactosidase, alkaline phosphatase, and 19 chromogenic/nonchromogenic substrate pairs . This method is equally valid in the absence or presence of competitive product inhibition and should be applicable to any enzyme-catalyzed, strongly exergonic reaction. J Bacteriol, 1989 Jun, 171(6), 3133 - 8 Multiple chromosomes of Azotobacter vinelandii; Nagpal P et al.; The number of copies of the genes leuB, nifH, nifD, and nifK per cell of Azotobacter vinelandii has been determined to be about 80 . A beta-lactamase gene was integrated into the A . vinelandii chromosome by single-point crossover . Subsequently, we have been able to detect nearly 80 copies of this beta-lactamase gene per cell of A . vinelandii when cultured for a large number of generations in the presence of ampicillin . The multiple copies of the beta-lactamase gene do not seem to be present on a single chromosome, as evident from the fragment obtained by digestion of cellular DNA with the appropriate restriction endonuclease . The kinetics of renaturation of DNA of A . vinelandii is suggestive of complexity similar to that of Escherichia coli . The DNA content of A . vinelandii, however, is 40 times that of E . coli . All these indicate the presence of multiple chromosomes, possibly as many as 80, in A . vinelandii. Obstet Gynecol Clin North Am, 1989 Jun, 16(2), 257 - 70 Penicillins; Faro S; Penicillins remain a class of antibiotics that are effective against various bacteria . Although newer penicillins continue to be developed and combinations of older penicillins with beta-lactamase inhibitors have become available, the use of these agents has not made them prohibitive for general use. Antimicrob Agents Chemother, 1989 Jun, 33(6), 805 - 9 Lincomycin increases the half-life of beta-lactamase mRNA; Matsushita O et al.; Escherichia coli K-12 strains isolates carrying plasmid pBR322 were grown in the presence of subinhibitory concentrations of lincomycin, which stimulated beta-lactamase synthesis about 2.5-fold, and the effects of the drug on the synthesis and degradation of bla mRNA were studied . The bla mRNA levels determined by 1-min pulse-labeling with {3H}uridine were significantly higher in a lincomycin-containing culture than in the control culture, indicating that stimulation of beta-lactamase synthesis is caused by an increase in the amount of bla mRNA . The enhancing effect of lincomycin was observed in strains harboring pBR322 delta P1 and pBR322 delta P3, which lacked the P1 or P3 promoter, respectively, as well as in the strain harboring pBR322 . S1 nuclease analysis showed that the half-life of bla mRNA increased about 2.7-fold when lincomycin was present . These results indicate that the increase in beta-lactamase synthesis caused by lincomycin is due to an increase in the stability of bla mRNA rather than activation of its synthesis. Mol Gen Genet, 1989 Jun, 217(2-3), 202 - 8 On the evolution of Tn21-like multiresistance transposons: sequence analysis of the gene (aacC1) for gentamicin acetyltransferase-3-I(AAC(3)-I), another member of the Tn21-based expression cassette; Wohlleben W et al.; The aminoglycoside-3-O-acetyltransferase-I gene (aacC1) from R plasmids of two incompatibility groups (R1033 {Tn1696}, and R135) was cloned and sequenced . In the case of R1033, it was shown that the aacC gene is coded by a precise insertion of 833 bp between the aadA promoter and its structural gene in a Tn21 related transposon (Tn1696) . This insertion occurs at the same target sequence as that of the OXA-1 beta-lactamase gene insertion in Tn2603 . Upstream of the aacC gene, we found an open reading frame (ORF) which is probably implicated in the site-specific recombinational events involved in the evolution of this family of genetic elements . These results provide additional confirmation of the role of Tn21 elements as naturally occurring interspecific transposition and expression cassettes. Gene, 1989 May 30, 78(2), 349 - 54 Substitution of serine for arginine in position 162 of TEM-type beta-lactamases extends the substrate profile of mutant enzymes, TEM-7 and TEM-101, to ceftazidime and aztreonam; Collatz E et al.; TEM-7 is a novel broad-spectrum beta-lactamase (Bla), selected in vivo, with a resistance profile similar to that of TEM-1 and TEM-2, but extended to ceftazidime (Caz) and aztreonam . Nucleotide sequencing revealed that the TEM-7 gene is almost identical with that of TEM-2 . There was 1 bp change which would result in the substitution of Ser (TEM-7) for Arg (TEM-2) in amino acid (aa) position 162 (i.e., aa position 139 of the mature enzyme) . This substitution, also found in TEM-101, a spontaneous in vitro derivative of TEM-1 selected on Caz, was assumed to be responsible for the extension of the substrate profile . The assumption was verified by exchange of a DNA fragment, carrying the mutation of the TEM-7-coding gene, with the homologous fragment of the TEM-1-coding gene in pBR322 . In the three-dimensional model of class-A Bla {Joris et al., Biochem . J . 250 (1988) 313-324}, aa 139 is located at the rim of the groove which contains the active center and adjacent to the evolutionarily conserved BoxV . It is speculated that extra free hydroxyl groups in this area may participate in the stabilization of otherwise non-substrate compounds. Anal Biochem, 1989 May 15, 179(1), 145 - 53 Synthesis and characterization of 6-O-(N-heptylcarbamoyl)-methyl-alpha-D-glucopyranoside, a new surfactant for membrane studies; Plusquellec D et al.; A new surfactant, 6-O-(N-heptylcarbamoyl)-methyl-alpha-D-glucopyranoside (HECAMEG, molar mass 335.38 g), was synthesized by a simple and low cost procedure from methyl-alpha-D-glucopyranoside . This surfactant is characterized by a high solubility in water (even at 0 degree C), ultraviolet light transparency in the region useful for protein detection, and a high critical micellar concentration (CMC = 19.5 mM), permitting fast elimination by dialysis . Furthermore, the surfactant is colorimetrically titratable by the anthrone technique and its weak interference in protein titration by the Lowry et al . procedure and the bicinchoninic method is easy to overcome . Two membrane proteins (NADH oxidase and succinate dehydrogenase) and a soluble enzyme (lactoperoxidase) retained full activity in the presence of HECAMEG below or above its CMC . The partial inhibition of beta-lactamase (soluble form) by HECAMEG above the CMC was probably only apparent and due to an interference of the surfactant with the substrate rather than a direct effect on the enzyme . HECAMEG was capable of extracting up to 75% of bacteriorhodopsin from the purple membrane of Halobacterium halobium in a nondenatured form as indicated by the spectral properties of the protein . It also solubilized spiralin from the Spiroplasma melliferum membrane with a great selectivity and efficiency, without detectable loss of antigenic properties . These data show that HECAMEG is a very mild surfactant, useful for membrane protein studies. EMBO J, 1989 May, 8(5), 1469 - 77 The precursor of beta-lactamase: purification, properties and folding kinetics; Laminet AA et al.; The precursor of Escherichia coli RTEM beta-lactamase was purified to homogeneity on a milligram scale by a procedure independent of the binding properties of the protein and refolded to an active, reduced form . For comparing the folding kinetics, the wild-type enzyme was reduced and a mutant was constructed, in which the two cysteines that form a very stable disulfide bond in the RTEM enzyme were both changed into alanines . The rate of folding was determined by directly measuring the increase in enzymatic activity . The reduced precursor folds at least 15 times more slowly than either the reduced mature enzyme or the mature Cys----Ala double mutant under identical conditions . The wild-type enzyme, the Cys----Ala double mutant and the precursor protein all had similar KM values, demonstrating a very similar native state . The slow folding of the precursor compared with the mature form may be an essential and general feature to secure a transport competent conformation necessary for the translocation through a membrane in protein export . This folding assay of a precursor by directly following its enzymatic activity may facilitate the characterization of putative folding modulators in bacterial membrane transport. FEMS Microbiol Lett, 1989 May, 50(1-2), 97 - 100 TEM-E1: a novel beta-lactamase conferring resistance to ceftazidime; Payne DJ et al.; A novel beta-lactamase, conferring resistance to ceftazidime, has been identified to be encoded by a 31 kb plasmid (pUK720) in a clinical E . coli strain isolated in Belgium . The beta-lactamase, new designated TEM-E1, has a pI of approximately 5.4 and lies in between the iso-electric focused bands of the beta-lactamases TEM-1 and TEM-7 . The TEM-E1 beta-lactamase has a similar molecular weight of 22,000 to the TEM-1 and it is also inhibited by clavulanic acid . However, the TEM-E1 enzyme differs from TEM-1 by its low rates and efficiency of hydrolysis for ceftazidime and cefotaxime, TEM-E1 has similar efficiency of hydrolysis values for ceftazidime and cefotaxime, but only confers resistance to ceftazidime. J Cell Biol, 1989 May, 108(5), 1647 - 55 The propeptide of preprosomatostatin mediates intracellular transport and secretion of alpha-globin from mammalian cells; Stoller TJ et al.; We have investigated the role of the somatostatin propeptide in mediating intracellular transport and sorting to the regulated secretory pathway . Using a retroviral expression vector, two fusion proteins were expressed in rat pituitary (GH3) cells: a control protein consisting of the beta-lactamase signal peptide fused to chimpanzee alpha-globin (142 amino acids); and a chimera of the somatostatin signal peptide and proregion (82 amino acids) fused to alpha-globin . Control globin was translocated into the endoplasmic reticulum as determined by accurate cleavage of its signal peptide; however, alpha-globin was not secreted but was rapidly and quantitatively degraded intracellularly with a t 1/2 of 4-5 min . Globin degradation was insensitive to chloroquine, a drug which inhibits lysosomal proteases, but was inhibited at 16 degrees C suggesting proteolysis occurred during transport to the cis-Golgi apparatus . In contrast to the control globin, approximately 30% of the somatostatin propeptide-globin fusion protein was transported to the distal elements of the Golgi apparatus where it was endoproteolytically processed . Processing of the chimera occurred in an acidic intracellular compartment since cleavage was inhibited by 25 microM chloroquine . 60% of the transported chimera was cleaved at the Arg-Lys processing site in native prosomatostatin yielding "mature" alpha-globin . Most significantly, approximately 50% of processed alpha-globin was sorted to the regulated pathway and secreted in response to 8-Br-cAMP . We conclude that the somatostatin propeptide mediated transport of alpha-globin from the endoplasmic reticulum to the trans-Golgi network by protecting molecules from degradation and in addition, facilitated packaging of alpha-globin into vesicles whose secretion was stimulated by cAMP. Agressologie, 1989 May, 30(5), 251 - 3 {Nosocomial pulmonary infections caused by Branhamella catarrhalis in intensive care units}; Denamur E et al.; Branhamella catarrhalis, a normal inhabitant of the human nasopharynx, is an opportunistic agent that usually infects patients with underlying diseases . B . catarrhalis has become increasingly recognized as an important respiratory pathogen . From november 1985 to february 1988 we have diagnosed four B . catarrhalis nosocomial pulmonary infections in the adult pulmonary intensive care unit (ICU) and eight in the pediatric ICU . The origin of the contamination has remained unproven because of the lack of a typing system for the strains . All isolates produced beta-lactamase. J Clin Microbiol, 1989 May, 27(5), 944 - 6 Evaluation of restriction endonuclease analysis as an epidemiologic typing system for Branhamella catarrhalis; Patterson JE et al.; Restriction endonuclease analysis (REA) was evaluated as an epidemiologic typing tool to distinguish Branhamella catarrhalis strains . Fourteen beta-lactamase-producing strains were collected over a 16-month period at a hospital where a nosocomial outbreak of this organism was previously documented by REA . REA produced 12 distinct patterns which correlated with epidemiologic data . Chromosomal REA appears to be a useful technique for distinguishing B . catarrhalis strains. Science, 1989 Apr 14, 244(4901), 182 - 8 A general method for site-specific incorporation of unnatural amino acids into proteins; Noren CJ et al.; A new method has been developed that makes it possible to site-specifically incorporate unnatural amino acids into proteins . Synthetic amino acids were incorporated into the enzyme beta-lactamase by the use of a chemically acylated suppressor transfer RNA that inserted the amino acid in response to a stop codon substituted for the codon encoding residue of interest . Peptide mapping localized the inserted amino acid to a single peptide, and enough enzyme could be generated for purification to homogeneity . The catalytic properties of several mutants at the conserved Phe66 were characterized . The ability to selectively replace amino acids in a protein with a wide variety of structural and electronic variants should provide a more detailed understanding of protein structure and function. J Gen Microbiol, 1989 Apr, 135 ( Pt 4), 761 - 5 Nucleotide sequence of an OXA-2 beta-lactamase gene from the R-plasmid R1767 derived plasmid pBP11 and comparison to closely related resistance determinants found in R46 and Tn2603; Nucken EJ et al.; Plasmid pBP11 contains a sequence homologous to Tn21-like element Tn2410 encoding dihydropteroate synthetase and beta-lactamase OXA-2 . The nucleotide sequence of a 1.5 kb segment of this region has been determined including the bla gene . It reveals strong sequence homology with the OXA-2 operon of plasmid R46 . The implications of an additional 319 bp segment in pBP11 for the different evolution of R46/pKM101 and pBP11 are discussed. J Hosp Infect, 1989 Apr, 13(3), 299 - 307 Nosocomial Branhamella catarrhalis in a paediatric intensive care unit: risk factors for disease; Cook PP et al.; There have been few reports on Branhamella catarrhalis as a nosocomial pathogen, and no risk factors for nosocomial infection have been identified . We report 11 cases (mean age 22 months) of nosocomial Branhamella catarrhalis respiratory tract infection in a paediatric intensive care unit (PICU) over a two-year period . There were 2 cases of pneumonia and 9 cases of bronchitis . Branhamella catarrhalis was the sole isolate recovered in 6 cases and was associated with other respiratory pathogens in 5 cases . A case-control study with two age-matched controls per patient (mean age 24.1 months) was undertaken to identify potential risk factors for infection; risk factors identified were the presence of an endotracheal tube (p less than 0.02) and frequent endotracheal tube suction (p less than 0.05) . Five of 6 tested strains from PICU patients produced beta-lactamase . DNA preparations of 4 B . catarrhalis isolates from PICU patients revealed no plasmids . B . catarrhalis should be considered a potential nosocomial pathogen. Chem Pharm Bull (Tokyo), 1989 Mar, 37(3), 824 - 5 Diketene analogs as beta-lactamase inhibitor; Tanizawa K et al.; Compounds having a structural analogy with diketene have been synthesized and their potencies as beta-lactamase inhibitors have been studied . Among six compounds so far tested, alpha-phenyl-beta-benzylidene-3-propanolide was shown to be an irreversible inhibitor of the enzyme . The availability of simple monocyclic compounds as beta-lactamase inhibitors is discussed. Jpn J Antibiot, 1989 Mar, 42(3), 612 - 22 {Clinical evaluation of sulbactam/ampicillin in the pediatric infections}; Meguro H et al.; Sulbactam/ampicillin (SBT/ABPC) was administered to 33 pediatric inpatients with 34 bacterial infections . Clinical efficacies were judged to be good in 33 cases (97.1%) out of 34 which included 13 cases of 14 with infections caused by beta-lactamase-producing strains of organisms, and successfully cured by this drug . There were no particular side effects to comment except some cases of diarrhea and loose stool . These results indicated that SBT/ABPC would be useful in the treatment of pediatric infections as a first choice of drug . Serum half-lives of ABPC and SBT were 0.79 hour and 1.02 hours, respectively, hence, q.i.d . dosage regimen would be appropriate. FEMS Microbiol Lett, 1989 Mar, 49(1), 25 - 31 Functioning of a hybrid BRP-beta-lactamase protein in the release of cloacin DF13 and lysis of Escherichia coli cells; Luirink J et al.; The gene encoding a hybrid BRP-Bla protein consisting of the pCloDF13 encoded BRP signal sequence, 25 of the 28 amino acid residues of the mature bacteriocin release protein (BRP) and the mature portion of beta-lactamase (Bla) was subcloned in the expression vector pEB112 . A similar construct was made using a mutant gene encoding a BRP-Bla protein in which the cysteine residue at the +1 position was changed into a glycine residue . The expression, processing, functioning and subcellular localization of the 'wild-type' and mutant hybrid protein at high-level expression conditions were studied . The 'wild-type' BRP-Bla protein was mainly found in the outer membranes and possessed all the activities of the BRP itself; the protein was able to bring about the release of cloacin DF13 and caused apparent cell-lysis after high-level synthesis . The mutant hybrid protein was predominantly located in the inner membranes, was inactive in the release of cloacin DF13, but caused apparent cell-lysis only after strong induction. Semin Respir Infect, 1989 Mar, 4(1), 12 - 8 Bacteremic Hemophilus influenzae pneumonia in the adult; Quinones CA et al.; Bacteremic Hemophilus influenzae pneumonia is an uncommon infection in the adult . It usually affects chronically ill patients, especially those with chronic obstructive lung disease, although healthy individuals can also be affected . The condition carries a significant mortality, reaching 57% in one series . We describe ten adult patients with bacteremic H influenzae pneumonia with some unique features: no deaths occurred, half of the involved patients were previously fit individuals, and a beta-lactamase producing strain was isolated in five out of ten patients . An interesting finding was the isolation of a beta-lactamase negative H influenza from the sputum of two patients whose blood cultures were positive for a beta-lactamase positive H influenza . The overall incidence of beta-lactamase production among bacteremic isolates was 50%--a finding with a great deal of impact on the treatment of this disease. J Bacteriol, 1989 Mar, 171(3), 1742 - 3 Temperature-sensitive sec mutants of Escherichia coli: inhibition of protein export at the permissive temperature; Ito K et al.; Phenotypes of secY and secA temperature-sensitive mutants at permissive (low) temperature have been examined . The secY24 mutant was found to be extremely susceptible to export inhibition by a basal-level synthesis of the MalE-LacZ 72-47 hybrid protein or to overproduction of a normal secretory protein such as maltose-binding protein or beta-lactamase . Comparison of this phenotype of secY24 with those of the secY100 and secA51 mutants under similar conditions suggested that MalE-LacZ protein and overproduced secretory protein do not nonspecifically enhance the partial secretion defect but act synergistically with secY24 to inhibit protein export. Rev Infect Dis, 1989 Mar-Apr, 11 Suppl 2, S479 - 83 New experimental drugs for the treatment of tuberculosis; Parenti F; New antitubercular agents are needed for two main purposes: to further simplify therapy (through reductions in the number of medicaments used, the number of doses administered, and the duration of treatment required), thus facilitating supervision and improving compliance, and to combat resistant mycobacteria . Reduction in the number of medicaments has been achieved by combining two or more drugs in a single tablet while retaining a degree of bioavailability similar to that of the single components . The adverse effects observed with once-weekly high doses of rifampin have limited the development of widely spaced intermittent regimens of treatment . For this reason new rifamycins have been developed that are as active as rifampin against mycobacteria but that also offer the advantage of high and prolonged serum levels and thus have the potential for once-weekly administration . The in vitro and in vivo properties of these drugs have been studied . Three classes of drugs show promise for the treatment of drug-resistant tuberculosis: spiropiperidyl rifamycin, the fluoroquinolones, and combinations of beta-lactam agents and beta-lactamase inhibitors. FEMS Microbiol Lett, 1989 Jan 15, 48(2), 219 - 21 SAR-2: identification of a novel plasmid-encoded beta-lactamase from India; Nandivada LS et al.; A novel beta-lactamase has been identified in an Escherichia coli strain isolated in South India . The beta-lactamase gene was carried on a plasmid (pUK734) along with resistance determinants to sulphonamides and tetracycline . The novel enzyme has a pI of 8.3 and an Mr of 36,000 . The enzyme has a broad-spectrum of activity against both penicillins and cephalosporins . It is also active against oxacillin and methicillin. Drugs Exp Clin Res, 1989, 15(11-12), 535 - 40 The kinetics of CAZ-5, a novel SHV-related plasmid-mediated beta-lactamase with enhanced hydrolytic activity against ceftazidime; Labia R et al.; The kinetic constants for "CAZ-5", a novel plasmid-mediated beta-lactamase with noticeable activity against third-generation cephalosporins and particularly ceftazidime have been determined . Two closely-related plasmid-mediated beta-lactamases have also been studied: SHV-2 and PIT-2 (also known as SHV-1) . These enzymes were synthesized constitutively; they were highly sensitive to the action of the inhibitors clavulanic acid and sulbactam and they lacked activity against the cephamycins and imipenem . PIT-2/SHV-1 had poor hydrolytic activity against the third-generation cephalosporins, SHV-2 was markedly active against cefotaxime and related compounds, whereas the new enzyme, which was also active against these cephalosporins, had a noticeably greater activity against ceftazidime . Aztreonam was slowly hydrolysed by CAZ-5 beta-lactamase, but demonstrated an unusually high affinity for this enzyme. Proteins, 1989, 6(3), 316 - 23 Chemical modification of the RTEM-1 thiol beta-lactamase by thiol-selective reagents: evidence for activation of the primary nucleophile of the beta-lactamase active site by adjacent functional groups; Knap AK et al.; The RTEM-1 thiol beta-lactamase (Sigal, I.S., Harwood, B.G., Arentzen, R., Proc . Natl . Acad . Sci . U.S.A . 79:7157-7160, 1982) is inactivated by thiol-selective reagents such as iodoacetamide, methyl methanethiosulfonate, and 4,4'-dipyridyldisulfide, which modify the active site thiol group . The pH-rate profiles of these inactivation reactions show that there are two nucleophilic forms of the enzyme, EH2 and EH, both of which, by analogy with the situation with cysteine proteinases, probably contain the active site nucleophile in the thiolate form . The pKa of the active site thiol is therefore shown by the data to be below 4.0 . This low pKa is thought to reflect the presence of adjacent functionality which stabilizes the thiolate anion . The low nucleophilicity of the thiolate in both EH2 and EH, with respect to that of cysteine proteinases and model compounds, suggests that the thiolate of the thiol beta-lactamase is stabilized by two hydrogen-bond donors . One of these, of pKa greater than 9.0, is suggested to be the conserved and essential Lys-73 ammonium group, while the identity of the other group, of pKa around 6.7, is less clear, but may be the conserved Glu-166 carboxylic acid . beta-Lactamase activity is associated with the EH2 form, and thus the beta-lactamase active site is proposed to contain one basic or nucleophilic group (the thiolate in the thiol beta-lactamase) and two acidic (hydrogen-bond donor) groups (one of which is likely to be the above-mentioned lysine ammonium group). APMIS Suppl, 1989, 5, 2 - 8 The incidence of beta-lactamase-producing pathogens; Acar JF et al.; In addition to the bacteria which naturally are able to enzymatically inactive penicillins and/or cephalosporins, a large number of species may develop this ability through mutation, acquisition of plasmids, or insertion of transposons . Characterization of the beta-lactamase activity of various pathogens has shown that a wide variety of enzymes exists and that new ones continue to evolve . The distribution of the genes for the numerous beta-lactamases vary according to geographic location and pathogen . Recently beta-lactamase inhibitors (sulbactam and clavulanic acid) have become available which, in combination with different beta-lactam antibiotics, expand the activity of those hydrolyzable antibiotics to pathogens producing beta-lactamases . The epidemiology of resistant pathogens and of the beta-lactamase genes that make them resistant are important factors in evaluating the role of these beta-lactam/beta-lactamase inhibitor combinations. Acta Leprol, 1989, 7 Suppl 1, 39 - 43 Isoenzymes as tools to discriminate various subdivisions in the Mycobacterium fortuitum complex; Blom-Potar MC et al.; Methods for the characterization of catalase, peroxidase, beta-glucosidase, esterase, and beta-lactamase mycobacterial isoenzymes were described . These methods were applied to examine strains of the M . fortuitum complex . M . fortuitum, M . peregrinum, M . chelonae- M . abscessus and an unnamed species had distinct isoenzyme profiles . M . chelonae and M . abscessus could not be satisfactorily differentiated using the described methods. J Enzyme Inhib, 1989, 2(4), 279 - 94 The structure of M-GTFI, an inhibitor of glucosyltransferase from S . mutans, and its inhibitory relationship with other sulfate ester-containing inhibitors; Uyeda M et al.; M-GTFI, an inhibitor of glucosyltransferase from S . mutans was produced by Micromonospora narashinoensis strain No . 731 . The isolation procedure for M-GTFI was improved and established for spectroscopic analyses, and some properties of the inhibitor were investigated . The structure of M-GTFI was shown to be trisodium {2-sulphonato-(E)-9-undecenyll-oxacyclotriacont-(E)-3- en-2-one, 16, 18-bis sulphonate . The chemical structure of M-GTFI was therefore similar to that of izumenolide which is a beta-lactamase inhibitor containing sulfate ester groups in its molecule . The inhibitory characteristics of M-GTFI were parallel to that of other inhibitory compounds containing sulphate esters but the spectrum of activity was wider. Vestn Dermatol Venerol, 1989, (6), 57 - 8 {beta-Lactamase-producing strains of the gonococcus}; Soina KK et al.; Analysis of the experimental findings permits a conclusion on the necessity of purposeful search for gonococcus strains producing f-lactamase. Drugs Exp Clin Res, 1989, 15(4), 151 - 4 Beta-lactamase evaluation of the amoxycillin-clavulanate association; Novelli A et al.; The capability of potassium clavulanate to protect amoxycillin from destruction by three partially purified beta-lactamases (from E . cloacae, B . cereus 569/H9 and E . coli R-TEM) was evaluated . The enzymatic inhibition was determined spectrophotometrically by assessing the ability of either amoxycillin or potassium clavulanate or their combination (at a 7:1 ratio by weight) to prevent the hydrolysis of a chromogenic substrate (nitrocefin) after different pre-incubation times, thus determining the IC50 and Ki values . Potassium clavulanate significantly reduced the IC50 values of amoxycillin for B . cereus and TEM enzymes by a factor ranging from 500 to 1200, by inhibiting these beta-lactamases at concentrations ranging from 4 to 0.008 microM . However, clavulanic acid was quite ineffective against type I enzymes (from E . cloacae), while amoxycillin was found to be sufficiently stable (IC50 of 0.8-1.4 microM). J Biol Chem, 1988 Dec 25, 263(36), 19690 - 6 Introduction of basic amino acid residues after the signal peptide inhibits protein translocation across the cytoplasmic membrane of Escherichia coli . Relation to the orientation of membrane proteins; Yamane K et al.; The introduction of positive charges at the amino terminus of the mature domain of secretory proteins resulted in strong inhibition of their translocation across the cytoplasmic membrane of Escherichia coli, both in vitro and in vivo . The model secretory proteins used were OmpF-Lpp chimeric proteins possessing a cleavable or uncleavable signal peptide, beta-lactamase (Bla) and Bla-Lpp chimeric proteins . It is suggested that positively charged residues preceding the hydrophobic domain of the signal peptide have a positive effect, and ones following the hydrophobic domain, a negative effect on the translocation . These findings are discussed in relation to the orientation of membrane proteins, of which positive charges are predominant on the cytoplasmic surface. Jpn J Antibiot, 1988 Dec, 41(12), 1954 - 8 {Clinical study of sultamicillin fine granules}; Okada K et al.; Sultamicillin, a mutual prodrug of a beta-lactam antibiotic and beta-lactamase inhibitor, was administered to 19 child patients with infectious diseases . The patients included 9 boys and 10 girls from 11 months to 13 years old and they were given orally a dosage of 15.4-40.8 mg/kg/day for 3 to 12 days . Clinical efficacies were excellent in 2 cases, good in 13 cases, fair in 3 cases, unknown in 1 case, and the total efficacy rate was 83.3% . Loose stool in 1 case and mild diarrhea in another occurred as side effects of the drug, but no abnormal laboratory test values were found upon the treatment. Jpn J Antibiot, 1988 Dec, 41(12), 1906 - 13 {Experiences with sultamicillin granules in pediatric patients}; Iguchi K et al.; Sultamicillin (SBTPC) is a combination drug of sulbactam (SBT) and ampicillin (ABPC) in an ester bonding at 1:1 ratio . SBT, a new semisynthetic beta-lactamase inhibitor restores and extends the spectrum of ABPC against resistant strains of bacteria . SBTPC granules were administered to 26 pediatric patients with infections, and clinical efficacies were studied in 23 patients . The clinical efficacy rate was 95.7% and the drug was evaluated to be highly effective for the treatment of infectious diseases in the pediatric field . SBTPC was safe and well tolerated. Jpn J Antibiot, 1988 Dec, 41(12), 1847 - 54 {Clinical evaluation of sultamicillin fine granules in pediatric infections}; Meguro H et al.; Sultamicillin fine granules (SBTPC), a mutual prodrug of sulbactam (SBT) and ampicillin (ABPC), were administered to 16 pediatric patients with bacterial infections . The efficacy rate was 93.3% . MICs (10(6) cells/ml) of SBTPC against beta-lactamase non-producing strains were not significantly different from those of ABPC, and ranged from less than or equal to 0.05 to 1.56 micrograms/ml . MICs of SBTPC, however, were 2-4 fold smaller than MICs of ABPC against beta-lactamase producing strains . Diarrhea and loose stool as side effects were observed in 4 (25%) of 16 patients, but none of them were severe . After oral administration of SBTPC (10 mg/kg), serum levels of ABPC and SBT peaked at 3.41 micrograms/ml and 2.43 micrograms/ml after 0.6 hour, and declined with half-lives of 1.79 and 1.00 hours, respectively. Gene, 1988 Nov 15, 71(1), 147 - 56 Selection for signal sequence mutations that enhance production of secreted human proinsulin by Escherichia coli; Stahl SJ et al.; This paper describes a method for the positive selection of signal sequence mutations that result in enhanced production of secreted human proinsulin by Escherichia coli . Coding sequences for the structural portion of beta-lactamase (EC 3.5.2.6) were substituted for those of the C terminus of proinsulin in a plasmid that normally directs the synthesis and secretion of proinsulin . The resulting plasmid directed the synthesis of a proinsulin/beta-lactamase fusion protein that was secreted into the periplasmic space and conferred resistance to low levels of ampicillin (Ap) . Beneficial changes to the signal sequence were selected by the host's ability to grow on high levels of Ap . The beta-lactamase coding sequences were then replaced with those of human proinsulin, resulting in plasmids which directed enhanced production of secreted proinsulin. N Z Med J, 1988 Nov 9, 101(857), 758 - 60 Bacterial meningitis in childhood: a 13 year review; Dawson KP et al.; One hundred and forty-five episodes of acute bacterial meningitis in children seen over a 13 year period are reviewed . The mortality rate was 1.4% . Over the study period H influenzae type b remained as the dominant causative organism, with 11% of the isolates being beta-lactamase positive . The difficulties in diagnosis in children, the sequelae of sensorineural deafness and continued morbidity in this disorder are stressed. Antimicrob Agents Chemother, 1988 Nov, 32(11), 1730 - 2 Rapid isoelectric focusing of plasmid-mediated beta-lactamases with Pharmacia PhastSystem; Huovinen S; A modified isoelectric focusing method for rapid semiquantitative identification of plasmid-mediated beta-lactamases by use of the Pharmacia PhastSystem (Uppsala, Sweden) is described . Sonication of bacterial colonies collected directly from growth plates decreased the time required for the procedure . With sonic extracts of known beta-lactamase-producing strains used as controls, the assay could be completed in less than 2 h. Presse Med, 1988 Oct 26, 17(37), 1890 - 4 {Behavior of ceftazidime in regard to different classes of beta-lactamases . The situation in 1988}; Labia R et al.; Following Ambler's observations, B-lactamases can be divided in four classes, probably derived from a very small number of ancestral genes . Class A includes the TEM-type beta-lactamases (TEM-1, TEM-2), PIT-2/SHV-1 and others which do not hydrolyse ceftazidime and other third-generation cephalosporins . The new plasmid-mediated beta-lactamases markedly active against third-generation cephalosporins also belong to class A and the primary structures of few of them are known . All the class A beta-lactamases are highly susceptible to the action of beta-lactamase inhibitors: clavulanic acid, sulbactam, YTR-830, and are devoid of any hydrolytic properties for cephamycins and imipenem . Third-generation cephalosporins are slowly hydrolyzed by the class C beta-lactamases: the inducible cephalosporinases . Thus only organisms which produce derepressed cephalosporinases are resistant to third-generation cephalosporins, but individual variations are known. J Biol Chem, 1988 Oct 5, 263(28), 14315 - 22 Membrane-bound beta-lactamase forms in Escherichia coli; Pluckthun A et al.; Frameshift pseudo-revertants of Escherichia coli RTEM beta-lactamase were obtained by a selection procedure, starting from frameshift mutants at the signal-processing site . These pseudo-revertant proteins, which have a totally altered COOH-terminal part of the signal sequence, are attached to the outer face of the inner membrane . The mutant proteins are enzymatically active in vitro and in vivo, and the membrane localization has no deleterious effect on cell growth . We conclude that initiation of transport across the membrane does not require the COOH-terminal part of the signal, but this part of the sequence determines whether the protein is released to the periplasm either with or without cleavage of the signal, or whether the protein remains anchored to the membrane . Mutants with two signals in series were used to show that a truncated signal is not refractory to transport per se . If neither signal contains a functional cleavage site, the protein is at least partially found on the outer face of the inner membrane . If both signals contain functional cleavage sites, both are removed and the protein is released to the periplasm . If only the first signal contains a cleavage site, a longer fusion protein is transported and released . The results presented here show that a pre-beta-lactamase-like protein can fold properly even as a membrane-bound species. Eur J Clin Microbiol Infect Dis, 1988 Oct, 7(5), 669 - 72 Rapid method for determining the beta-lactamase-inducing potency of drugs; Mett H et al.; A rapid semiquantitative method for determining the beta-lactamase inducing potency of drugs was developed . Bacteria carrying a gene for inducible beta-lactamase expression were inoculated at a concentration of 10(8) CFU/ml into microtiter plates for determination of MICs, which were recorded after 4 h of incubation . A suitable chromogenic beta-lactamase substrate was then added, and after incubation for another 3 h colour changes were monitored. Biochem J, 1988 Sep 15, 254(3), 923 - 5 Kinetic characterization of the acyl-enzyme mechanism for beta-lactamase I; Martin MT et al.; beta-Lactamase I catalyses the hydrolysis of penicillins by an acyl-enzyme mechanism . A procedure was developed for determining the rate constants for the acylation and deacylation steps for the good substrates benzylpenicillin and phenoxymethylpenicillin; this depends on determining the fraction of enzyme that is present as acyl-enzyme in the steady state. Antimicrob Agents Chemother, 1988 Sep, 32(9), 1347 - 9 New sensitive bioassay for sulbactam in bovine tissues; Keeler GH et al.; A quantitative cylinder plate bioassay for sulbactam has been developed which can detect concentrations as low as 0.01 microgram/g in bovine muscle, fat, kidney, and liver tissues . This procedure may also be applicable to human tissues and fluids . In addition to the improved sensitivity, this method differs from all previously described systems because it is based on inhibition by sulbactam of a cell-free beta-lactamase incorporated in the assay agar . The assay is unaffected by the presence of ampicillin even at concentrations 10 times that of the sulbactam concentration . This report describes the analytical technique as well as the accuracy and precision of the method . The assay can be applied to tissue depletion studies. Gene, 1988 Aug 15, 68(1), 159 - 62 pACYC184-derived cloning vectors containing the multiple cloning site and lacZ alpha reporter gene of pUC8/9 and pUC18/19 plasmids; Martinez E et al.; A new series of vectors, pSU2716, pSU2717, pSU2718, and pSU2719, has been constructed . The plasmids contain (i) the P15A replicon, (ii) the chloramphenicol acetyl transferase (CAT)-coding gene from Tn9, and (iii) the HaeII fragment which carries the multiple cloning site and the lacZ alpha reporter gene of pUC8, pUC9, pUC18 and pUC19, respectively . These vectors allow rapid and simple transfer of inserts from pUC plasmids, have an intermediate copy number (which allows regulated expression from the lac promoter), and are compatible with ColE1-derived vectors (and, therefore, can be used in studies requiring the joint expression of two genes, for example, in genetic complementation analysis) . Furthermore, the accumulation of CAT instead of beta-lactamase, allows an easy visualization in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of proteins of 28-35 kDa, which can otherwise be obscured by the beta-lactamase. Arzneimittelforschung, 1988 Jul, 38(7), 863 - 5 In vitro activity of mecillinam and amoxicillin/clavulanic acid against strains of Escherichia coli producing TEM-1, Oxa-1 and chromosomal beta-lactamases; Marre R et al.; The in vitro activity of mecillinam and amoxicillin/clavulanic acid against Escherichia coli strains producing beta-lactamases of the TEM-1, Oxa-1 and chromosomal type were studied using the broth and agar dilution technique . In addition the beta-lactamase activity was determined . The experiments indicated that the beta-lactamase types differed in their influence on the minimum inhibitory concentrations . Both, mecillinam and amoxicillin/clavulanic acid were considerably more active against TEM-1 strains than ampicillin alone, but, with increasing specific beta-lactamase activity, a decrease of susceptibility was found . Strains producing Oxa-1 and chromosomal beta-lactamases usually where highly susceptible to mecillinam but only moderately sensitive or resistant to the combination of amoxicillin/clavulanic acid . In vitro activity of TEM-1 E . coli to mecillinam was strongly reduced when tested in a broth dilution technique . These test systems, however, did not affect the susceptibility of Oxa-1 and chromosomal beta-lactamase producing E . coli strains to either antibiotic. Cancer, 1988 Jun 1, 61(11), 2315 - 7 Branhamella catarrhalis septicemia in patients with leukemia; Saito H et al.; During a 10-year period, four patients with leukemia were identified who had Branhamella catarrhalis septicemia . Two patients had acute leukemia and the remaining two had chronic myelogenous leukemia with blastic transformation . All patients were febrile and neutropenic at the onset of the septicemia . After appropriate antibiotic therapy, they recovered from their infection despite persistence of neutropenia . Because beta-lactamase-producing bacteria are an increasing cause of nosocomial infections, treatment should be selected to cover them. J Mol Biol, 1988 May 20, 201(2), 451 - 4 Evidence for a unique first position codon-anticodon mismatch in vivo; Toth MJ et al.; The Ser68(AGC) codon of the beta-lactamase gene was changed to the glycine codons GGA and GGC . With glycine at position 68, beta-lactamase is inactive because it does not have a nucleophilic side-chain to function in the reaction mechanism . The mutant SG68(GGA) allele had no detectable beta-lactamase activity; however, the mutant SG68(GGC) did produce a small amount of activity . Both mutant alleles produce comparable amounts of beta-lactamase protein in a maxi-cell system . To identify why these two "same-sense" beta-lactamase mutants differ phenotypically, we introduced the alleles into Escherichia coli strains with mutations that affect translational fidelity . The rpsD mutation, which decreases fidelity, significantly increased activity with the SG68(GGC) allele, while the rpsL mutation, which increases translational fidelity, had little effect on the beta-lactamase activity . The rpsD and rpsL alleles had no effect on the SG68(GGA) allele . From the allele specificity of the activity produced by the bla mutants, and from the differential effect of translational fidelity on the activity of the SG68(GGC) allele, we infer that tRNA(GCU)Ser, the AGU/C reading tRNA(Ser), mistranslates SG68(GGC) at a frequency of about 0.1%, and subsequently produces active beta-lactamase . This is the first observation of an A/G wobble with a wild-type tRNA at the first position of the codon-anticodon interaction. Cell, 1988 May 6, 53(3), 423 - 32 A single amino acid determinant of the membrane localization of lipoproteins in E . coli; Yamaguchi K et al.; When beta-lactamase was fused with the signal peptide plus the amino-terminal 9 amino acid residues of the major outer membrane lipoprotein, the resultant lipo-beta-lactamase (LL-1) was shown to be localized to the outer membrane . However, when the 9 residue sequence was replaced with the amino-terminal 12 residue sequence of lipoprotein-28, an inner membrane protein, the resultant lipo-beta-lactamase (LL-2) was found exclusively in the inner membrane . The localization of LL-2 was shifted to the outer membrane simply by substituting the second amino acid residue (Asp) of LL-2 with Ser . Conversely, the alteration of the second residue (Ser) of LL-1 to Asp resulted in the localization of LL-1 to the inner membrane . These results suggest that the second amino acid residue of the lipoproteins plays a crucial role in determining their final locations in the E . coli envelope. J Antibiot (Tokyo), 1988 May, 41(5), 667 - 74 Effects of lincomycin on synthesis of TEM beta-lactamase by Escherichia coli; Okabe A et al.; Sub-inhibitory concentrations of lincomycin slightly inhibit growth of Escherichia coli carrying plasmid RP4 and cause a 2-fold increase in TEM-2 beta-lactamase . To analyze this effect, cultures were pulse-labeled with {3H}leucine, chased with non-radioactive leucine and immunoprecipitated with anti-beta-lactamase antiserum . The synthesis rate of beta-lactamase was two times higher in inhibited cultures than in control cultures . No significant decrease of labeled enzyme occurred during the 30 minutes chase, indicating no degradation of beta-lactamase . The rate of maturation of pre-beta-lactamase was determined by measuring the decrease in the amount of pre-beta-lactamase after a 1-minute labeling interval . There was no significant difference between the control and lincomycin-treated cultures, indicating that posttranslational translocation is not involved in the stimulation . Both plasmid encoded and chromosomally encoded TEM-1 beta-lactamase increased in the presence of lincomycin . The effects of other protein synthesis inhibitors on the synthesis of TEM-1 beta-lactamase were examined . The stimulation of beta-lactamase synthesis by lincomycin appears to be specific for macrolide and related antibiotics and is not a general phenomenon resulting from partial inhibition of protein synthesis. Pathol Biol (Paris), 1988 May, 36(5), 366 - 9 {Titration curves (ph gradient electrophoresis) of SHV-1 and SHV-2 beta-lactamases and a new type}; Vedel G et al.; The molecular structures of the SHV-1 (p 453) and SHV-2 (pBP 60-1) beta-lactamases and of a new enzyme, a SHV-2 like extended broad-spectrum beta-lactamase (86-4), were compared by analysis of their titration curves (pH gradient electrophoresis) . The titration curves of SHV-1 and SHV-2, which have the same isoelectric points (pI = 7.7) . were completely superimposable for the whole of the pH gradient (pH 3.5-10), indicating a close homology between the two proteins, with perhaps the substitution of several amino acids by ones having the same charge . The curves of SHV-1 (pI = 7.7) and the new SHV-2-like enzyme (pI = 6.98) indicated that a basic residue in SHV-1 has been replaced by an acidic residue in the new SHV-2-like enzyme . These results show that, like SHV-2, the new beta-lactamase is a variant of SHV-1, and that the structural differences are probably limited to a very small number of amino acid residues . Nevertheless, this new beta-lactamase (SHV-3) may have arisen directly from SHV-1, indirectly via SHV-2, or even from another beta-lactamase. FEBS Lett, 1988 Apr 11, 231(1), 217 - 20 Single amino acid substitution between SHV-1 beta-lactamase and cefotaxime-hydrolyzing SHV-2 enzyme; Barthelemy M et al.; SHV-2 beta-lactamase was purified from an overproducing variant of a clinical isolate of Escherichia coli resistant to cefotaxime . Pure protein was digested by trypsin and Lys-C endoproteinase . Proteolytic peptides, isolated by reverse-phase HPLC, were submitted to manual Edman degradation and aligned by homology with the sequence of SHV-1 beta-lactamase . A putative amino acid sequence was deduced . Structural comparison revealed that SHV-2 differed from SHV-1 by only one amino acid, Gly----Ser, at position 213 of the mature protein. South Med J, 1988 Apr, 81(4), 507 - 14 Adenotonsillectomy in children: indications and contraindications; Kavanagh KT et al.; Adenoidectomy and tonsillectomy are the most common major operations done on children . The indications for tonsillectomy in certain clinical situations are constantly being debated in the literature and among professionals . We studied the efficacy (or lack of it) of adenotonsillectomy for chronic tonsillitis (recurrent throat infections), oral nasal obstruction, peritonsillar abscess, elimination of a bacterial carrier state, biopsy, and prevention of tongue thrusting with resultant anterior open bite . Adenoidectomy has been advocated in the literature for the treatment of nasal obstruction, sinusitis, and chronic serous otitis media . Complications of tonsillectomy and adenoidectomy include hemorrhage, anesthetic death, infection, nasopharyngeal stenosis, patulous eustachian tube, and hypernasality . Children at risk for hypernasality are those with mental retardation, cerebral palsy, neuromuscular disorders, and submucous cleft of the soft palate . Because of the severity of the complications that can be encountered in any child, medical and conservative therapy should be attempted before operation is done . Proper antibiotic therapy will often control chronic serous otitis, sinusitis, and chronic, recurrent tonsillitis . Bacterial synergy is important to consider when selecting antibiotic therapy, since beta-lactamase production may protect pathogens commonly considered susceptible to standard antibiotic therapy. Mol Microbiol, 1988 Mar, 2(2), 209 - 17 Defective Escherichia coli signal peptides function in yeast; Pines O et al.; To investigate structural characteristics important for eukaryotic signal peptide function in vivo, a hybrid gene with interchangeable signal peptides was cloned into yeast . The hybrid gene encoded nine residues from the amino terminus of the major Escherichia coli lipoprotein, attached to the amino terminus of the entire mature E . coli beta-lactamase sequence . To this sequence were attached sequences encoding the nonmutant E . coli lipoprotein signal peptide, or lipoprotein signal peptide mutants lacking an amino-terminal cationic charge, with shortened hydrophobic core, with altered potential helicity, or with an altered signal-peptide cleavage site . These signal-peptide mutants exhibited altered processing and secretion in E . coli . Using the GAL10 promoter, production of all hybrid proteins was induced to constitute 4-5% of the total yeast protein . Hybrid proteins with mutant signal peptides that show altered processing and secretion in E . coli, were processed and translocated to a similar degree as the non-mutant hybrid protein in yeast (approximately 36% of the total hybrid protein) . Both non-mutant and mutant signal peptides appeared to be removed at the same unique site between cysteine 21 and serine 22, one residue from the E . coli signal peptidase II processing site . The mature lipo-beta-lactamase was translocated across the cytoplasmic membrane into the yeast periplasm . Thus the protein secretion apparatus in yeast recognizes the lipoprotein signal sequence in vivo but displays a specificity towards altered signal sequences which differs from that of E . coli. Mol Cell Probes, 1988 Mar, 2(1), 83 - 5 Reliability of biotinylated DNA probes in colony hybridization: evaluation of an improved colony lysis method for detection of TEM-1 beta-lactamase; Huovinen S et al.; Utilizing an improved method for colony hybridization developed by Haas & Fleming, biotin and 32P-labelled TEM-1 probes were compared for sensitivity and specificity in identifying the type of beta-lactamase made by over 100 clinical bacterial isolates . The new procedure was more reliable than a standard one, but still gave more than 20% false positive and false negative reactions. J Gen Microbiol, 1988 Mar, 134 ( Pt 3), 835 - 40 Molecular cloning of the Shv-1 beta-lactamase gene and construction of an Shv-1 hybridization probe; Bisessar U et al.; We have cloned the Shv-1 beta-lactamase gene from the R1010 plasmid into pACYC184 . By subcloning and transposon mutagenesis we have localized the gene to a 1.6 kb BscI-SalI fragment of R1010, which is present in the recombinant plasmid pUB8 . A 900 bp PstI fragment of pUB8 was shown to be a specific hybridization probe by testing against plasmids which encode 17 different beta-lactamase enzymes . A comparison was made of the sensitivity of the Shv-1 probe labelled with either {35S}dCTP or with photobiotin. Antimicrob Agents Chemother, 1988 Feb, 32(2), 175 - 9 Detection of plasmid-mediated beta-lactamases with DNA probes; Huovinen S et al.; beta-Lactamase identification by colony hybridization with 32P-labeled DNA probes for TEM-1, SHV-1, OXA-1, OXA-2, PSE-1, PSE-2, and PSE-4 was compared with isoelectric focusing in 122 clinical isolates making a variety of enzyme types . All strains producing a probe-type enzyme gave a positive hybridization reaction . Cross-hybridization was observed between TEM-1 and TEM-2 or TLE-1, between SHV-1 and SHV-2, between OXA-1 and OXA-4, between OXA-2 and OXA-3 (weak), between PSE-2 and OXA-6 or OXA-5 (weak), and among PSE-1, PSE-4, and CARB-3 . With allowance for such cross-hybridization, only six strains gave false-positive reactions, and the procedure was 99% specific. J Med Chem, 1988 Feb, 31(2), 370 - 4 N-aryl 3-halogenated azetidin-2-ones and benzocarbacephems, inhibitors of beta-lactamases; Joyeau R et al.; N-(3-Carboxy-6-methylphenyl)-3-fluoroazetidin-2-one and a series of related N-aryl-3-halo- and -3,3-dihaloazetidinones 3, in which the halo substituent is a fluorine or a bromine atom, were prepared by using the Wasserman procedure of cyclization of beta-bromopropionamides as a key step . Their affinities for the TEM-1 beta-lactamase were determined and compared with those of a series of tricyclic azetidinones, the benzocarbacephems 2, and known beta-lactamase inhibitors . The beta-lactams 2 and 3 behave as competitive inhibitors and not as substrates of the enzyme; neither halogen substitution (series 3) nor ring strain (series 2) induces enzymatic hydrolysis. Antimicrob Agents Chemother, 1988 Jan, 32(1), 134 - 6 Sequence of PSE-2 beta-lactamase; Huovinen P et al.; The nucleotide sequence of PSE-2 beta-lactamase, an enzyme that readily hydrolyzes both carbenicillin and oxacillin, has been determined . The deduced sequence of 266 amino acids contained 93 residues identical to those of OXA-2 beta-lactamase and the Ser-Thr-Phe-Lys tetrad also found in the active site of TEM-1 beta-lactamase. J Enzyme Inhib, 1988, 2(2), 73 - 90 The potential use of mechanism-based enzyme inactivators in medicine; Silverman RB; Mechanism-based enzyme inactivator, alanine racemase, S-adenosylhomocysteine hydrolase, D-amino acid aminotransferase, gamma-aminobutyric acid aminotransferase, arginine decarboxylase, aromatase, L-aromatic amino acid decarboxylase, dihydrofolate reductase, dihydroorotate dehydrogenase DNA polymerase I, dopamine beta-hydroxylase, histidine decarboxylase, beta-lactamase, monoamine oxidase, ornithine decarboxylase, serine proteases, testosterone 5 alpha-reductase, thymidylate synthetase, xanthine oxidase. Drugs Exp Clin Res, 1988, 14(5), 335 - 9 The kinetics of SHV-2 plasmid-mediated beta-lactamase compared to those of the parent enzyme from which it is derived; Labia R et al.; Kinetic constants were determined for two closely related plasmid-mediated beta-lactamases: SHV-2 and PIT-2 (also known as SHV-1) . These enzymes were synthesized constitutively . They were highly sensitive to the action of the inhibitors clavulanic acid and sulbactam, and they lacked activity against cephamycins and also imipenem . Both enzymes were significantly active against penicillins and first-generation cephalosporins, and the main difference concerned the third-generation cephalosporins: SHV-2 was highly active against these compounds whereas PIT-2 was not. Plasmid, 1988 Jan, 19(1), 21 - 9 Molecular structure and interrelationships of multiresistance beta-lactamase transposons; Levesque RC et al.; Transposons coding for beta-lactamases OXA-3, OXA-4, OXA-5, LCR-1, and CARB-3 have been isolated and compared functionally and structurally with transposons for TEM-1, OXA-1, PSE-1, PSE-2, and PSE-4 enzymes . Each beta-lactamase gene type occurred in a unit together with resistance to other antibiotics, particularly streptomycin and sulfonamide but also chloramphenicol, mercuric ion, or gentamicin, kanamycin, and tobramycin . Restriction mapping, gene cloning, and DNA hybridization were used to compare the transposons and to localize their functional components . Although the multiresistance beta-lactamase transposons varied in size from 8 to 25 kb, the similarity of some of their restriction maps suggested a common derivation . Six of 12 transposons contained DNA segments homologous to the tnpR gene of transposon Tn21 and could complement a tnpR- Tn21 derivative . Consequently, these six transposons appear to have evolved from a common progenitor by acquisition of DNA coding for various beta-lactamases and other resistance genes. Antimicrob Agents Chemother, 1988 Jan, 32(1), 9 - 14 Dissemination of the novel plasmid-mediated beta-lactamase CTX-1, which confers resistance to broad-spectrum cephalosporins, and its inhibition by beta-lactamase inhibitors; Kitzis MD et al.; The novel beta-lactamase CTX-1 (pI 6.3) encoded on a transferable 84-kilobase plasmid was found in six different bacterial species . It was responsible for a significant decrease in susceptibility towards most penicillins and cephalosporins, except imipenem, temocillin, and cephalosporins which have a 7-alpha-methoxy substituent . Synergy between either ampicillin, piperacillin, cefotaxime, ceftazidime, or aztreonam and three beta-lactamase inhibitors (clavulanic acid, sulbactam, and YTR 830) was generally found for different strains harboring CTX-1 . This enzyme may be related to or derived from the TEM enzyme, since an intragenic probe of the TEM-1 gene hybridized with a fragment of the plasmid carrying CTX-1. J Biol Chem, 1987 Dec 25, 262(36), 17591 - 5 Controls on the biosynthesis of the manganese-containing superoxide dismutase of Escherichia coli . Effects of thiols; Gardner PR et al.; In vitro synthesis of Escherichia coli manganese-containing superoxide dismutase, directed by the plasmid pDT1-5, has been achieved . The Mn superoxide dismutase polypeptide was identified by electrophoresis on polyacrylamide gels, immunoprecipitation, and the competitive immunoprecipitation effect of pure, active E . coli Mn superoxide dismutase . Dithiothreitol and glutathione, but not cysteine, suppressed in vitro synthesis of Mn superoxide dismutase . The parallel syntheses of beta-lactamase and of another unidentified polypeptide were not suppressed by thiols . In vitro transcription of the E . coli Mn superoxide dismutase gene was similarly suppressed by glutathione, dithiothreitol, and beta-mercaptoethanol; but not by L-cysteine or thioglycolate . Compounds, such as diamide, 1-chloro-2,4-dinitrobenzene, potassium ferricyanide, and methylene blue, which are expected to deplete intracellular glutathione, caused the induction of Mn superoxide dismutase in anaerobic E . coli. J Biol Chem, 1987 Dec 5, 262(34), 16433 - 8 Localization and membrane topology of EnvZ, a protein involved in osmoregulation of OmpF and OmpC in Escherichia coli; Forst S et al.; OmpR and EnvZ, the protein products of the ompB locus, are regulatory components required for osmoexpression of outer membrane porin proteins, OmpF and OmpC, in Escherichia coli . EnvZ is considered to be an osmosensor which transmits signals across the membrane to OmpR, a transcriptional activator for ompF and ompC . We inserted the envZ gene into a high expression vector, pIN-III . Following cellular fractionation, EnvZ was found to be localized in the inner membrane . Sequence analysis revealed that the signal peptide-like N-terminal sequence was not removed from the purified EnvZ . A genetic approach using EnvZ/beta-lactamase fusion proteins was taken to determine the topology of EnvZ in the inner membrane . When beta-lactamase was fused after the N-terminal signal peptide-like sequence, ampicillin resistance, conferred by the beta-lactamase moiety of the fusion protein, was expressed . However, when beta-lactamase was fused after the second downstream apolar sequence, the cells showed very poor ampicillin resistance indicating that the enzyme was localized on the cytoplasmic side of the inner membrane . The results of this approach reveal that the hydrophilic region of EnvZ between the two apolar sequences is periplasmically localized and that the hydrophilic region downstream of the second apolar sequence is cytoplasmically directed . These results were confirmed by partial proteolysis of the fusion proteins in intact cells. Antimicrob Agents Chemother, 1987 Dec, 31(12), 2007 - 9 Molecular epidemiology of OHIO-1 beta-lactamase; Kron MA et al.; A total of 31 plasmids, all bearing a gene that encodes a novel, plasmid-mediated Richmond-Sykes class III beta-lactamase designated OHIO-1 and a gene that encodes aminoglycoside 2"-adenyltransferase, have been collected from hospitals in Ohio . By using restriction endonuclease digestion and Southern hybridization, we were able to demonstrate that all these plasmids have a common genetic origin. FEBS Lett, 1987 Nov 16, 224(1), 213 - 8 Export and secretion of overproduced OmpA-beta-lactamase in Escherichia coli; Bolla JM et al.; The export of beta-lactamase to the periplasm of Escherichia coli can be directed by the OmpA signal peptide in the secretion cloning vector pIN-III . The overproduction of the hybrid precursor specifically induces a delay in the onset of processing of newly synthesized polypeptide chains . However, when the processing starts, no alteration in the rate of cleavage itself is observed . Our results suggest that the temporal mode of processing (which reflects translocation) does not depend on the nature of the signal peptide but rather depends on the nature of the polypeptide chain exported. J Gen Microbiol, 1987 Oct, 133 ( Pt 10), 2915 - 20 Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase from Streptomyces cacaoi; Lenzini MV et al.; A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number plasmid pIJ702 as vector . A 30-fold higher yield of beta-lactamase was obtained from S . lividans strain ML1, carrying the recombinant plasmid pDML51, than from S . cacaoi grown under optimal production conditions . In all respects (molecular mass, isoelectric point, kinetics of inhibition by beta-iodopenicillanate) the overproduced S . lividans ML1 beta-lactamase was identical to the original S . cacaoi enzyme . A considerable reduction of beta-lactamase production was caused by elimination of a 12.8 kb portion of the 19 kb DNA fragment by cleavage at an internal SphI site located more than 3 kb upstream of the beta-lactamase structural gene . The beta-lactamase gene was located within a 1.8 NcoI-BclI fragment but when this fragment was cloned in S . lividans pIJ702, the resulting strain produced hardly any more beta-lactamase than the original S . cacaoi. Eur J Clin Microbiol, 1987 Oct, 6(5), 570 - 1 Beta-lactamase hydrolysis and inhibition studies of the new 1-carbacephem LY163892; Jones RN et al.; A novel 1-carbacephem, LY163892, was determined to be more stable to plasmid-mediated beta-lactamases than cefaclor . Chromosomal-mediated Type Ia and IVc enzymes destroyed LY163892 at rates ranging from 16 to 93% that of nitrocefin . LY163892 showed minimal ability to inhibit beta-lactamases other than Type Ia (P99). Mikrobiyol Bul, 1987 Oct, 21(4), 284 - 8 {A comparison of the in vitro susceptibility of three bacteria to beta-lactamase inhibitors and to ampicillin and cefazolin}; Acar N et al.; In this study in vitro susceptibility of three different bacteria, which are able to release various Beta-lactamases, against combinations of clavulanic acid + amoxicillin and sulbactam + ampicillin were compared with susceptibility to ampicillin alone and cefazolin of same strains. Chemioterapia, 1987 Oct, 6(5), 337 - 40 The effects of three beta-lactamase inhibitors: YTR830H, sulbactam and clavulanic acid on the growth of human cells in culture; Yamabe S et al.; Three beta-lactamase inhibitors: YTR83OH (YTR), sulbactam and clavulanic acid were studied for their effects on the growth of human amnion FL line cells . The numbers of viable cells after the 3-day culture were almost unaffected by YTR at concentrations up to 250 mg/l . The effects of sulbactam on FL cells were similar to those of YTR . The growth inhibitory effects of clavulanic acid were significantly different and were effective down to a concentration of 50 mg/l . The morphological effects were also observed microscopically. Biochem J, 1987 Oct 1, 247(1), 29 - 33 Inactivation of the thiol RTEM-1 beta-lactamase by 6-beta-bromopenicillanic acid . Identity of the primary active-site nucleophile; Knap AK et al.; The thiol RTEM-1 beta-lactamase {Sigal, Harwood & Arentzen (1982) Proc . Natl . Acad . Sci . U.S.A . 79, 7157-7160} is inactivated by 6-beta-bromopenicillanic acid with formation of a characteristic chromophore, absorbing maximally at 350 nm, which is covalently bound to the enzyme . Model studies suggest that the chromophore is that of a 6-carboxylate thiol ester of 2,3-dihydro-2,2-dimethyl-1,4-thiazine-3,6-dicarboxylate, which can arise by rearrangement of the thiol-penicilloate obtained by thiolysis of the beta-lactam of 6-beta-bromopenicillanate . Loss of activity of the enzyme is also concerted with disappearance of its single (cysteine) thiol group . These results indicate that the thiol group of this enzyme is indeed a nucleophilic catalyst in beta-lactam turnover . The thiol beta-lactamase is also inactivated by clavulanic acid with formation of a chromophore, presumably a 3-aminoacrylate thiol ester, at 308 nm . Both 6-beta-bromopenicillanate and clavulanate are hydrolysed more slowly by the thiol enzyme than by the native serine beta-lactamase, but, probably as a consequence of this, both compounds inactivate the former enzyme more efficiently . Cefoxitin, a poor substrate of the native enzyme, does not appear to interact covalently with the thiol beta-lactamase. Vopr Virusol, 1987 Sep-Oct, 32(5), 551 - 4 {Determination of antibodies to the AIDS virus and the viral antigen by an immunoenzyme method using peroxidase and beta-lactamase}; Vengerov IuIu et al.; The ELISA test using beta-lactamase for the detection of anti-AIDS virus antibodies and virus antigen in serum is described . The properties of this test system are compared with those of the system based on horseradish peroxidase conjugates. Plasmid, 1987 Sep, 18(2), 127 - 34 Effects of recombinant plasmid size on cellular processes in Escherichia coli; Cheah UE et al.; The effects of recombinant plasmid size on cell growth and viability, plasmid copy number, and synthesis of plasmid-encoded protein were investigated in Escherichia coli using plasmid pUC8 and four recombinant derivatives containing inserts of Drosophila melanogaster DNA of 1.7-6.0 kb . Growth in log phase was unaffected by plasmid size, but as plasmid size increased, maximum cell density decreased and, with the largest plasmid, cell death was accelerated after the stationary phase was reached . There was also a correlation between increasing plasmid size and decreased viability at high ampicillin concentrations, resistance to which is conferred by the plasmids . These effects were shown not to be due to transcription or translation of Drosophila sequences carried on the recombinant plasmids . Cells harboring the largest plasmid, pBS5 (8.7 kb), fared poorly in competition with plasmid-free cells in mixed cultures, compared with cells harboring pUC8 (2.7 kb) . In addition, pBS5 was harbored at significantly fewer copies per cell than pUC8 at all phases of growth and supported much less production of the plasmid-encoded protein, beta-lactamase, than did pUC8 . The results suggest that recombinant plasmid size may be an important parameter in the optimization of large-scale production of plasmid-encoded proteins. Mol Gen Genet, 1987 Sep, 209(2), 391 - 5 tolA, tolB and excC, three cistrons involved in the control of pleiotropic release of periplasmic proteins by Escherichia coli K12; Fognini-Lefebvre N et al.; Mutants of Escherichia coli K12 carrying exc mutations inducing the release of the plasmid pBR322-encoded beta-lactamase (EC 3.5.2.6) into the extracellular medium were analysed and compared with previously described excretory mutants carrying lky mutations associated with the release of alkaline phosphatase and to tolA and tolB mutants, originally described as tolerant towards various colicins . The exc, lky and tol mutations mapped near the gal operon at min 16.5 of the E . coli linkage map . A genetic analysis presented in this paper showed that some exc and lky mutations belonged to the tolA and tolB complementation groups . Furthermore, we identified a third cistron, excC, involved in the excretion of periplasmic enzymes but distinct from the two others. Proc Natl Acad Sci U S A, 1987 Sep, 84(18), 6349 - 53 Uptake and expression of bacterial and cyanobacterial genes by isolated cucumber etioplasts; Daniell H et al.; The uptake and expression by plastids isolated from dark-grown cucumber cotyledons (etioplasts) of two pUC derivatives, pCS75 and pUC9-CM, respectively carrying genes for the large small subunits of ribulose bisphosphate carboxylase/oxygenase of Anacystis nidulans or chloramphenicol acetyltransferase, is reported . Untreated etioplasts take up only 3% as much DNA as that taken up by EDTA-washed etioplasts after 2 hr of incubation with nick-translated {32P}-pCS75 . The presence or absence of light does not affect DNA uptake, binding, or breakdown by etioplasts . Calcium or magnesium ions inhibit DNA uptake by 86% but enhance binding (23-200%) and breakdown (163-235%) of donor DNA by EDTA-treated etioplasts . Uncouplers that abolish membrane potential (delta psi), transmembrane proton gradient (delta pH), or both do not affect DNA uptake, binding, or breakdown by etioplast . However, both DNA uptake and binding are severely inhibited by ATP . Presumably this results from the hydrolysis of ATP, because the poorly hydrolyzable analog adenyl-5'-yl imidodiphosphate does not inhibit the uptake or binding of DNA by etioplasts . beta-Lactamase specified by the ampicillin resistance gene of pCS75 can be detected only in EDTA-treated etioplasts that have been incubated with the plasmid pCS75 . After the incubation of EDTA-treated etioplasts with pCS75, immunoprecipitation using antiserum to the small subunit of ribulose bisphosphate carboxylase/oxygenase from A . nidulans reveals the synthesis of small subunits; these are smaller by 2 kDa than the cucumber small subunit encoded by the nuclear genome . Treatment of etioplasts with 10 mM EDTA shows a 10-min duration to be optimal for the expression of chloramphenicol acetyltransferase encoded by pUC9-CM . A progressive increase in the expression of this enzyme is observed with an increase in the concentration of pUC9-CM in the DNA uptake medium . The plasmid-dependent incorporation of {35S}methionine by EDTA-treated organelles declines markedly during cotyledon greening in vivo. Anal Biochem, 1987 Aug 15, 165(1), 75 - 87 The analysis of enzyme progress curves by numerical differentiation, including competitive product inhibition and enzyme reactivation; Koerber SC et al.; A new method for analyzing steady-state enzyme kinetic data is presented . The technique, which is based on the numerical differentiation of the complete reaction curve, has several advantages over initial velocity and integrated Michaelis-Menten equation methods . The differentiated data are fit to the differential equation describing the appropriate kinetic scheme . This approach is particularly valuable in cases of strong competitive product inhibition and of changing concentrations of active enzyme . The method assumes a reversible reaction and is applicable to a very wide variety of steady-state kinetic schemes . A particular advantage of this approach over integrated methods is that it is independent of {S0} and hence of errors in {S0} . The combination of complete progress curve and computer analysis makes this approach very efficient with respect to both time and materials . Running on an IBM PC XT or equivalent microcomputer with an 8087 coprocessor, the analyses are very fast, the complete process usually being complete in a minute or two . The utility of the technique is demonstrated by application to both simulated and real data . We show that the differentiation of the progress curve for the ribonuclease-catalyzed hydrolysis of 2',3'-cyclic cytidine monophosphate reveals strong product inhibition by 3'-CMP, and this product inhibition accounts for the large discrepancies reported in the literature for the value of Km for this substrate . The method was also applied to determine the rate of reactivation of beta-lactamase which had been reversibly inactivated by cloxacillin . Since large numbers of data points are required for the numerical differentiation the method has become practical only with the advent of computer-acquired data systems. Biochem J, 1987 Aug 1, 245(3), 911 - 3 The crystal structure of the beta-lactamase of Streptomyces albus G at 0.3 nm resolution; Dideberg O et al.; The crystal structure of the beta-lactamase of Streptomyces albus G has been solved at 0.3 nm resolution by X-ray-diffraction methods . The enzyme is a typical two-domain protein . One domain consists of five alpha-helices, and the other is five-stranded beta-sheet with alpha-helices on both sides of the sheet . The active-site serine residue (Ser-48) is within a cleft located between the two domains. EMBO J, 1987 Aug, 6(8), 2463 - 8 Extracellular release of colicin A is non-specific; Baty D et al.; The possible involvement of topogenic export sequences within the colicin A polypeptide chain has been investigated . Different constructs have been made using various techniques to introduce deletions in the central and NH2-terminal regions of colicin A . Together, these deletions span the region from amino acid 15 to the end of the protein . None of these regions was found to be required for extracellular release or had any effect on the efficiency of this process . By inserting a termination codon, a Shine-Dalgarno sequence and an initiation codon into the gene for colicin A, the NH2-terminal and central plus COOH-terminal domains could be demonstrated to be released to the same extent when produced as separate polypeptides as when produced as linked ones . The introduction into the COOH-terminal domain of mutations promoting cytoplasmic aggregation had no effect on the secretion of the NH2-terminal polypeptide . These results demonstrated that no specific interaction between the NH2- and COOH-terminal regions of the colicin A polypeptide chain is involved in the release of colicin A . We are led to conclude that there is no topogenic export signal in the polypeptide chain of colicin A involved in the release mechanism . Thus the process is non-specific with respect to the colicin itself and depends solely on the expression of the colicin A lysis protein (Cavard et al., 1985, 1987) . The expression of the protein causes the release of not only the colicin but also many other cellular proteins, including beta-lactamase, EF-Tu, and chloramphenicol acetyltransferase. Eur J Clin Microbiol, 1987 Aug, 6(4), 474 - 6 Beta-lactamase alteration of beta-lactam inhibitory zones; Tausk F et al.; A disk approximation test, in which the inhibitory zone of one beta-lactam antibiotic is truncated under influence of another beta-lactam (e.g . cefoxitin), was used as a screen for the presence of inducible beta-lactamase . By using beta-lactamase extracts and a specific inhibitor, it was shown that chromosomal beta-lactamase can indeed be the sole cause of truncated beta-lactam inhibitory zones. J Med Chem, 1987 Aug, 30(8), 1469 - 74 Synthesis and beta-lactamase inhibitory properties of 2 beta-{(1,2,3-triazol-1-yl)methyl}-2 alpha-methylpenam-3 alpha-carboxylic acid 1,1-dioxide and related triazolyl derivatives; Micetich RG et al.; Benzhydryl 2 beta-{(1,2,3-triazol-1-yl)methyl}-2 alpha-methylpenam- 3 alpha-carboxylate 1,1-dioxide was prepared by heating benzhydryl 2 beta-(azidomethyl)-2 alpha-methylpenam-3 alpha-carboxylate 1,1-dioxide with (trimethylsilyl)acetylene . The ester group was removed by hydrogenolysis to give sodium 2 beta-{(1,2,3-triazol-1-yl)methyl}-2 alpha-methylpenam-3 alpha-carboxylate 1,1-dioxide (3i, YTR-830), which was found to be a potent inhibitor of various bacterial beta-lactamases . A series of related compounds was prepared in a similar way, and all of these compounds show excellent beta-lactamase inhibitory properties. Antimicrob Agents Chemother, 1987 Aug, 31(8), 1266 - 70 Structure and mobilization of an ampicillin and gentamicin resistance determinant; Martin C et al.; A DNA segment originally found in an epidemic plasmid of Escherichia coli encoding an aminoglycoside-(3)-N-acetyltransferase gene (aacC5) and a TEM-type beta-lactamase gene was characterized . The two genes were adjacent and constituted a single transcriptional unit . In addition, these genes were simultaneously mobilized through the action of an insertion sequence related to IS26, IS140, and IS15-delta . This DNA segment is a composite transposon which has been called Tn2922. J Biol Chem, 1987 Jul 15, 262(20), 9463 - 8 Wild type and mutant signal peptides of Escherichia coli outer membrane lipoprotein interact with equal efficiency with mammalian signal recognition particle; Garcia PD et al.; The signal peptide of the outer membrane lipoprotein (OMLP) of Escherichia coli was shown to be capable of promoting protein translocation across mammalian microsomal membranes in vitro . We assayed translocation of a fusion protein containing the OMLP signal peptide and nine amino acids of OMLP fused in frame to beta-lactamase . The efficiency with which the mammalian translocation machinery recognizes and accepts the OMLP signal peptide as substrate is indistinguishable from that of mammalian secretory proteins . Upon translocation mammalian signal peptidase processes the pre-OMLP-beta-lactamase protein at different sites than are utilized in vivo by E . coli OMLP signal peptidase (signal peptidase II) but that can be predicted as mammalian signal peptidase cleavage sites . Mutants in the OMLP signal peptide were tested for their ability to promote translocation of the fusion protein in this assay system . It has been shown previously that mutants in the positively charged amino acids at the amino terminus of the signal peptide severely delay the translocation of OMLP in vivo in E . coli . However, these mutants had no detectable effect either on signal recognition by mammalian signal recognition particle or on the efficiency of translocation itself. Eur J Biochem, 1987 Jul 15, 166(2), 345 - 50 Nucleotide sequence of the gene encoding the Streptomyces albus G beta-lactamase precursor; Dehottay P et al.; A 1400-base DNA fragment, which contains the gene encoding the extracellular active-site serine beta-lactamase of Streptomyces albus G previously cloned into Streptomyces lividans {Dehottay et al . (1986) Gene 42, 31-36}, was sequenced . The gene codes for a 314-amino-acid precursor, the N-terminal region of which has the characteristics of a signal peptide . The beta-lactamase as excreted by the host strain S . lividans PD6 has a ragged N-terminus, indicating either the presence of a leader peptidase of poor specificity or the action of an aminopeptidase . The primary structure (as deduced from the nucleotide sequence) was confirmed by amino acid sequencing of a 16-residue stretch at the amino terminus of the protein, a 12-residue stretch containing the active-site serine {De Meester et al . (1987) Biochem . J . 244, 427-432} and a 23-residue stretch obtained by trypsin digestion of the protein . The beta-lactamase belongs to class A, has three half-cystine residues (one of which occurs on the amino side of the active-site serine) and is inactivated by thiol reagents . Putative ribosome binding site and terminator region were identified. Biochim Biophys Acta, 1987 Jul 14, 909(2), 115 - 25 Effects of ppGpp on transcription by DNA-dependent RNA polymerase from Escherichia coli: circular dichroism, absorption and specific transcription studies; Woody AY et al.; Concrete evidence is presented for conformational changes elicited in RNA polymerase upon binding ppGpp by circular dichroism measurements . In the presence of 100 microM ppGpp, the molar ellipticity of RNA polymerase at 220 nm is reduced by 14% from the initial value of - 11,100 deg X cm2 X dmol-1 at 25 degrees C . In vitro transcription on templates containing the beta-lactamase promoter and colicin E1 promoter on poly{d(A-T)} is inhibited by ppGpp . None of these templates had GC-rich nucleotide sequence near the transcription initiation site, and yet they were influenced by ppGpp . Comparison of the effect on the synthesis of mRNAs for beta-lactamase and colicin E1 and the synthesis of the proteins themselves indicates that the effect of ppGpp is at the level of transcription for the former case and involves coupled transcription-translation for the latter case . Difference absorption, polyacrylamide gel electrophoresis, and nitrocellulose filter-binding studies show that the binding of ppGpp to RNA polymerase does not impair the extent of the interaction between enzyme and DNA . Kinetic studies suggest that ppGpp affects transcription initiation on beta-lactamase promoter . On poly{d(A-T)}, ppGpp affects the rate of open complex formation and is a mixed inhibitor with respect to the incorporation of nucleotides . Our results are consistent with the idea that ppGpp acts as a regulator by binding at a site different from the active site and changes the RNA polymerase conformation, causing altered transcriptional behavior on different DNA templates. Nucleic Acids Res, 1987 Jul 10, 15(13), 5157 - 68 Cloning and mapping of infA, the gene for protein synthesis initiation factor IF1; Sands JF et al.; The gene for translation initiation factor IF1, infA, has been identified by using two synthetic oligonucleotides to screen a Charon 30 library of Escherichia coli DNA . A recombinant lambda phage, C1921, was purified from a plaque positive for both probes . A 2.8 kb BglII fragment and a 2.0 kb HindIII fragment isolated from C1921 were subcloned into the BamHI and HindIII sites of pBR322 to yield pTB7 and pTH2 respectively . Synthesis of IF1 in maxicells transformed with pTB7 or pTH2 indicates the presence of inf A in both inserts . This was confirmed by DNA sequencing: a region was found that codes for a 8,119 dalton protein with an amino acid sequence corresponding to IF1 . The chromosomal location of inf A was determined by mapping the closely linked beta-lactamase gene (Ampr) in pTB7 and pTH2 . pTB7 and pTH2 were transformed into polA Hfr hosts, and integration of the plasmid by homologous recombination near inf A was selected on the basis of ampicillin resistance . The site of integration was confirmed by Southern blot analysis of restriction nuclease digested wild type and transformed genomic DNA . The Ampr marker (and therefore inf A) was mapped to about 20 minutes by Hfr interrupted matings and P1 transduction experiments . The structure and regulation of the inf A operon currently are being investigated. Diagn Microbiol Infect Dis, 1987 Jul, 7(3), 219 - 23 Single-dose surgical prophylaxis using ticarcillin/clavulanic acid (Timentin): a prospective, randomized comparison with cefotaxime; Jones RN et al.; Single 3.1-g dose surgical prophylaxis with ticarcillin/clavulanic acid (Timentin) was compared to single-dose cefotaxime . Among 224 evaluable cases, the Timentin associated wound infection rate (0.7%), nonwound infectious morbidity (8.8%), adverse reactions (1.9%), and total costs ($14.15) were not statistically different than that of the control drug . The beta-lactamase {corrected} inhibitor combination reported herein should be considered along with other beta-lactams {corrected} for use as a cost-effective, single-dose surgical prophylaxic agent on a wide variety of operative procedures. Mikrobiyol Bul, 1987 Jul, 21(3), 178 - 80 {The effect of sulbactam-ampicillin on renal function}; Kaya IS et al.; In this study, the effect of a beta-lactamase inhibitor sulbactam-ampicillin on renal function of albino mice was investigated and compared with the control and ampicillin groups. Infection, 1987 Jul-Aug, 15(4), 257 - 9 Novel R-factor borne beta-lactamase of Escherichia coli confering resistance to cephalosporins; Bauernfeind A et al.; Strains of Escherichia coli resistant to ceftazidime but susceptible to other third generation cephalosporins were detected in three patients at two different locations (Munchen, Bremen) . The resistance was self-transmissible to other E . coli strains . Resistance against ampicillin, kanamycin, chloramphenicol and sulfonamide was co-transferred . The isoelectric point (pI) of the beta-lactamase was similar to the pI of the AER-1 and LCR-1 beta-lactamases . These enzymes, however, do not confer resistance to ceftazidime . Therefore the beta-lactamase described is the first example for a ceftazidimase. J Biol Chem, 1987 Jun 15, 262(17), 8318 - 24 Effects of prolipoprotein signal peptide mutations on secretion of hybrid prolipo-beta-lactamase in Escherichia coli; Lunn CA et al.; Hybrid proteins were constructed by coupling beta-lactamase to the signal sequence (plus nine amino acids) of selected mutant prolipoproteins of Escherichia coli . The mutant prolipoprotein signal peptides contained lesions in two structural domains of the signal peptide, the basic amino-terminal domain and the hydrophobic core domain . We then compared the processing and localization of the mutant prolipo-beta-lactamases to the processing and localization of the comparable mutant prolipoproteins . We show that a mutant signal sequence with an anionic amino terminus exhibits similar limitations in the processing of prolipo-beta-lactamase as previously observed in prolipoprotein . Deletion of four hydrophobic residues from hydrophobic core results in a signal peptide which slowly translocates a fraction of the total mutant hybrid protein synthesized . This signal peptide was previously shown to translocate lipoprotein efficiently . Alteration of this hydrophobic core, which stimulated synthesis of mutant prolipoproteins, does not stimulate synthesis of prolipo-beta-lactamase . Finally mutations that slowed processing of prolipoprotein by affecting the proposed helical structure of the signal peptide had no significant effect on the processing of prolipo-beta-lactamase . These results suggest that the positively charged amino-terminal domain of the signal peptide has a common role in protein secretion regardless of the secretory protein . On the other hand, other domains of the signal peptide exhibit different phenotypes when the secretory protein is changed. Eur J Biochem, 1987 Jun 15, 165(3), 559 - 64 Covalent flavinylation of 6-hydroxy-D-nicotine oxidase analyzed by partial deletions of the gene; Brandsch R et al.; The expression of the enzymatically active 6-hydroxy-D-nicotine oxidase (6-HDNO) from Arthrobacter oxidans requires the covalent attachment of FAD to the polypeptide chain . How this modification takes place and at what time during the synthesis of the polypeptide is not known . We investigated the possibility of cotranslational flavinylation by generating various deletions of the 6-HDNO gene carried on appropriate plasmid vectors . The polypeptides expressed from these plasmids were analyzed for their ability to incorporate {14C}FAD covalently in an Escherichia coli-derived coupled transcription/translation system . The data show that removal of approximately 40% from the carboxy-terminal part of the 6-HDNO polypeptide did not inhibit the covalent flavinylation of the truncated protein . A fusion protein, consisting of the truncated 6-HDNO polypeptide and the beta-lactamase of pBR322, was also covalently flavinylated . The amino acid sequence surrounding the histidine residue, assumed to bind FAD, was shown to be situated approximately 70 amino acid residues from the amino-terminal end of the 6-HDNO polypeptide . Removal of the first 30 amino acids did not abolish covalent flavinylation . Flavinylation could no longer be detected, however, if a short amino acid sequence, consisting of seven residues, replaced the amino acid sequence upstream of the histidine . These findings prove, in our opinion, that cotranslational flavinylation takes place in the synthesis of 6-HDNO. J Infect Dis, 1987 Jun, 155(6), 1220 - 5 Selective ceftazidime resistance in Escherichia coli: association with changes in outer membrane protein; Bakken JS et al.; A strain of Escherichia coli (MG/32) was recovered from the blood of a patient who had received ceftazidime for eight weeks . The isolate was resistant to ceftazidime but susceptible to other third-generation cephalosporins . Alterations in outer membrane proteins were implicated in this selective ceftazidime resistance . As ceftazidime susceptibility was regained, the quantity of outer membrane proteins of 37,000 and 39,000 molecular weight increased . Although the isolate possessed a TEM-1 beta-lactamase, this enzyme was not involved in the selective resistance to ceftazidime; it did not disappear on reacquisition of ceftazidime susceptibility and did not hydrolyze the drug . Potassium clavulanate enhanced the activity of ceftazidime against E . coli strain MG/32, but this enhancement was due to a direct effect on outer membrane proteins and not to beta-lactamase inhibition. Mol Cell Probes, 1987 Jun, 1(2), 159 - 68 Sensitive detection of genes by sandwich hybridization and time-resolved fluorometry; Dahlen P et al.; Europium has been used as a non-radioactive marker in immunoassays as this metal can be detected with high sensitivity by time-resolved fluorometry . In this work streptavidin labeled with europium was used to detect biotinylated probes in a sandwich nucleic-acid hybridization assay with microtitration strips as the solid phase . pBR 322 plasmids were detected with a sensitivity of 4 x 10(5) molecules . As the sample is added in solution in sandwich hybridization, fast and simple sample pre-treatment can be used without encountering background problems . The method was applied to test bacterial samples of uropathogenic Escherichia coli strains for the presence of the beta-lactamase gene. J Bacteriol, 1987 Jun, 169(6), 2476 - 81 Creation of a test plasmid for detecting G-C-to-T-A transversions by changing serine to arginine in the active site of beta-lactamase; Foster PL et al.; Oligonucleotide-directed mutagenesis of the beta-lactamase gene, bla, on pBR322 was used to change the codon for the active-site serine 70, AGC, to CGC, coding for arginine . Escherichia coli cells carrying the mutant plasmid, pGD104, were sensitive to ampicillin, indicating that the arginine-containing enzyme is inactive . We characterized the reversion of the mutant bla gene by a number of mutagens and in different genetic backgrounds and demonstrated that full ampicillin resistance can be restored only by a G-C-to-T-A transversion occurring at the first base of the codon . Thus, reversion of the mutant bla gene is diagnostic for G-C-to-T-A transversions, and bacteria carrying pGD104 can be used as test strains to detect the occurrence of this mutation. J Bacteriol, 1987 May, 169(5), 2294 - 7 Response to temperature shifts of expression of the amp gene on pBR322 in Escherichia coli K-12; Kuriki Y; Synthesis of beta-lactamase, the product of the amp gene on pBR322, in Escherichia coli K-12 was reversibly repressed with a shift-up of the growth temperature from 30 to 42 degrees C . The temperature shift, however, did not affect the level of mRNA encoding beta-lactamase, which suggested the involvement of translational control. J Bacteriol, 1987 May, 169(5), 2245 - 50 Modification, processing, and subcellular localization in Escherichia coli of the pCloDF13-encoded bacteriocin release protein fused to the mature portion of beta-lactamase; Luirink J et al.; A fusion between the pCloDF13-derived bacteriocin release protein and beta-lactamase was constructed to investigate the subcellular localization and posttranslational modification of the bacteriocin release protein in Escherichia coli . The signal sequence and 25 of the 28 amino acid residues of the mature bacteriocin release protein were fused to the mature portion of beta-lactamase . The hybrid protein (Mr, 31,588) was expressed in minicells and whole cells and possessed full beta-lactamase activity . Immunoblotting of subcellular fractions revealed that the hybrid protein is present in both the cytoplasmic and outer membranes of E . coli . Radioactive labeling experiments in the presence or absence of globomycin showed that the hybrid protein is modified with a diglyceride and fatty acids and is processed by signal peptidase II, as is the murein lipoprotein . The results indicated that the pCloDF13-encoded bacteriocin release protein is a lipoprotein which is associated with both membranes of E . coli cells. Proc Natl Acad Sci U S A, 1987 May, 84(9), 2683 - 7 Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells; Zambetti G et al.; The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis . Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton . We report here the construction of an Escherichia coli pBR322 beta-lactamase signal peptide-human H3 histone fusion gene . The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication . Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression. Biull Eksp Biol Med, 1987 May, 103(5), 627 - 30 {Immunoenzyme demonstration of viruses and virus-specific antibodies by using conjugates based on gene engineering-produced beta-lactamase}; Kharitonenkov IG et al.; Enzyme immunoassays for the detection of viral antigens and virus-specific antibodies in biological samples have been described . Molecular complexes of antibodies and beta-lactamase (penicillinase) have been used as anti-specific conjugates . To synthesize the conjugate, the enzyme obtained with the aid of genetic engineering has been used . Enzyme immunoassays have been tested for the indication of the influenza virus and virus-induced specific antibodies . Enzyme immunoassays were shown to possess certain advantages (e.g., the use of simple and nontoxic substrate) along with the sensitivity identical to that of other methods, employing peroxidase-based conjugates. Antimicrob Agents Chemother, 1987 May, 31(5), 728 - 34 Development of natural and synthetic DNA probes for OXA-2 and TEM-1 beta-lactamases; Boissinot M et al.; Cloning of a 6.3-kilobase BglII DNA fragment from plasmid R46 permitted the isolation of the OXA-2 beta-lactamase gene . Selected DNA fragments internal and adjacent to the OXA-2 beta-lactamase structural gene were used as probes in homology studies with other plasmid-mediated beta-lactamases . Under conditions of high stringency, no cross hybridization could be detected with DNA probes from within the open reading frame of the OXA-2 structural gene . At a lower stringency, one of two DNA fragments used as probes cross hybridized weakly with the OXA-3 bla gene . Other DNA fragments tested and known to contain sequences flanking the OXA-2 determinant cross hybridized with OXA-3 and PSE-4 plasmid DNA . From the known nucleotide sequence of OXA-2 and TEM-1, we synthesized a series of oligonucleotides corresponding to sequences internal to their respective structural genes . A 12-mer oligonucleotide containing the OXA-2-active-site nucleotide sequences cross hybridized only with OXA-3 . All other oligonucleotides tested were found to be specific for their respective OXA-2 or TEM-1 gene . Such beta-lactamase gene probes should facilitate studies of beta-lactamase molecular epidemiology and beta-lactamase gene polymorphism. Schweiz Med Wochenschr, 1987 Apr 4, 117(14), 509 - 17 {Quantitative sensitivity determination of bacteria . Indications and methods}; von Graevenitz A et al.; The authors describe the indications for quantitative susceptibility tests and also the methods themselves (agar dilution, macrodilution, combination tests, tests for beta-lactamase production) . This study forms part of a series on susceptibility methods which started with a paper on disk susceptibility testing (this journal, 1984; 114: 1079-1086). J Biol Chem, 1987 Mar 25, 262(9), 3951 - 7 The consequences of stepwise deletions from the signal-processing site of beta-lactamase; Pluckthun A et al.; Amino acids have been deleted from the processing site of pre-beta-lactamase, either into the signal sequence or into the mature protein . Whereas the loss of more than 2 amino acid residues from the C-terminal end of the signal sequence prevents the translocation of the protein into the periplasm, the removal of two or more amino acids from the beginning of the mature protein has no effect on the translocation of the truncated protein . The insertion of an additional one to three amino acids at the processing site has no detectable phenotypic consequence either . It appears that many sequences for the first few residues of the mature protein allow successful translocation and processing . In sharp contrast, the removal of one (but not both) of the amino acids that flank the processing site results in a severe growth defect in the host cell and very low expression of the protein . Yet removal of two amino acids from either side of the processing site, or removal of both the flanking residues of the processing site, results in normal secretion and signal cleavage . These results illustrate the limits on the amino acid sequence around the processing junction and suggest that interference with the signal cleavage step can lead not only to aborted secretion but also to pleiotropic consequences for the growth of the host organism. Pediatr Infect Dis J, 1987 Mar, 6(3), 265 - 71 Amoxicillin-clavulanate potassium compared with cefaclor for acute otitis media in infants and children; Kaleida PH et al.; One hundred thirty-three infants and children with documented acute otitis media (OM) were randomized to receive the oral suspension of either amoxicillin-clavulanate potassium or cefaclor . Beta-lactamase-producing bacteria were found in 10.9 and 14.5% of subjects treated with amoxicillin-clavulanate potassium and cefaclor, respectively . Subjects were reexamined at 5, 10, 30, 60 and 90 days after the initiation of therapy and whenever signs/symptoms of acute otitis media recurred . All but two children had resolution of otalgia/otorrhea during the initial treatment period . The drug groups were not significantly different in the percentage of evaluable subjects with otitis media with effusion at each scheduled follow-up visit . Recurrence of acute OM/otorrhea {corrected} developed in a similar percentage of subjects in both treatment categories . Both subjects with and those without middle ear effusion at 10 days had approximately a 50% recurrence rate of subsequent middle ear disease . Adverse side effects/complaints, which occurred in significantly more children treated with amoxicillin-clavulanate potassium, were generally mild and primarily gastrointestinal. J Gen Microbiol, 1987 Mar, 133 ( Pt 3), 761 - 5 beta-Lactamase production by intestinal spirochaetes; Tompkins DS et al.; beta-Lactamase production was demonstrated in four of nineteen strains of intestinal spirochaetes isolated from human subjects . The enzyme was preferentially active against penicillins and was inhibited by clavulanic acid; it was membrane bound and non-inducible . No plasmids were detected in the intestinal spirochaetes and the beta-lactamase-production characteristic was not transferable to non-producing strains. J Antimicrob Chemother, 1987 Mar, 19(3), 285 - 95 Rapid and automated measurement of Km and specific Vmax values of beta-lactamases in bacterial extracts; Nichols WW et al.; A method is reported for automating measurements of Km and specific Vmax values from single progress curves of hydrolysis of beta-lactam antibiotics by crude (or purified) beta-lactamase preparations . The method is based on the half-time analysis procedure of Wharton & Szawelski (1982) . The specific Vmax is obtained in units of mols of substrate hydrolysed /min/mg dry wt of cells, but could be expressed as easily in terms of mols of enzyme or mg of protein . We propose that the method will allow reporting of Km and specific Vmax values, rather than relative rates of hydrolysis, in surveys of bacterial beta-lactamases. Can J Microbiol, 1987 Mar, 33(3), 205 - 11 Oligonucleotide probes for the detection of TEM-1 and TEM-2 beta-lactamase genes and their transposons; Ouellette M et al.; We describe the use of molecular probes to detect the TEM-type beta-lactamase genes . As a general probe, we prepared a 656 base pair restriction fragment, entirely within the TEM structural gene . This probe was specific for the TEM family, hybridizing only with TEM-1 and TEM-2 . The TEM-1 and TEM-2 beta-lactamases differ by only one amino acid . We synthesized two oligonucleotides whose central bases correspond to this difference . The use of these oligonucleotides enables us to discriminate between TEM-1 and TEM-2 genes . Using oligonucleotides homologous to parts of Tn3, we also monitored the presence of TnA-like transposons in bacteria harboring different beta-lactamase genes . Only the TEM-1 and TEM-2 genes were found to be on transposons with terminal sequences identical to those of Tn3 . All hybridization experiments were performed with both dot-blot and colony-hybridization techniques, and the suitability of these two methods for epidemiological studies is compared. Mol Gen Genet, 1987 Feb, 206(2), 252 - 8 Molecular cloning and DNA homology of plasmid-mediated beta-lactamase genes; Levesque RC et al.; Molecular cloning of DNA fragments between 1.5 and 8 kb from BamHI, EcoRI, HindIII, SalI, or Sau3A digests permitted the isolation of structural genes coding for TEM-1, ROB-1, OXA-1, OXA-3, OXA-4, OXA-5, PSE-1, PSE-2, PSE-3, PSE-4, CARB-3, CARB-4, AER-1, and LCR-1 beta-lactamases . Ampicillin-resistant clones were selected and it was confirmed that they contained the respective beta-lactamase genes by isoelectric focusing . Detailed physical maps of 14 different recombinant plasmids were constructed using 8 restriction endonucleases . Plasmid deletions and lacZ fusions were used to localize the beta-lactamase structural genes . DNA probes were constructed for the TEM-1, ROB-1, OXA-1, and PSE-1 genes . Under conditions of high stringency, hybridization was observed between the genes for TEM-1 and TEM-2 or TLE-1, OXA-1 and OXA-4, and PSE-1 and PSE-4 or CARB-3, while the ROB-1 gene probe showed no cross-hybridization . Such bla gene probes should facilitate studies of beta-lactamase molecular epidemiology. Infect Immun, 1987 Jan, 55(1), 206 - 10 Formation of transmural complement pores in serum-sensitive Escherichia coli; Bhakdi S et al.; The binding of C9 at 0 and 37 degrees C to viable Escherichia coli K-12 cells carrying C5b-8 complexes was quantified . At low temperature, limited average binding of only 1 to 1.4 molecules of C9 per C8 molecule occurred, whereas 6 to 8 C9 molecules were bound per C8 molecule at 37 degrees C . Despite incorporation of C9 into C5b-9 complexes at 0 degrees C, these terminal complexes caused no loss of bacterial viability even when present in very large numbers (1,000 to 1,500 per CFU) on the bacterial cells . In contrast, generation of 50 to 100 C5b-9 complexes carrying multiple C9 molecules per CFU caused loss of viability . The failure of C5b-81C91 complexes to generate transmural pores was confirmed by measurements of o-nitrophenyl-beta-D-galactoside influx into the cells . Whereas treatment of C5b-8-laden cells with C9 at 32 degrees C caused virtually instantaneous influx of the marker, almost no influx was registered in cells receiving C9 at 0 degrees C . When cells carrying C5b-7 were brought into the stationary phase and given C8 and C9 at 32 degrees C, a C9-dependent disruption of the outer membrane permeability barrier immediately occurred as demonstrated by cleavage of a chromogenic substrate by periplasmic beta-lactamase . In sharp contrast, o-nitrophenyl-beta-D-galactoside influx was markedly retarded over a prolonged period, with abrupt permeability increases of the inner membrane toward this molecule being noted just before bacterial cell division occurred . We conclude that killing of E . coli requires binding of C5b-9 complexes containing C9 oligomers to the outer membrane and suggest that formation of pores in the inner membrane occurs when these complexes are "hit" by transiently forming zones of bioadhesion . Formation of the latter may be a dynamic process that is accentuated during cell division and quiescent during the stationary phase. Proteins, 1987, 2(4), 290 - 7 Stability of wild-type and mutant RTEM-1 beta-lactamases: effect of the disulfide bond; Schultz SC et al.; Uniquely among class A beta-lactamases, the RTEM-1 and RTEM-2 enzymes contain a single disulfide bond between Cys 77 and Cys 123 . To study the possible role of this naturally occurring disulfide in stabilizing RTEM-1 beta-lactamase and its mutants at residue 71, this bond was removed by introducing a Cys 77----Ser mutation . Both the wild-type enzyme and the single mutant Cys 77----Ser confer the same high levels of resistance to ampicillin in vivo to Escherichia coli; at 30 degrees C the specific activity of purified Cys 77----Ser mutant is also the same as that of the wild-type enzyme . Also, neither wild-type enzyme nor the Cys 77----Ser mutant is inactivated by brief exposure to p-hydroxymercuribenzoate . However, above 40 degrees C the mutant enzyme is less stable than wild-type enzyme . After introduction of the Cys 77----Ser mutation, none of the double mutants (containing the second mutations at residue 71) confer resistance to ampicillin in vivo at 37 degrees C; proteins with Ala, Val, Leu, Ile, Met, Pro, His, Cys, and Ser at residue 71 confer low levels of resistance to ampicillin in vivo at 30 degrees C . The use of electrophoretic blots stained with antibodies against beta-lactamase to analyze the relative quantities of mutant proteins in whole-cell extracts of E . coli suggests that all 19 of the doubly mutant enzymes are proteolyzed much more readily than their singly mutant analogues (at Thr 71) that contain a disulfide bond.(ABSTRACT TRUNCATED AT 250 WORDS) Rev Infect Dis, 1987 Jan-Feb, 9(1), 16 - 27 Acute otitis media caused by Branhamella catarrhalis: biology and therapy; Van Hare GF et al.; Since 1980, we have observed an epidemic of otitis media caused by Branhamella catarrhalis . This event was characterized by studying the nasopharyngeal colonization of infants and children with B . catarrhalis and the clinical presentation and therapeutic outcome of acute otitis media caused by this organism . Pharyngeal colonization with B . catarrhalis was commoner in winter than summer . B . catarrhalis was present in middle-ear fluid (MEF) of 17% of children with otitis media, and was commoner in fall and winter (20%) than in spring and summer (11%, P less than .05) . Seventy-five percent of isolates produced beta-lactamase (Ravasio type) . In five of 20 patients, treatment with beta-lactamase-susceptible agents failed to sterilize B . catarrhalis-infected MEF . All of these five patients were infected with beta-lactamase-producing strains . The increasing prominence of antibiotic-resistant B . catarrhalis in acute otitis media may lead to a reevaluation of initial antibiotic therapy for acute otitis media, particularly in winter or in areas where colonization with such strains is prevalent. Gene, 1987, 55(1), 141 - 6 Measurement of cat expression from growth-rate-regulated promoters employing beta-lactamase activity as an indicator of plasmid copy number; Klotsky RA et al.; Many promoter-fusion vectors contain an intact beta-lactamase (BLA) gene (bla) to allow measurement of BLA activity as an internal control for plasmid copy number . This approach rests on the assumption that bla is constitutively expressed . To use such vectors for comparison of promoter activity at different growth rates it was necessary to confirm that this is the case under all physiological conditions . The relationship between plasmid copy number and BLA activity at different steady-state growth rates in Escherichia coli HB101 transformed with a ColE1-type plasmid (pBR325) was examined . Both BLA activity and plasmid copy number decreased in a parallel fashion as growth rate increased . This finding was tested further by measuring the growth-rate-regulated expression of the chloramphenicol acetyltransferase (CAT) gene under the control of the rrnB P1 promoter in a plasmid pKK231-1 fusion . The results indicate that BL