Pediatr Surg Int . 2005 Jan 4; {Epub ahead of print} One-stage correction of intermediate imperforate anus in males; Adeniran JO et al.; This prospective study was designed to assess the safety, cost-effectiveness, and advantages of performing posterior sagittal anorectoplasty (PSARP) without colostomy on males with intermediate imperforate anus in a developing country . Fifteen consecutive males with intermediate imperforate anus were entered into the study . Chest and abdominal x-rays, skeletal surveys, renal ultrasound scans, and invertograms were done . Patients were resuscitated and Pena's PSARP done in prone positions . A 2-ml syringe vent was inserted into the new anus for 10 days . Babies were nursed prone postoperatively . Cephalosporin and metronidazole were given as perioperative antibiotics . All patients had intermediate anomalies . There were no other major associated congenital anomalies . A urethral catheter could not be inserted in one patient, and one patient who presented with septicemia and jaundice was deemed too ill to withstand a major operation; these two patients therefore had diverting colostomies . There were no problems with PSARP in the other 13 patients . One patient's father discharged him against medical advice on the 5th postoperative day; the mother had had postpartum hemorrhage, so they opted for traditional treatment because they could not provide blood donors . The skin wounds of 10 patients healed completely with removal of stitches; two boys had superficial wound infection . Parents who lived far from the hospital were taught how to dilate the anus . Follow-up has ranged between 3 months and 2 years . This prospective study shows that it is feasible for males with intermediate imperforate anus to have safe PSARP without colostomy . The advantages of one instead of three major operations are many, especially in developing countries . If this result can be reproduced in cases of high anomalies, colostomy may be unnecessary in many cases of anorectal malformations, with many benefits to these children and their families. Drug Metab Pharmacokinet, 2002, 17(4), 363 - 6 Lack of Interaction Between Cefdinir and Calcium Polycarbophil: In vitro and In vivo Studies; Kato R et al.; The effect of calcium polycarbophil on the absorption of cefdinir, cephalosporin derivative, was evaluated in both in vitro and in vivo studies . In the in vitro study, the release of cefdinir from a cellulose membrane in the presence or absence of metal cations was measured using the dissolution test procedure . In the in vivo study, volunteers and a randomized crossover design with two phases were used . In the first phase, the volunteers received 200 mg of cefdinir alone (Study 1); in the other phase, they received 200 mg of cefdinir and 1200 mg of fine calcium polycarbophil granules concomitantly (Study 2) . The cefdinir concentrations in the samples or serum were measured by an UV-VIS spectrophotometer or high-performance liquid chromatography . Release in the presence of iron ions was slower than that in the absence of metal ions, however no difference was observed between release in the presence of calcium ions and that in the absence of metal ions . No difference was observed in AUC(0-10), C(max) and t(max) between Study 1 and Study 2 . The absorption of cefdinir was not affected by co-administration of calcium polycarbophil . Moreover, the in vitro study on the release of drugs from a cellulose membrane may predict the absorption of a drug caused by the formation of chelate complexes between the drug and metal ions. Clin Exp Allergy, 2004 Nov, 34(11), 1768 - 75 The diagnostic interpretation of basophil activation test in immediate allergic reactions to betalactams; Torres MJ et al.; BACKGROUND: Basophil activation by allergens, including drugs, has been used to determine sensitivity and to study IgE recognition and cross-reactivity . OBJECTIVE: We sought to determine the sensitivity and specificity of a basophil activation test (Basotest) in patients with immediate allergic reactions to betalactams, with a later comparison between patients who were selective (those recognizing the culprit drug excluding benzylpenicillin (BP)) and cross-reactors (those recognizing several penicillin determinants including BP) . METHODS: Basotest to different haptens was performed in 70 patients with immediate allergic reactions to betalactams, classified into three groups: (A) skin test positive independently of CAP/RAST immunoassay value, (B) skin test negative and CAP/RAST positive, and (C) skin test and CAP/RAST negative but drug provocation test positive . Basotest was carried out by flow cytometry following the manufacturer's instructions using different betalactam determinants and results expressed as a stimulation index . RESULTS: Of the 70 patients, 34 (48.6%) were positive to Basotest (sensitivity: 48.6%), 31 (44.3%) to CAP/RAST and 46 (65.7%) to either one or the other . Considering the different groups, Basotest was positive in 50.9% of patients in Group A, 60% in Group B and 14.3% in Group C . The specificity was 91.3% . Positivity to the haptens was 28.6% to amoxicillin (AX), 21.7% to BP, 20% to benzylpenicilloyl-poly-l-lysine, 12.5% to ampicillin and 2.2% to minor determinant mixture . In patients with cephalosporin reactions, Basotest to the culprit cephalosporin was positive in 77.7% . There were differences between the two reactor groups in the sensitivity of Basotest (selective to AX=50%, cross-reactors=28.6%; chi(2)=10.809, P=0.004) and in the CAP/RAST (selective to AX=28.6%, cross-reactors=61.9%; chi(2)=8.944, P=0.011) . CONCLUSIONS: The sensitivity of Basotest is similar to immunoassays (CAP/RAST) . Sensitivity is improved when used in combination . Although further studies are required, Basotest results for cephalosporin allergy seem very promising . This technique does not help differentiate between selective reactors and cross-reactors. Gene, 2004 Nov 24, 342(2), 269 - 81 AcFKH1, a novel member of the forkhead family, associates with the RFX transcription factor CPCR1 in the cephalosporin C-producing fungus Acremonium chrysogenum; Schmitt EK et al.; In the filamentous fungus Acremonium chrysogenum, a complex regulatory network of transcription factors controls the expression of at least seven cephalosporin C biosynthesis genes . The RFX transcription factor CPCR1 binds to regulatory sequences in the promoter region of cephalosporin C biosynthesis genes, and is involved in the transcriptional regulation of the pcbC gene which encodes isopenicillin N synthase . In this study, we used CPCR1 in a yeast two-hybrid screen to identify potential protein interaction partners . A cDNA was identified, encoding the C-terminal part (pos . 438-665) of the novel forkhead protein, AcFKH1 . The full-length AcFKH1 amino acid sequence is 665 residues and shares between 31% and 60% identity with forkhead protein sequences in the genomes of Aspergillus nidulans, Fusarium graminearum, and Neurospora crassa . AcFKH1 is characterized by two conserved domains, the N-terminal forkhead-associated domain (FHA), which might be involved in phospho-protein interactions, and the C-terminal DNA-binding domain (FKH) of the winged helix/forkhead type . The two-hybrid system was also used to map the protein domains required for the interaction of transcription factors CPCR1 and AcFKH1 . The observed interaction between CPCR1 and the C-terminus of AcFKH1 in the yeast system was verified in vitro in a GST pulldown assay . Using gel retardation analysis, the DNA-binding properties of the fungal forkhead protein AcFKH1 were investigated . AcFKH1 recognizes two forkhead consensus binding sites within the 1.2 kb promoter region of the divergently oriented cephalosporin biosynthesis gene pair pcbAB-pcbC from A . chrysogenum . Additionally, AcFKH1 is able to bind with high affinity to the SWI5-binding site of the yeast FKH2 protein. J Org Chem, 2004 Nov 12, 69(23), 7965 - 70 Toward the development of a cephalosporin-based dual-release prodrug for use in ADEPT; Grant JW et al.; In previous work we have shown that a cephalosporin structure bearing an S-aminosulfenimine at the 7-position behaved as a beta-lactamase-dependent dual-release prodrug . Scission of the beta-lactam ring of such a structure led to the rapid loss of the sulfur-attached side chain moiety via an intramolecular displacement, while the 3'-group was lost via the well-established elimination process at that position . In the present work we report on an evaluation of the scope and limitations of exploiting the S-aminosulfenimine functionality to generate a cephalosporin-based prodrug incorporating two biologically active components . Starting from 7-ACA, a viable synthetic cycle was put in place that avoided formation of the Delta(2) isomer throughout and that allowed incorporation of aminoglutethimide at the 3'-position and of a tosyl S-aminosulfenimine at the 7-position . The direct incorporation of a biologically active sulfonamide (ethoxzolamide) or a sulfamate (coumate) at this latter position was not achieved as a result of the difficulty of generating the corresponding sulfur diimides . An indirect route for the formation of an S-aminosulfenimine was put in place, as was a general method of alkylation (Mitsunobu reaction) of the tosyl S-aminosulfenimine following its incorporation. Vnitr Lek, 2004 Aug, 50(8), 619 - 23 {The latest trends in the treatment peritonitis of the patients on peritoneal dialysis}; Hajkova B et al.; Peritonitis is still one of the major complications ambulatory peritoneal dialysis (CAPD) . The initial empiric therapy for peritonitis should be effective against most grampositive organisms as well as gramnegative organisms . Intraperitoneal administration antibiotics has the advantage of a high concentration of the antibiotics at the site of infection . The treatment should provide broad coverage of all organisms, without side-effects or risk for the patient and should not provoke the emergence of resistant germs . The present guidelines of the ad hoc advisory committee are of great value in this regard . The ISPD guidelines for treatment of peritonitis recommended an empirical therapy based on the association of a first-generation cephalosporin with an aminoglycoside or ceftazidime. J Wound Care, 2004 Oct, 13(9), 363 - 6 Infection prophylaxis of gunshot wounds using probiotics; Nikitenko VI; OBJECTIVE: To report a new method of surgical infection prophylaxis for postoperative gunshot wounds to the extremities . METHOD: Gunshot wounded animals were divided into three groups: treatment (probiotic Sporobacterin), antibiotic (cephalosporin cefamezin) and control (no treatment) . Histological studies of wound-bed tissue were taken on days 1, 3, 5 and 10 of the study . RESULTS: The probiotic administered per os was more effective than antibiotics for prophylaxis of surgical infection . CONCLUSION: The probiotic's effect is based on the natural defence mechanism activated after injury--the bacterial translocation of saprophytic bacteria from the gut to the wound . DECLARATION OF INTEREST: None. Appl Environ Microbiol, 2004 Oct, 70(10), 6324 - 8 A single amino acid substitution converts gamma-glutamyltranspeptidase to a class IV cephalosporin acylase (glutaryl-7-aminocephalosporanic acid acylase); Suzuki H et al.; The aspartyl residue at position 433 of gamma-glutamyltranspeptidase of Escherichia coli K-12 was replaced by an asparaginyl residue . This substitution enabled gamma-glutamyltranspeptidase to deacylate glutaryl-7-aminocephalosporanic acid, producing 7-aminocephalosporanic acid, which is a starting material for the synthesis of semisynthetic cephalosporins. Indian J Dent Res, 2003 Oct-Dec, 14(4), 220 - 3 Steven Johnson syndrome due to I.V Ceftriaxone--a case report; Narayanan VS et al.; Steven-Johnson syndrome (SJS) is a rare vesiculobullous disease characterized by an acute cutaneous eruption that ivolves the skin and mucous membranes including those of the oral cavity . A rare case of Steven-Johnson syndrome, an unexpected treatment response, in a 25-year-old female patient due to administration of intravenous Cefriaxone (1 gm), a third generation cephalosporin has been reported and literature reviewed. J Biol Chem, 2004 Oct 29, 279(44), 46295 - 303 Epub 2004 Aug 23. Site-saturation mutagenesis of Tyr-105 reveals its importance in substrate stabilization and discrimination in TEM-1 beta-lactamase; Doucet N et al.; The conserved Class A beta-lactamase active site residue Tyr-105 was substituted by saturation mutagenesis in TEM-1 beta-lactamase from Escherichia coli in order to clarify its role in enzyme activity and in substrate stabilization and discrimination . Minimum inhibitory concentrations were calculated for E . coli cells harboring each Y105X mutant in the presence of various penicillin and cephalosporin antibiotics . We found that only aromatic residues as well as asparagine replacements conferred high in vivo survival rates for all substrates tested . At position 105, the small residues alanine and glycine provide weak substrate discrimination as evidenced by the difference in benzylpenicillin hydrolysis relative to cephalothin, two typical penicillin and cephalosporin antibiotics . Kinetic analyses of mutants of interest revealed that the Y105X replacements have a greater effect on K(m) than k(cat), highlighting the importance of Tyr-105 in substrate recognition . Finally, by performing a short molecular dynamics study on a restricted set of Y105X mutants of TEM-1, we found that the strong aromatic bias observed at position 105 in Class A beta-lactamases is primarily defined by a structural requirement, selecting planar residues that form a stabilizing wall to the active site . The adopted conformation of residue 105 prevents detrimental steric interactions with the substrate molecule in the active site cavity and provides a rationalization for the strong aromatic bias found in nature at this position among Class A beta-lactamases. Curr Med Chem, 2004 Jul, 11(14), 1951 - 64 The discovery and development of modified penicillin- and cephalosporin-derived beta-lactamase inhibitors; Buynak JD; While beta-lactam antibiotics remain among the most commonly prescribed pharmaceutical products, their effectiveness is currently threatened by the development of bacterial resistance . One key resistance mechanism is the ability to destroy the antibiotic through utilization of one or more types of beta-lactamase . An effective countermeasure is to employ a combination product, consisting of both a beta-lactam antibiotic and a beta-lactamase inhibitor . Unfortunately, currently available inhibitors narrowly target only class A beta-lactamases . This review will detail our research, directed toward the development of a useful broad-spectrum beta-lactamase inhibitor . In the process, we have discovered new inhibitors capable of simultaneously inactivating class A, C, and D beta-lactamase, produced conjugate siderophore/beta-lactamase inhibitors, and explored the SAR's of tunable, cephalosporin-derived beta-lactamase inactivators . Useful synthetic methodology will be described, which simplifies the large scale production of many known inhibitors and which allows the rapid preparation of libraries of prospective inhibitors. Biotechnol Lett, 2004 Jun, 26(11), 939 - 45 Construction and application of fusion proteins of D-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase for direct bioconversion of cephalosporin C to 7-aminocephalosporanic acid; Luo H et al.; Two fusion proteins of D-amino acid oxidase (DAAO) and glutaryl-7-aminocephalosporanic acid acylase (GLA) were designed to simplify the bioconversion process of cephalosporin C to 7-aminocephalosporanic acid (7-ACA), which is conventionally produced in a two-step enzymatic process . Two recombinant plasmids, pET-DLA and pET-ALD, were constructed to express fusion proteins of DAAO-linker-GLA (DLA) and GLA-linker-DAAO (ALD), respectively . When the recombinant plasmids were expressed in E . coli, the fusion protein DLA was not correctly folded and only DAAO activity could be detected . ALD, however, possessed activities of both DAAO and GLA, which directly catalyze the conversion of cephalosporin C into 7-ACA. J Clin Microbiol, 2004 Jul, 42(7), 3329 - 32 Epidemiological investigation of bloodstream infections by extended spectrum cephalosporin-resistant Escherichia coli in a Taiwanese teaching hospital; Yan JJ et al.; In an epidemiologic and case-control study including 30 case patients over a 3.5-year period in a Taiwanese university hospital, only beta-lactamase inhibitor use and extended-spectrum cephalosporin use were identified as independent risk factors for nosocomial CMY-2-producing Escherichia coli bloodstream infection, and CMY-2 producers were found more prevalent than extended-spectrum beta-lactamase-producing isolates. Immunol Allergy Clin North Am, 2004 Aug, 24(3), 463 - 76, vi-vii Cephalosporin allergy; Madaan A et al.; Allergic reactions to cephalosporins may occur because of sensitization to cephalosporin determinants shared with penicillin or to unique cephalosporin haptens . The exact nature of the haptenic determinants resulting from the degradation of currently available cephalosporins is incompletely understood . Cephalosporin skin testing or specific IgE immunoassays have limited clinical utility . Patients with a history of allergy to cephalosporins or penicillin may be at increased risk for a reaction to cephalosporins . Skin testing for an allergy to penicillin may be helpful in patients with a history of penicillin allergy who have a clinical indication for cephalosporin use . Most of these patients have negative tests and should not be at increased risk for a reaction to cephalosporins. Ann Intern Med, 2004 Jul 6, 141(1), 16 - 22 Cross-reactivity and tolerability of cephalosporins in patients with immediate hypersensitivity to penicillins; Romano A et al.; BACKGROUND: In patients with documented IgE-mediated hypersensitivity to penicillins, data on sensitization to cephalosporins vary . Administering cephalosporins to such patients is often deferred because of the risk for cross-reactivity . OBJECTIVE: To assess the cross-reactivity with cephalosporins and its potential determinants in patients with documented penicillin allergy . DESIGN: Prospective study in patients without clinical indications for cephalosporin treatment . SETTING: Italy . PATIENTS: 128 consecutive patients who sustained anaphylactic shock (n = 81) or urticaria (n = 47) and had positive results on skin tests for at least 1 of the penicillin reagents tested . MEASUREMENTS: All patients were skin tested with cephalothin, cefamandole, cefuroxime, ceftazidime, ceftriaxone, and cefotaxime . Patients with negative results for the last 4 cephalosporins were challenged with cefuroxime axetil and ceftriaxone . RESULTS: 14 patients (10.9% {95% CI, 6.1% to 17.7%}) had positive results on skin tests for cephalosporins, mostly for cephalothin or cefamandole . Skin test results for the minor determinant mixture were positive in 10 of 14 patients (71.4%) with cross-reactivity and 44 of 114 patients (38.6%) without cross-reactivity (odds ratio, 3.90 {CI, 1.17 to 13.40}; P = 0.0189) . All 101 patients with negative results on skin tests for cefuroxime, ceftazidime, ceftriaxone, and cefotaxime tolerated cefuroxime axetil and ceftriaxone (tolerability rate, 100% {CI, 96.4% to 100%}) . LIMITATIONS: Challenges were not followed by full therapeutic courses . Twenty-two patients declined challenges; positive responses in those patients would have decreased the tolerability rate to 82.1% (CI, 74.2% to 88.4%) . CONCLUSIONS: These data confirm the advisability of avoiding cephalosporin treatment in patients with positive results on skin tests for penicillin . In patients who especially require cephalosporin treatment, we recommend skin tests with cephalosporins before assessing the tolerability of the cephalosporin with a graded challenge. Transfusion, 2004 Jul, 44(7), 1033 - 40 Ceftriaxone causes drug-induced immune thrombocytopenia and hemolytic anemia: characterization of targets on platelets and red blood cells; Grossjohann B et al.; BACKGROUND: Ceftriaxone, a third-generation cephalosporin, has been reported to occasionally cause fatal drug-induced immune hemolytic anemia (DIHA) . A clinical and serologic analysis of the first two patients with severe drug-induced thrombocytopenia (DITP) due to ceftriaxone and one patient with fatal DIHA is reported . STUDY DESIGN AND METHODS: Sera were assessed by the IAT, EIA, glycoprotein (GP)-specific immunoassay, flow cytometry, and immunoprecipitation using transfectants expressing GPIIb/IIIa and GPIb/IX and with different cephalosporins . RESULTS: Sera from Patients 1 and 2 reacted strongly with PLTs in the presence of the drug, but not with RBCs . The binding sites of the drug-dependent antibodies (DDAbs) could be localized to GPIIb/IIIa and GPIb/IX, respectively . Inhibition studies indicated that DDAbs recognized epitopes residing on the GPIIb/IIIa complex and on the GPIX subunit, respectively . No cross-reactivity was observed with other cephalosporin derivatives . Serum 3 showed strong agglutination with RBCs of Rh(null) phenotype in the presence of ex-vivo metabolites of ceftriaxone, but no cross-reactivity with PLTs . CONCLUSIONS: The first two cases of severe DITP and a third patient with DIHA are reported . DDAbs from all patients showed individual reaction patterns and clear cell lineage specificity . In addition, the DDAbs were dependent on the substitution at position 3 of the ceftriaxone molecule . Epitopes on GPIIb/IIIa and GPIX were involved on PLTs . The Rh protein was not the only target of DDAbs on RBCs. Pharmacotherapy, 2004 Jun, 24(6), 808 - 11 Cephalexin-induced acute tubular necrosis; Longstreth KL et al.; A 24-year-old woman with a history of penicillin allergy developed reversible acute renal failure after receiving cephalexin for 4 days . The patient experienced nausea, vomiting, diarrhea, pruritus, cough, and an elevated creatinine level of 2.2 mg/dl . The patient's creatinine level continued to rise, peaking at 5.3 mg/dl on hospital day 3 . Nephrotoxic acute tubular necrosis was confirmed by electron microscopy . Within 1 month of discharge from the hospital, the patient's creatinine level decreased to 0.6 mg/dl . Although the renal injury most commonly associated with the cephalosporin class of antibiotics is allergic interstitial nephritis, currently available cephalosporins infrequently can cause direct tubular toxicity. Cutis, 2004 May, 73(5 Suppl), 25 - 6 Impetigo in pediatrics; Epps RE; Impetigo is a common skin infection in children . If not treated promptly, it can spread rapidly through a student population and become a significant health problem . Whereas uncomplicated, localized lesions typically are treated with topical mupirocin, widespread infections involving multiple pathogens require systemic treatment with a cephalosporin or a beta-lactamase-resistant antibiotic. Biochem Biophys Res Commun, 2004 Jun 25, 319(2), 486 - 92 Modifying the substrate specificity of penicillin G acylase to cephalosporin acylase by mutating active-site residues; Oh B et al.; The penicillin G acylase (PGA) and cephalosporin acylase (CA) families, which are members of the N-terminal (Ntn) hydrolases, are valuable for the production of backbone chemicals like 6-aminopenicillanic acid and 7-aminocephalosporanic acid (7-ACA), which can be used to synthesize semi-synthetic penicillins and cephalosporins, respectively . Regardless of the low sequence similarity between PGA and CA, the structural homologies at their active-sites are very high . However, despite this structural conservation, they catalyze very different substrates . PGA reacts with the hydrophobic aromatic side-chain (the phenylacetyl moiety) of penicillin G (PG), whereas CA targets the hydrophilic linear side-chain (the glutaryl moiety) of glutaryl-7-ACA (GL-7-ACA) . These different substrate specificities are likely to be due to differences in the side-chains of the active-site residues . In this study, mutagenesis of active-site residues binding the side-chain moiety of PG changed the substrate specificity of PGA to that of CA . This mutant PGA may constitute an alternative source of engineered enzymes for the industrial production of 7-ACA. FEMS Microbiol Lett, 2004 Jun 1, 235(1), 43 - 9 Strain improvement for cephalosporin production by Acremonium chrysogenum using geneticin as a suitable transformation marker; Rodriguez-Saiz M et al.; An Acremonium chrysogenum strain improvement program based on the transformation with cephalosporin biosynthetic genes was carried out to enhance cephalosporin C production . Best results were obtained with cefEF and cefG genes, selecting transformants with increased cephalosporin C production and lower accumulation of biosynthetic intermediates . Phleomycin resistant transformants, designated B1 and C1, showed a single copy random integration event, higher levels of cefEF transcript and, according to immunoblotting analyses, higher amounts of deacetylcephalosporin C acetyltransferase (DAC-AT) protein than their parental strains . Moreover, DAC-AT activity was higher in the transformants . Plasmids carrying geneticin resistance markers based on the nptII gene from Tn5 and the aphI gene from Tn903 were constructed to transform again B1 and C1, showing that the cassette Pgdh-nptII-trpC was able to confer geneticin resistance to A . chrysogenum and demonstrating that geneticin is a helpful selection marker. Transfusion, 2004 Jun, 44(6), 849 - 52 Persistence of cefotetan on red blood cells; Davenport RD et al.; BACKGROUND: Cefotetan can cause severe immune hemolytic anemia that may persist long after the drug is discontinued . To study the binding of cefotetan to RBCs, patients who received cefotetan were followed and tested for the presence of antibody to cefotetan . STUDY DESIGN AND METHODS: Patients receiving cefotetan were identified from pharmacy and nursing records . Blood samples obtained for routine hematology tests were analyzed . Cefotetan binding to patients' RBCs was tested using a previously characterized high-titer anticefotetan serum by gel technique . To determine the minimum amount of drug necessary for binding to occur, RBCs were incubated with serial dilutions of cefotetan at pH 7.4 . RESULTS: Sixty patients receiving 1 to 25 g i.v . (median, 2 g) of cefotetan were followed for 1 to 123 days (median, 18 days) . All were initially positive, for cefotetan on RBCs . Positivity persisted for up to 98 days after the last dose of drug . Fifteen patients became negative during follow-up . The first negative sample occurred at Day 30 to 123 . Using the midpoint between the last positive and first negative to estimate of the duration of positivity, we estimate that cefotetan remains RBC-bound for 16.5 to 92 days (median, 67.5 days) . During the follow-up period, five patients developed anticefotetan detectable in the serum . Twenty patients receiving other cephalosporin antibiotics showed no specific reactivity of their RBCs with anticefotetan . In vitro studies showed a minimum necessary drug concentration of 1 micromol/L at physiologic pH, which was not significantly altered by RBC pretreatment with ficin, sialydase, or DTT . CONCLUSIONS: Cefotetan is tightly bound to RBCs after intravenous administration and remains detectable for weeks after the last dose . Antibodies to cefotetan may occur in about 8 percent of patients receiving the drug . The minimum necessary concentration for RBC binding is low compared to an estimated plasma concentration of 240 micromol/L from a single i.v . dose of 1 g. Neuropsychobiology, 2004, 49(4), 218 - 22 Nonconvulsive status epilepticus during cephalosporin therapy; Primavera A et al.; Cephalosporins may induce nonconvulsive status epilepticus (NCSE), a potentially reversible condition . Despite the wide use of these antibiotics, there are only few reported cases, because this condition is probably underestimated . We report two new cases of NCSE occurring during treatment with cefepime and ceftazidime, and emphasize the utility of emergent electroencephalogram in patients with an acute altered state of consciousness while receiving treatment with cephalosporins, particularly when there is evidence of impaired renal function . Expert Opin Investig Drugs, 2004 Apr, 13(4), 393 - 401 New therapies for pneumococcal meningitis; Cottagnoud PH et al.; The treatment of pneumococcal meningitis remains a major challenge, as reflected by the continued high morbidity and case fatality of the disease . The worldwide increase of penicillin-resistant pneumococci and more recently cephalosporin- and vancomycin-tolerant pneumococci has jeopardised the efficacy of standard treatments based on extended spectrum cephalosporins alone or in combination with vancomycin . This review provides a summary of newly developed antibiotics tested in the rabbit meningitis model . In particular, newer beta-lactam monotherapies (cefepime, meropenem, ertapenem), recently developed quinolones (garenoxacin, gemifloxacin, gatifloxacin, moxifloxacin) and a lipopeptide antibiotic (daptomycin) are discussed . A special emphasis is placed on the potential role of combination treatments with some of the new compounds, which are of interest based on the background of increasing resistance problems due to their often synergistic activity in the rabbit model of pneumococcal meningitis. J Pharm Pharmacol, 2004 Apr, 56(4), 485 - 93 Influence of enhancers on the absorption and on the pharmacokinetics of cefodizime using in-vitro and in-vivo models; Mrestani Y et al.; In the development of novel antibiotics, more and more compounds have been found that cannot be absorbed orally and, therefore, must be administered intravenously or intramuscularly . Because of the obvious drawbacks of drug delivery by injection, the development of alternatives with enhanced oral bioavailability has received much attention in pharmaceutical research . Cefodizime, a novel third-generation cephalosporin with significant advantages in the parenteral treatment of common infections, was used as a model drug . Cefodizime behaves as a highly hydrophilic compound, as shown from its extremely low partition coefficient . The effect of cationic absorption enhancers (hexadecyldimethylbenzylammonium chloride, N-hexadecylpyridinium bromide, dodecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide) on the lipophilicity of cefodizime was investigated by means of the n-octanol/water system . Results showed that the counter-ions had a positive influence on the solubility of cefodizime . These results on partitioning coefficients in the n-octanol/buffer system were confirmed using an in-vitro transport model with artificial and biological membranes (Caco-2-cells) . Furthermore, the physiological compatibility of the absorption enhancers was investigated using the active D-glucose transport . The pharmacokinetic profile of cefodizime was evaluated in rabbits after intraduodenal administration with and without an absorption enhancer. Rapid Commun Mass Spectrom, 2004, 18(6), 707 - 10 Determination of cefuroxime in human serum or plasma by liquid chromatography with electrospray tandem mass spectrometry; Viberg A et al.; Cefuroxime is a second-generation cephalosporin used against different kinds of bacterial infections . To be able to optimize the dosing it is necessary to characterize the pharmacokinetics of cefuroxime which requires a selective and sensitive analytical method for cefuroxime in plasma or serum . A new rapid liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) method, using cefotaxime as internal standard, was developed for analysis of cefuroxime in human serum . The work-up procedure consisted of protein precipitation with acetonitrile/cefotaxime, and after centrifugation the supernatant was dissolved in mobile phase . The sample was injected on a SB-CN column and the detection was performed using tandem mass spectrometry (MS/MS) . The limit of quantification was determined to 0.025 microg/mL . The method was linear in the range 0.025-50 microg/mL with a coefficient of correlation >0.999 . The limit of quantification and intra-day variability were found to be the same for plasma samples, which indicates that the method is valid for serum as well as plasma samples . Bioorg Med Chem Lett, 2004 Apr 5, 14(7), 1737 - 9 The inhibition of metallo-beta-lactamase by thioxo-cephalosporin derivatives; Tsang WY et al.; The 8-thioxocephalosporins are poor substrates for the B . cereus metallo beta-lactamase (k(cat)/K(m)=61.4M(-1) s(-1)) and act as weak competitive inhibitors (K(i) approximately 700 microM) . The hydrolysis product of thioxocephalosporin, a thioacid, also inhibits the enzyme competitively with a K(i)=96 microM, whereas the cyclic thioxo-piperazinedione, formed by intramolecular aminolysis of thioxocephalexin has a K(i) of 29 microM. Bioorg Med Chem Lett, 2004 Feb 23, 14(4), 1007 - 10 Doxorubicin prodrug on the basis of tert-butyl cephalosporanate sulfones; Veinberg G et al.; Doxorubicin-cephalosporin prodrug adapted to the development of elastases for the liberation of parent drug was synthesized on the basis of cephalosporanate sulfone esters. Pediatr Neurol, 2004 Feb, 30(2), 135 - 9 Cephalosporin-induced nonconvulsive status epilepticus in a uremic child; Chedrawi AK et al.; The temporal relationship between convulsive seizures and the administration of beta-lactams has long been recognized . A specific form of seizures, nonconvulsive status epilepticus, is less common and is often manifested by alterations in mental status without associated seizures . It is most commonly encountered in uremic patients and poses a diagnostic challenge because of its nonspecific clinical manifestations . In this report, we describe a child with chronic renal failure who developed nonconvulsive status epilepticus on two separate occasions after administration of a third-generation cephalosporin . Awareness of this potentially treatable condition is crucial to ensure appropriate and prompt medical therapy . To our knowledge, this is the first report of cephalosporin-induced nonconvulsive status epilepticus in a child with chronic renal failure. Eukaryot Cell, 2004 Feb, 3(1), 121 - 34 Winged helix transcription factor CPCR1 is involved in regulation of beta-lactam biosynthesis in the fungus Acremonium chrysogenum; Schmitt EK et al.; Winged helix transcription factors, including members of the forkhead and the RFX subclasses, are characteristic for the eukaryotic domains in animals and fungi but seem to be missing in plants . In this study, in vitro and in vivo approaches were used to determine the functional role of the RFX transcription factor CPCR1 from the filamentous fungus Acremonium chrysogenum in cephalosporin C biosynthesis . Gel retardation analyses were applied to identify new binding sites of the transcription factor in an intergenic promoter region of cephalosporin C biosynthesis genes . Here, we illustrate that CPCR1 recognizes and binds at least two sequences in the intergenic region between the pcbAB and pcbC genes . The in vivo relevance of the two sequences for gene activation was demonstrated by using pcbC promoter-lacZ fusions in A . chrysogenum . The deletion of both CPCR1 binding sites resulted in an extensive reduction of reporter gene activity in transgenic strains (to 12% of the activity level of the control) . Furthermore, Acremonium transformants with multiple copies of the cpcR1 gene and knockout strains support the idea of CPCR1 being a regulator of cephalosporin C biosynthesis gene expression . Significant differences in pcbC gene transcript levels were obtained with the knockout transformants . More-than-twofold increases in the pcbC transcript level at 24 and 36 h of cultivation were followed by a reduction to approximately 80% from 48 to 96 h in the knockout strain . The overall levels of the production of cephalosporin C were identical in transformed and nontransformed strains; however, the knockout strains showed a striking reduction in the level of the biosynthesis of intermediate penicillin N to less than 20% of that of the recipient strain . We were able to show that the complementation of the cpcR1 gene in the knockout strains reverses pcbC transcript and penicillin N amounts to levels comparable to those in the control . These results clearly indicate the involvement of CPCR1 in the regulation of cephalosporin C biosynthesis . However, the complexity of the data points to a well-controlled or even functional redundant network of transcription factors, with CPCR1 being only one player within this process. Clin Microbiol Infect, 2004 Feb, 10(2), 98 - 118 Actinobacillus actinomycetemcomitans endocarditis; Paturel L et al.; Among the bacteria of the HACEK group, Actinobacillus actinomycetemcomitans is the organism involved most commonly in infective endocarditis . However, the epidemiological and clinical features specifically associated with this species have not been evaluated adequately . Three patients with infective endocarditis caused by A . actinomycetemcomitans seen at the Hospital La Timone (Marseille, France) between 1994 and 2001 are reported . Of 99 cases in the literature, 75% of patients had previous heart disease before infective endocarditis, the portal of entry of which was usually the oral cavity . Among the total of 102 cases, 27 had prosthetic valves . Intermittent fever was observed in all cases, and weight loss and peripheral signs of endocarditis were noteworthy in this study . Anaemia and microscopic haematuria were frequently noted . The disease is insidious, with a mean duration of symptoms of 13 weeks before diagnosis, as confirmed by blood cultures incubated for > 5 days . The aortic valve is most commonly involved, and echocardiographic findings were non-specific . Complications occurred in 63% of patients, with emboli being the most common . The surgery rate was 23.5% . The overall mortality rate was 18% . Of the cases, 76.5% were cured with antibiotics alone, including a simple third-generation cephalosporin or a combination of ampicillin and an aminoglycoside . An antibiotic therapy duration of at least 4 weeks is recommended . Surgical therapy is usually required for haemodynamic reasons . Prophylaxis of A . actinomycetemcomitans endocarditis relies on antibiotic therapy for all cardiac patients at risk before dental procedures . Among 17 patients undergoing dental manipulations, only eight received amoxycillin before the procedure, demonstrating that prophylaxis is far from being systematically prescribed . In conclusion, A . actinomycetemcomitans endocarditis should be highly suspected in patients with previous cardiac disease and for whom symptoms have evolved over a number of weeks or even months. J Pharm Pharmacol, 2003 Dec, 55(12), 1601 - 6 In-vitro and in-vivo studies of cefpirom using bile salts as absorption enhancers; Mrestani Y et al.; Cephalosporins have to be administered by injection because of the poor intestinal absorption of the orally delivered drugs . Because of the obvious drawbacks of drug delivery by injection, the development of alternatives with enhanced oral bioavailability is receiving much attention in pharmaceutical research . Cefpirom (Cp) is a new semi-synthetic amino-2-thiazolyl-methoxyimino cephalosporin that has been substituted in position 3 with a cyclopenteno-pyridinium group in order to create a zwitterionic compound . It exhibits highly hydrophilic properties, as shown from its extremely low partition coefficient, and therefore its lipophilicity was increased using bile salts . The effect of this on the partition coefficients determined in the n-octanol/buffer system was confirmed using an in-vitro transport model with artificial and biological membranes . The pharmacokinetic properties of Cp were investigated in rabbits after intraduodenal administration with and without bile salts . Furthermore, the physiological compatibility of the bile salts was investigated using active D-glucose transport. J Biol Chem, 2004 Apr 9, 279(15), 15420 - 6 Epub 2004 Jan 20. Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase; Lloyd MD et al.; Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutarate-dependent oxygenase involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium . It catalyzes two oxidative reactions, oxidative ring-expansion of penicillin N to deacetoxycephalosporin C, and hydroxylation of the latter to give deacetylcephalosporin C . The enzyme is closely related to deacetoxycephalosporin C synthase (DAOCS) and DACS from Streptomyces clavuligerus, which selectively catalyze ring-expansion or hydroxylation reactions, respectively . In this study, structural models based on DAOCS coupled with site-directed mutagenesis were used to identify residues within DAOC/DACS that are responsible for controlling substrate and reaction selectivity . The M306I mutation abolished hydroxylation of deacetylcephalosporin C, whereas the W82A mutant reduced ring-expansion of penicillin G (an "unnatural" substrate) . Truncation of the C terminus of DAOC/DACS to residue 310 (Delta310 mutant) enhanced ring-expansion of penicillin G by approximately 2-fold . A double mutant, Delta310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS . The Delta310/N305L/M306I triple mutant selectively catalyzed ring-expansion of penicillin G and had improved kinetic parameters (K(m) = 2.00 +/- 0.47 compared with 6.02 +/- 0.97 mm for the wild-type enzyme) . This work demonstrates that a single amino acid residue side chain within the DAOC/DACS active site can control whether the enzyme catalyzes ring-expansion, hydroxylation, or both reactions . The catalytic efficiency of mutant enzymes can be improved by combining active site mutations with other modifications including C-terminal truncation and modification of Asn-305. AAPS PharmSciTech . 2001 Nov 19;2(4):23. Stability of a second-generation cephalosporin veterinary mastitis formulation after electron beam irradiation; Johns PJ et al.; This study focused on the chemical stability of the cephalosporin (6R, 7R)-7-(1-pentafluorophenoxyacetamido)-3-{2-(5-methyl-1,3,4-thiodiazolyl)thiomethyl}-Delta(3)-cephem-4-carboxylic acid, sodium salt (cephem 1) formulation after electron beam (e-beam) irradiation . The cephem 1 concentrations of samples irradiated at 5, 10, and 15 kilograys for glass vials and low-density polyethylene (LDPE) cannula syringes were not statistically different from the concentrations of the nonirradiated control samples . Samples from each irradiation dose stored in controlled-temperature chambers at 5 degrees C and 30 degrees C for 24 months did not show any concentration changes within statistical limits compared with the nontreated samples . Samples from each irradiation dose stored at 40 degrees C for 12 months also did not show any concentration changes within statistical limits compared with the nontreated samples . The percentage of related substances increased slightly with the increase in e-beam irradiation level and storage temperature, but this increase was within the proposed label claim of 90% to 110% (45-55 mg/g) . In conclusion, e-beam sterilization did not affect the chemical stability of cephem 1 intramammary formulation in LDPE cannula syringes, suggesting that e-beam irradiation may be a feasible method for terminal sterilization of this cephem 1 formulation. J Org Chem, 2004 Jan 23, 69(2), 339 - 44 Kinetics and mechanisms of hydrolysis and aminolysis of thioxocephalosporins; Tsang WY et al.; The effect of replacing the beta-lactam carbonyl oxygen in cephalosporins by sulfur on their reactivity has been investigated . The second-order rate constant for alkaline hydrolysis of the sulfur analogue is 2-fold less than that for the natural cephalosporin . The thioxo derivative of cephalexin, with an amino group in the C7 side chain, undergoes beta-lactam ring opening with intramolecular aminolysis by a reaction similar to that for cephalexin itself . However, the rate of intramolecular aminolysis for the S-analogue is 3 orders of magnitude greater than that for cephalexin . Furthermore, unlike cephalexin, intramolecular aminolysis in the S-analogue occurs up to pH 14 with no competitive hydrolysis . The rate of intermolecular aminolysis of natural cephalosporins is dominated by a second-order dependence on amine concentration, whereas that for thioxocephalosporins shows only a first-order term in amine . The Bronsted beta(nuc) for the aminolysis of thioxo-cephalosporin is +0.39, indicative of rate-limiting formation of the tetrahedral intermediate with an early transition state with relatively little C-N bond formation. Nat Struct Mol Biol, 2004 Jan, 11(1), 95 - 101 Epub 2003 Dec 29. The structural basis of cephalosporin formation in a mononuclear ferrous enzyme; Valegard K et al.; Deacetoxycephalosporin-C synthase (DAOCS) is a mononuclear ferrous enzyme that transforms penicillins into cephalosporins by inserting a carbon atom into the penicillin nucleus . In the first half-reaction, dioxygen and 2-oxoglutarate produce a reactive iron-oxygen species, succinate and CO2 . The oxidizing iron species subsequently reacts with penicillin to give cephalosporin and water . Here we describe high-resolution structures for ferrous DAOCS in complex with penicillins, the cephalosporin product, the cosubstrate and the coproduct . Steady-state kinetic data, quantum-chemical calculations and the new structures indicate a reaction sequence in which a 'booby-trapped' oxidizing species is formed . This species is stabilized by the negative charge of succinate on the iron . The binding sites of succinate and penicillin overlap, and when penicillin replaces succinate, it removes the stabilizing charge, eliciting oxidative attack on itself . Requisite groups of penicillin are within 1 A of the expected position of a ferryl oxygen in the enzyme-penicillin complex. J Coll Physicians Surg Pak, 2003 Nov, 13(11), 637 - 9 Is mechanical bowel preparation really necessary in colorectal surgery? Ahmad M, Abbas S, Asghar MI. OBJECTIVE: To determine the outcome of colorectal surgery without mechanical bowel preparation . DESIGN: A descriptive, analytical and observational study . PLACE AND DURATION OF STUDY: Combined Military Hospital, Kharian and Pano Aqil, from September 1998 to April 2003 . SUBJECTS AND METHODS: Forty-seven patients underwent debridement/resection and repair/primary anastomosis of colon and upper rectum without bowel preparation . Of these, 16 patients were operated in emergency . The anastomosis was carried out with polyglactin (vicryl) interrupted, full thickness single layer and no patient had defunctioning colostomy . Third generation cephalosporin, cefotaxime or ceftazidime and metronidazole were given perioperatively, repeated during surgery if lasted for more than 2 hours and continued for 3-5 days postoperatively . RESULTS: Anastomoses were ileocolic in 29.7%, colicocolic in 61.7% and colorectal in 14.8% cases . Anastomotic failure was seen in 4.2% and wound infection in 8.5% cases . There was one mortality (2.1%) due to unrelated cause . CONCLUSION: Mechanical bowel preparation is not necessary for safe colorectal surgery. J Am Chem Soc, 2003 Dec 31, 125(52), 16322 - 6 A mechanism-based inhibitor targeting the DD-transpeptidase activity of bacterial penicillin-binding proteins; Lee M et al.; Penicillin-binding proteins (PBPs) are responsible for the final stages of bacterial cell wall assembly . These enzymes are targets of beta-lactam antibiotics . Two of the PBP activities include dd-transpeptidase and DD-carboxypeptidase activities, which carry out the cross-linking of the cell wall and trimming of the peptidoglycan, the major constituent of the cell wall, by an amino acid, respectively . The activity of the latter enzyme moderates the degree of cross-linking of the cell wall, which is carried out by the former . Both these enzymes go through an acyl-enzyme species in the course of their catalytic events . Compound 6, a cephalosporin derivative incorporated with structural features of the peptidoglycan was conceived as an inhibitor specific for DD-transpeptidases . On acylation of the active sites of dd-transpeptidases, the molecule would organize itself in the two active site subsites such that it mimics the two sequestered strands of the bacterial peptidoglycan en route to their cross-linking . Hence, compound 6 is the first inhibitor conceived and designed specifically for inhibition of DD-transpeptidases . The compound was synthesized in 13 steps and was tested with recombinant PBP1b and PBP5 of Escherichia coli, a dd-transpeptidase and a dd-carboxypeptidase, respectively . Compound 6 was a time-dependent and irreversible inhibitor of PBP1b . On the other hand, compound 6 did not interact with PBP5, neither as an inhibitor (reversible or irreversible) nor as a substrate. Biochem Biophys Res Commun, 2003 Dec 19, 312(3), 755 - 60 Analysis of a substrate specificity switch residue of cephalosporin acylase; Sio CF et al.; Residue Phe375 of cephalosporin acylase has been identified as one of the residues that is involved in substrate specificity . A complete mutational analysis was performed by substituting Phe375 with the 19 other amino acids and characterising all purified mutant enzymes . Several mutations cause a substrate specificity shift from the preferred substrate of the enzyme, glutaryl-7-ACA, towards the desired substrate, adipyl-7-ADCA . The catalytic efficiency ( {Formula: see text} (cat)/ {Formula: see text} (m)) of mutant SY-77(F375C) towards adipyl-7-ADCA was increased 6-fold with respect to the wild-type enzyme, due to a strong decrease of {Formula: see text} (m) . The {Formula: see text} (cat) of mutant SY-77(F375H) towards adipyl-7-ADCA was increased 2.4-fold . The mutational effects point at two possible mechanisms by which residue 375 accommodates the long side chain of adipyl-7-ADCA, either by a widening of a hydrophobic ring-like structure that positions the aliphatic part of the side chain of the substrate, or by hydrogen bonding to the carboxylate head of the side chain. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2003 Oct, 25(5), 547 - 9 {Clinical diagnosis, treatment and prognosis of elderly SARS patients}; Cao B et al.; OBJECTIVE: To discuss the clinical manifestations, therapeutic strategy and prognosis of patients with severe acute respiratory syndrome (SARS) older than 60 years . METHODS: Elderly patients diagnosed as SARS in Peking Union Medical College Hospital were compared with younger patients . RESULTS: Twenty-four elderly patients and 53 younger patients were analysed . Elderly patients had more coexisting conditions, such as hypertension, diabetes, coronary heart disease, and renal disease than control group (P < 0.05) . Rate of respiratory failure in elderly patients was higher than that in control group (P < 0.05) . Elderly patients had more respiratory symptoms, such as cough, sputum, and shortness of breath (P < 0.05) . Rate of lymphocytopenia and thrombocytopenia in elderly patients was higher than that in control group . All patients were given ribavirin and antibiotics . More patients in elderly group were given 3rd generation cephalosporin and imipenem . Mortality rate in elderly group was higher than that in control group (33.3% vs 3.8%, P < 0.05) . Univariate analysis showed that age, respiratory failure, and thrombocytopenia were risk factors of death, but logistic analysis did not find any independent risk factor . CONCLUSIONS: Though the elderly patients have a lower morbidity of SARS, they have more coexisting conditions . The therapy of elderly patients is more difficult than that of control group, and the mortality in elderly patients is high. Antibiot Khimioter, 2003, 48(7), 16 - 9 {Experience with maxipime in neurosurgical clinic}; Kondrat'ev AN et al.; Cefepime (Maxipime, Bristol-Myers Squibb), a 4th generation cephalosporin was used in the postoperative treatment of 121 patients of Anesthesiology and Intensive Care Unit of Neurosurgical Clinics . The patients were divided into groups by the risk factor of pyoseptic complications . The results were estimated by the number and nature of the complications such as increasing liquor neutrophilic cytosis, systemic inflammations and others . The findings (increasing liquor neutrophilic cytosis only in 2 patients and endobronchitis in 4 patients) and good tolerance of cefepime (Maxipime) were in favour of its use in a dose of 1 g administered intravenously dropwise during initial narcosis and in 12 hours as an efficient agent for perioperative prophylaxis in neurosurgical patients. Bol Asoc Med P R, 2000 Sep-Dec, 92(9-12), 110 - 4 Efficacy of ceftibuten for pediatric patients undergoing adenotonsillectomy; Juarbe C; A survey between Otolaryngologist Head and Neck Surgeons in Puerto Rico and a prospective study was done, to evaluate the efficacy of ceftibuten in pediatric patients undergoing adenotonsillectomy . Surgery of the tonsils and adenoids is the most common operation performed in the pediatric age by the Otolaryngologist Head and Neck Surgeons in Puerto Rico . Over 70% are performed in a ambulatory setting and almost all of the patients are given antibiotics after surgery . Ceftibuten was given to 112 pediatrics patients after surgery . Adenotonsillectomy is a painful operation and children do not take medication well after surgery . One of the benefits of this third generation cephalosporin, is that is given once a day . Twenty events were reported taking the medication, but only seven patients had to discontinue its use . Ceftibuten had a tolerance rate of 94% . Ceftibuten seems to be a safe antibiotic to use in pediatric patients undergoing adenotonsillectomy and has the convenience of being given once a day. J Biol Chem, 2004 Jan 2, 279(1), 341 - 7 Epub 2003 Oct 08. A bound water molecule is crucial in initiating autocatalytic precursor activation in an N-terminal hydrolase; Yoon J et al.; Cephalosporin acylase is a member of the N-terminal hydrolase family, which is activated from an inactive precursor by autoproteolytic processing to generate a new N-terminal nucleophile Ser or Thr . The gene structure of the precursor cephalosporin acylases generally consists of a signal peptide that is followed by an alpha-subunit, a spacer sequence, and a beta-subunit . The cephalosporin acylase precursor is post-translationally modified into an active heterodimeric enzyme with alpha- and beta-subunits, first by intramolecular cleavage and, second, by intermolecular cleavage . Intramolecular autocatalytic proteolysis is initiated by nucleophilic attack of the residue Ser-1beta onto the adjacent scissile carbonyl carbon . This study determined the precursor structure after disabling the intramolecular cleavage . This study also provides experimental evidence showing that a conserved water molecule plays an important role in assisting the polarization of the OG atom of Ser-1beta to generate a strong nucleophile and to direct the OG atom of the Ser-1beta to a target carbonyl carbon . Intramolecular proteolysis is disabled as a result of a mutation of the residues causing conformational distortion to the active site . This is because distortion affects the existence of the catalytically crucial water at the proper position . This study provides the first evidence showing that a bound water molecule plays a critical role in initiating intramolecular cleavage in the post-translational modification of the precursor enzyme. Biochem Biophys Res Commun, 2003 Oct 10, 310(1), 19 - 27 Deacylation activity of cephalosporin acylase to cephalosporin C is improved by changing the side-chain conformations of active-site residues; Oh B et al.; Semisynthetic cephalosporins are primarily synthesized from 7-aminocephalosporanic acid (7-ACA), mainly by environmentally toxic chemical deacylation of cephalosporin C (CPC) . Thus, the enzymatic conversion of CPC to 7-ACA by cephalosporin acylase (CA) would be very interesting . However, CAs use glutaryl-7-ACA (GL-7-ACA) as a primary substrate and the enzymes have low turnover rates for CPC . The active-site residues of a CA were mutagenized to various residues to increase the deacylation activity of CPC, based on the active-site conformation of the CA structure . The aim was to generate sterically favored conformation of the active-site to accommodate the D-alpha-aminoadipyl moiety of CPC, the side-chain moiety that corresponds to the glutaryl moiety of GL-7-ACA . A triple mutant of the CA, Q50betaM/Y149alphaK/F177betaG, showed the greatest improvement of deacylation activity to CPC up to 790% of the wild-type . Our current study is an efficient method for improving the deacylation activity to CPC by employing the structure-based repetitive saturation mutagenesis. Bioprocess Biosyst Eng, 2002 Sep, 25(3), 193 - 203 Epub 2002 Aug 14. Continuous cephalosporin C purification: dynamic modelling and parameter validation; Barboza M et al.; A mathematical kinetic model for the adsorption and desorption of cephalosporin C on Amberlite XAD-2 resin is proposed . The model can represent Langmuir, Freundlich or linear isotherms at equilibrium . The intrinsic kinetic parameters and adsorption isotherms as well as physical parameters such as the effective diffusivity and the external mass transfer coefficient were obtained at different temperatures and ethanol concentrations . An unfavourable cephalosporin C adsorption occurred when ethanol was present in the solution . It has been shown that at 25 degrees C the ethanol, at concentrations from 1.5% to 2.5%, decreases the cephalosporin C adsorption . However, this behaviour was not observed at 10 degrees C . The kinetic model fitted the experimental data well under different conditions . The model was validated in a continuous process of cephalosporin C purification using the same resin . The model with the validated parameters is able to predict the behaviour of the reactor system . The continuous process is composed of two stirred tank reactors with adsorber recycle . The adsorption occurs in the first stage, and elution of the product takes place in the second stage with ethanol as eluent . The dynamic behaviour of the process was described using the following parameters: hydraulic residence time for the first (theta(h1)) and second stage (theta(h2)), solid residence time (theta(s)), initial concentration of CPC (C(0)), inlet ethanol concentration (C(ET0)) and kinetics parameters. Pediatrics, 2003 Sep, 112(3 Pt 1), 543 - 7 Empirical therapy for neonatal candidemia in very low birth weight infants; Benjamin DK Jr et al.; OBJECTIVE: Neonatal candidemia is often fatal . Empirical antifungal therapy is associated with improved survival in neonates and patients with fever and neutropenia . Although guidelines for empirical therapy exist for patients with fever and neutropenia, these do not exist for neonates . METHODS: A multicenter, retrospective, cohort study was conducted of neonatal intensive care unit patients (N = 6172) who had a blood culture (N = 21,233) after day of life 3 and whose birth weight was <or=1250 g . We performed multivariable conditional logistic regression of risk factors for candidemia . From the regression modeling coefficients, we developed a candidemia score . RESULTS: In multivariable modeling, thrombocytopenia (odds ratio {OR}: 3.56; 95% confidence interval {CI}: 2.68-4.74) and cephalosporin or carbapenem use in the 7 days before obtaining the blood culture (OR: 1.77; 95% CI: 1.33-2.29) were risk factors for subsequent candidemia . Children who were 25 to 27 weeks' estimated gestational age (OR: 2.02; 95% CI: 1.52-3.05) and children who were born at <25 weeks (OR: 4.15; 95% CI: 3.12-6.29) were at higher risk of developing candidemia than were children who were born at >or=28 weeks . We developed a candidemia score on the basis of the ORs from the multivariable model . Children with a candidemia score >or=2 points were classified as having a "positive" score, and a score of >or=2 points had a sensitivity of 85% and a specificity of 47% . CONCLUSIONS: We developed a clinical predictive model for neonatal candidemia with high sensitivity and moderate specificity for candidemia . On the basis of our model, when a physician obtains a blood culture, the physician should consider providing antifungal therapy to neonates who are <25 weeks' estimated gestational age and to neonates who have thrombocytopenia at the time of blood culture . In addition, if a physician obtains a blood culture from a child who is 25 to 27 weeks' estimated gestational age and is not thrombocytopenic but has a history of third-generation cephalosporin or carbapenem exposure in the 7 days before the blood culture, then the physician should consider administration of empirical antifungal therapy. Transfusion, 2003 Sep, 43(9), 1317 - 21 Thrombotic thrombocytopenic purpura after cephalosporin administration: a possible relationship; Baron BW et al.; BACKGROUND: Acquired thrombotic thrombocytopenic purpura (TTP) is an autoimmune disorder . The pathogenesis is believed to be mediated by an autoantibody directed against the metalloproteinase responsible for the degradation of the very-high-molecular-weight multimers of the vWF . The syndrome can be precipitated by a variety of conditions, and certain medications also have been implicated . CASE REPORTS: The cases of two patients who took a cephalosporin antibiotic, cephalexin (Keflex, Eli Lilly), and then developed TTP are reported . One patient subsequently received a third-generation cephalosporin, ceftriaxone (Rocephin, Roche), without adverse reaction . Of interest, one patient had taken cefaclor (Ceclor, Eli Lilly) 8 years before and had also developed TTP at that time . The other patient also took cefaclor for approximately 3 weeks before taking cephalexin . In addition, she had had a dose of clarithromycin (Biaxin, Abbott Laboratories) the day before the onset of the TTP symptoms . CONCLUSIONS: To our knowledge, TTP has not been reported previously after administration of cephalosporin antibiotics . Attention is called to the possibility that this syndrome may occur after exposure to some of these drugs, although the incidence is very rare or, alternatively, underdiagnosed. J Pediatr, 2003 Jul, 143(1), 130 - 2 Cefuroxime-induced immune hemolysis; Malloy CA et al.; Drug-induced immune hemolytic anemia (IHA) is rare but is being reported with increasing frequency . The most commonly cited cause of drug-induced IHA has been receipt of second and third generation cephalosporin antibiotics . We report the first case of IHA associated with cefuroxime administration. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2001 Apr, 23(2), 196 - 8 {The factors that influence the consistency of international normalized ratio of prothrombin time}; Su W et al.; OBJECTIVE: To investigate the factors that influence the consistency of international normalized ratio (INR) of prothrombin time . METHODS: Using the same automated blood coagulation analyzer and INR calibration plasma with two different types of prothrombin time (PT) reagent to assay the INR values of 19 controls (group 1), 12 patients who took long-term anticoagulant orally (group 2), 12 patients who took it at the first time (group 3) and 8 patients who took long-term anticoagulant together with cephalosporin N (group 4) . RESULTS: The two kinds of reagents gave quite different INR values in all four groups (P < 0.01) . The INR difference in group 3 (0.23 +/- 0.07, n = 12) was larger than that in group 2 (0.51 +/- 0.20, n = 12) (P < 0.01) . The difference in group 4 (0.61 +/- 0.21, n = 8) was larger than that in group 2 (P < 0.01) . CONCLUSIONS: The factors that may influence INR consistency are the source and sensitivity of the reagents, as well as the period of the anti coagulant been taken and the other drugs taken at the same time. Dtsch Med Wochenschr, 2003 Aug 1, 128(31-32), 1649 - 52 {Rhabdomyolysis, hyponatremia and fever in a patient with Schmidt's syndrome}; Kordish I et al.; HISTORY: A 38-year-old man had fever (40 degrees C) and a swollen right leg . Two weeks before admission he had received non-steroid anti-inflammatory drugs (paracetamol and ibuprofen) after a tooth extraction . Some weeks before he had noticed a rough voice and a dry skin . INVESTIGATIONS: The patient had a sinus tachycardia of 145/min (blood pressure of 130/80 mm Hg) . Laboratory data revealed a CK of 16,650 U/l (< 80 U/l), myoglobin of 2420 U/l (< 90 microg/l) and LDH of 1250 U/l (<240 U/l), leukocyte count of 12,000 / microl and C-reactive protein of 3.0 mg/dl (<0.5 mg/dl) . Sodium was markedly decreased (110 mmol/l (135 - 145 mmol/l)) . Determination of thyroid hormone showed primary hypothyroidism with an elevated TSH of 62.6 mU/l (0.3 - 4.0 mU/l); T4 of 38 nmol/l (58 - 154 nmol/l), T3 1.17 of nmol/l (1.23 - 3.08 nmol/l) and fT4 of 6 pmol/l (10 - 25 pmol/l) . The thyroid autoantibodies were increased (thyroxine peroxidase antibodies of 684 U/l (<35 U/l) and thyroglobulin antibodies of 173 U/l (<40 U/l)) . Ultrasound of the thyroid revealed an nonhomogeneous structure . Cortisol at 8.00 a . m . was reduced by 63 mmol/l (180 - 640 mmol/l) and did not increase after administration of ACTH (60 min . cortisol at 90 mmol/l (>550 mmol/l)) . ACTH was increased (141 pg/ml; normal range 17 - 52 pg/ml) . TREATMENT AND COURSE: The initial therapy consisted of hydrocortisone (100 mg i.v as bolus and 100 mg during the next 24 hours) and levothyroxine replacement (200 micro g) was initiated . During the following 8 days clinical symptoms regressed . Values of sodium, myoglobin and LDH decreased . After therapy with cephalosporin (Ceftriaxon) and penicillin (Flucloxacillin) fever and inflammation parameters decreased . CONCLUSION: This is a rare case of a rhabdomyolysis and hyponatriaemia due to hypothyroidism and Addison's disease (Schmidt's syndrome). Biotechnol Lett, 2003 Feb, 25(3), 227 - 33 Cross-linked cell aggregates of Trigonopsis variabilis: D-amino acid oxidase catalyst for oxidation of cephalosporin C; Becka S et al.; Trigonopsis variabilis CBS 4095 was treated with alkali (pH 11, 30 min), heated (65 degrees C, 60 s) and immobilized . Glutaraldehyde, polyethyleneimine and a cross-linking reagent formed by reaction of polyethyleneimine with glutaraldehyde were used for stabilization of D-amino acid oxidase in the cells, as well as for aggregation and binding of the cells . A specific activity of 82-98 U of D-amino acid oxidase per g dry mass was produced with a yield of about 20% . The half-life time of 142 repeated conversion cycles corresponds to a productivity of 130 kg cephalosporin C oxidized per kg catalyst dry mass. Neuropharmacology, 2003 Sep, 45(3), 304 - 14 Evidence for the involvement of GABA(A) receptor blockade in convulsions induced by cephalosporins; Sugimoto M et al.; There is accumulating evidence that most beta-lactam antibiotics (i.e., cephalosporins and penicillins) have some degree of convulsive activity, both in laboratory animals as well as in clinical settings . The proposed mechanism is suppression of inhibitory postsynaptic responses, mainly mediated by gamma-amino butyric acid (GABA)(A)-receptors (GABA(A)-R) . However, comprehensive studies on the convulsive activities of various beta-lactam antibiotics in vivo and in vitro have not been performed . We have therefore examined the convulsive activities of seven different cephalosporins using both in vivo and in vitro models: intracerebroventricular (ICV) administration in mouse; {(3)H}muscimol binding assay (BA) in mouse brain synaptosome; and inhibition of recombinant mouse alpha1beta2gamma2s GABA(A)-Rs in Xenopus oocyte (GR) . The rank orders of convulsive activities in mouse (cefazolin>cefoselis>cefotiam>cefpirome>cefepime>ceftazidime>cefozopran) correlated with those of inhibitory potencies on {(3)H}muscimol binding and GABA-induced currents of GABA(A)-R in vitro, with correlation coefficients of ICV:GR, ICV:BA and BA:GR of 0.882, 0.821 and 0.832, respectively . In contrast, none of the antibiotics had affinities for N-methyl-D-aspartate (NMDA) receptors nor facilitatory actions on NMDA receptor-mediated current in oocytes . These results clearly demonstrate that the mechanism of cephalosporin-induced convulsions is mediated predominantly through the inhibition of GABA(A)-R function and not through NMDA receptor modulation. Curr Med Chem, 2003 Sep, 10(17), 1741 - 57 Design of beta-lactams with mechanism based nonantibacterial activities; Veinberg G et al.; The majority of nonantibacterial activities discovered for beta-lactam derivatives during the last 15 years are based on their ability to form a stable covalent complex with nucleophile in the active site of enzymes regulating fundamental physiological processes in mammalian organism such as serine and cysteine proteases, LDL phospholipase A(2), A-independent transacylase and some still indeciphered enzymes . Regulation of their catalytic activity both in vitro and in vivo by compounds designed on the cephalosporin, penicillin and 2-azetidinone base was successfully exploited in the treatment of inflammatory, respiratory, cardiovascular disorders, cancer and other pathologic processes . Availability of X-ray crystallographic data for target enzymes and computational molecular modelling in combination with wide possibilities of structural modifications for commercial natural and synthetic beta-lactams and the chiral blocks allow to consider this class of organic compounds as a perspective source of mechanism based nonantibacterial drugs. J Chemother, 2003 Jun, 15(3), 211 - 9 Ceftriaxone in febrile neutropenia; Karthaus M et al.; Standard management of febrile neutropenia requires prompt administration of empirical, broad-spectrum antibiotic therapy, since febrile neutropenia is associated with a significant risk of infectious complications and mortality . Risk-assessment models have been developed that differentiate febrile patients with neutropenia according to their risk for infectious complications and/or mortality and have prompted a change in the management of these patients . Ceftriaxone is a long-lasting, broad spectrum cephalosporin which has demonstrated efficacy in this indication in many publications . The role of ceftriaxone in febrile neutropenia will be discussed based on literature analysis and on the author's experience. Bioconjug Chem, 2003 Jul-Aug, 14(4), 797 - 804 PDEPT: polymer-directed enzyme prodrug therapy . 2 . HPMA copolymer-beta-lactamase and HPMA copolymer-C-Dox as a model combination; Satchi-Fainaro R et al.; Polymer-directed enzyme prodrug therapy (PDEPT) is a novel two-step antitumor approach that uses a combination of a polymeric prodrug and polymer-enzyme conjugate to generate a cytotoxic drug rapidly and selectively at the tumor site . Previously we have shown that N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound cathepsin B can release doxorubicin intratumorally from an HPMA copolymer conjugate PK1 . Here we describe for the first time the synthesis and biological characterization of a PDEPT model combination that uses an HPMA-copolymer-methacryloyl-glycine-glycine-cephalosporin-doxorubicin (HPMA-co-MA-GG-C-Dox) as the macromolecular prodrug and an HPMA copolymer conjugate containing the nonmammalian enzyme beta-lactamase (HPMA-co-MA-GG-beta-L) as the activating component . HPMA-co-MA-GG-C-Dox had a molecular weight of approximately 31 600 Da and a C-Dox content of 5.85 wt % . Whereas free beta-L has a molecular weight of 45 kDa, the HPMA-co-MA-GG-beta-L conjugate had a molecular weight in the range of 75-150 kDa, and following purification no free enzyme was detectable . Against the cephalosporin C or HPMA-co-MA-GG-C-Dox substrates, the HPMA-co-MA-GG-beta-L conjugate retained 70% and 80% of its activity, respectively . In vivo (125)I-labeled HPMA-co-MA-GG-beta-L showed prolonged plasma concentration and greater tumor targeting than (125)I-labeled beta-L due to the enhanced permeability and retention (EPR) effect . Moreover, administration of HPMA-co-MA-GG-C-Dox iv to mice bearing sc B16F10 melanoma followed after 5 h by HPMA-co-MA-GG-beta-L led to release of free Dox . The PDEPT combination caused a significant decrease in tumor growth (T/C = 132%) whereas neither free Dox nor HPMA-co-MA-GG-C-Dox alone displayed activity . The PDEPT combination displayed no toxicity at the doses used, so further evaluation of this approach to establish the maximum tolerated dose (MTD) is recommended. J Mol Recognit, 2003 May-Jun, 16(3), 148 - 56 Synthesis, characterization and immunochemical evaluation of cephalosporin antigenic determinants; Sanchez-Sancho F et al.; Lack of knowledge of the exact chemical structure of cephalosporin antigenic determinants has hindered clinical interpretation of adverse reactions to these drugs and delayed understanding of the mechanisms involved in the specific recognition and binding of IgE molecules to these antigenic determinants . We further resolve the relationship between structure and activity of proposed antigenic chemicals, including the rational design and synthesis of these haptenic structures . Comparative RAST inhibition studies of the synthesized molecules revealed that they were recognized by IgE antibodies induced by cephalosporin antibiotics . Thus, these data indicate that recognition is mainly directed to the acyl side chain and to the beta-lactam fragment that remains linked to the carrier protein in the cephalosporin conjugation course . Pediatr Radiol, 2003 Sep, 33(9), 648 - 51 Epub 2003 Jun 26. Ceftriaxone-associated nephrolithiasis and biliary pseudolithiasis in a child; Prince JS et al.; Ceftriaxone is a widely used third-generation cephalosporin . It is generally very safe, but complications of biliary pseudolithiasis and, rarely, nephrolithiasis have been reported in children . These complications generally resolve spontaneously with cessation of the ceftriaxone therapy; however, they may symptomatically mimic more serious clinical problems, such as cholecystitis . We report a case of both ceftriaxone-induced biliary pseudolithiasis and nephrolithiasis. Protein Eng, 2003 May, 16(5), 341 - 50 Insight into the mechanism of the IMP-1 metallo-beta-lactamase by molecular dynamics simulations; Oelschlaeger P et al.; Two models, a purely nonbonded model and a cationic dummy atom approach, were examined for the modeling of the binuclear zinc-containing IMP-1 metallo-beta-lactamase in complex with a mercaptocarboxylate inhibitor . The cationic dummy atom approach had substantial advantages as it maintained the initial, experimentally determined geometry of the metal-containing active site during molecular dynamics simulations in water . The method was extended to the modeling of the free enzyme and the enzyme in complex with a cephalosporin substrate docked in an intermediate structure . For all three systems, the modeled complexes and the tetrahedral coordination of the zinc ions were stable . The average zinc-zinc distance increased by approximately 1 A in the substrate complex compared with the inhibitor complex and the free enzyme in which a hydroxide ion acts as a bridging ligand . Thus, the zinc ions are predicted to undergo a back and forth movement upon the cycle of hydrolysis . In contrast to previous assumptions, no interaction of the Asn167 side chain with the bound cephalosporin substrate was observed . Our observations are in agreement with quantum-mechanical calculations and experimental data and indicate that the cationic dummy atom approach is useful to model zinc-containing metallo-beta-lactamases as free proteins, in complex with inhibitors and in complex with substrates. Antimicrob Agents Chemother, 2003 Jul, 47(7), 2138 - 44 High-level expression of ampC beta-lactamase due to insertion of nucleotides between -10 and -35 promoter sequences in Escherichia coli clinical isolates: cases not responsive to extended-spectrum-cephalosporin treatment; Siu LK et al.; Two Escherichia coli isolates were recovered from the blood of two cancer patients and were demonstrated to produce high levels of the AmpC beta-lactamase with isoelectric points of >9.0 . The hypertranscription of ampC RNA was observed by Northern blot hybridization in both isolates . One isolate (isolate EC44) had a point mutation (G-->A at position -28) and insertion of thymidine between positions -20 and -19 of the ampC promoter gene (GenBank accession no . AE000487) . The single nucleotide insertion of T between positions -19 and -20 created an optimal distance (17 bp) in the Pribnow box for ampC hyperproduction . The other isolate (isolate EC38) had two point mutations (G-->A at position -28 and C-->T at position +58) and a 2-base (GT) insertion between positions -14 and -15 . Although the insertion of GT between positions -14 and -15 may create a new promoter next to the original promoter, cloning of the ampC region with truncated nucleotides of the original -35 region of EC38 failed to verify the hypothesis that a new promoter would be created by such a nucleotide insertion . Instead, multiple start sites for ampC transcription at -1, +1, +2, and +3 were observed in an S1 nuclease protection assay . These results suggest that the RNA polymerase is flexible in the selection of a start site in ampC hypertranscription . In conclusion, nucleotide insertions between the -35 and -10 ampC promoter sequences was the mechanism for the hyperproduction of AmpC beta-lactamase and resistance to oxyimino-cephalosporins . The failure of the two patients to respond to treatment with oxyimino-cephalosporins highlights the important role of such a resistance mechanism in the clinical setting. Folia Microbiol (Praha), 2003, 48(2), 149 - 55 Glutathione metabolism of Acremonium chrysogenum in relation to cephalosporin C production: is gamma-glutamyltransferase in the center? Nagy MA, Emri T, Fekete E, Sandor E, Springael JY, Penninckx MJ, Pocsi I. Methionine increased the intracellular glutathione (reduced) (GSH) pool and the specific gamma-glutamyltransferase (gamma-GT) activity in the cephalosporin C (CPC) producer Acremonium chrysogenum . The accelerated turnover of GSH might be indicative of the existence of a functioning gamma-glutamate cycle, and might supply the CPC biosynthetic machinery with L-cysteine . The gamma-GT was not subject to nitrogen metabolic repression but was more active in cells exposed to different oxidative-stress-generating agents . Exogenous cysteine hindered both the uptake of methionine and the induction of gamma-GT, and was not beneficial for CPC production . There was no causal relationship between the redox status of the cells and the observed cell morphology. Clin Nephrol, 2003 May, 59(5), 388 - 90 Neurotoxicity induced by Cefepime in a very old hemodialysis patient; Ferrara N et al.; Neurotoxicity is an unusual complication of cephalosporin therapy . Only few cases of neurotoxicity induced by Cefepime have been described and probably the frequency of Cefepime-induced status epilepticus is underestimated . We report a case of an 82 year-old male, ESRD patient on chronic hemodialysis program affected by pneumonia, who received a treatment with intravenous Cefepime (1 g/day) and developed a seizure 4 days after the starting antibiotic therapy . Cefepime-induced neurotoxicity was suspected and its administration was immediately discontinued . In order to increase Cefepime clearance a hemodialysis session was urgently started and an improvement of his conscious level was observed . On the following day, after a second hemodialysis session his clinical condition and the status of neurotoxicity were completely recovered . The patient was discharged from the hospital in stable clinical condition one week later . At variance with the cases previously reported, the daily dose of Cefepime administrated to our patient was 50% lower and respected drug prescription dosage . Thus, we speculate on the hypothesis that advanced age of our patient and metabolic encephalopathy induced by chronic uremia made him more sensitive to the neurotoxicity induced by the drug . In conclusion, our case suggests that, in very old patients on long-term hemodialysis, it should be considered, to avoid neurotoxicity, to monitor the clinical neurological status, to use Cefepime at lower dosage than that allowed in patients with severe renal impairment (1 g/day) and, when possible, to evaluate Cefepime plasma levels . However, in these patients, other agents of the same class should be considered such as Cefotaxime and Ceftriaxone which are characterized by both an hepatic and renal excretion . In alternative to cephalosporins, antibiotics with the same action spectrum in the absence of neurological toxicity (i.e . Meropenem) should be recommended. Farmaco, 2003 Jun, 58(6), 409 - 18 An alternative procedure for preparation of cefdinir; Gonzalez M et al.; Cefdinir, a broad spectrum third-generation cephalosporin for oral administration, was prepared by the following synthetic pathway: synthesis of diphenylmethyl 7beta-amino-3-vinyl-3-cephem-4-carboxylate hydrochloride from 7-aminocephalosporanic acid (7-ACA), preparation of sodium 2-(2-tritylaminothiazol-4-yl)-(Z)-2-(tritylhydroxyimino) acetate from ethyl acetoacetate, coupling of both intermediaries to obtain diphenylmethyl 7beta-{2-(2-tritylaminothiazol-4-yl)-(Z)-2-tritylhydroxyimino-3-vinyl-3-cephem-4-carboxylate and final cleavage of trityl and diphenylmethyl protective groups . This procedure allows to obtain better yields of cefdinir and to avoid the use of diketene during the synthesis of this antibiotic by the previously reported method. Vet Ther, 2003 Spring, 4(1), 83 - 93 A comprehensive review of ceftiofur sodium and hydrochloride formulations for treatment of acute bovine foot rot; Kausche FM et al.; Seven well-controlled studies conducted under multiple management conditions demonstrated that ceftiofur, a late-generation veterinary parenteral cephalosporin, is effective for the treatment of bovine foot rot in beef and dairy cattle . Two preliminary dosage titration studies using a challenge model compared the efficacy of ceftiofur (1.1 mg or 2.2 mg ceftiofur equivalents {CE}/kg administered once daily for 3 days) with placebo . One preliminary clinical study evaluated the efficacy of ceftiofur sodium (1.0 mg CE/kg once daily for 3 days) in lactating dairy cows . Two clinical trials evaluated the efficacy of ceftiofur sodium versus placebo for naturally occurring foot rot, and two trials compared the efficacy of ceftiofur sodium or hydrochloride (1.0 mg CE/kg) with oxytetracycline (6.6 or 10 mg/kg), each administered once daily for 3 days, for treatment of acute foot rot in beef cattle . All trials demonstrated the efficacy of ceftiofur for treatment of acute bovine foot rot . Ceftiofur and oxytetracycline were comparable in efficacy, with ceftiofur having excellent injection-site tolerance and short or no milk discard or preslaughter withdrawal. Yakushigaku Zasshi, 2003, 37(2), 119 - 27 {The origin of cephalosporins}; Nakajima S; The origin of cephalosporins is investigated . In 1945, Giuseppe Brotzu, who was the rector of the University of Cagliari in Sardina, Italy, isolated a cephalosporin-producing strain, Cephalosporium acremonium . Although as many as 48 cephalosporin derivatives have been developed in Japan, how a cephalosporin-producing organism was discovered is not widely known here . This article contains the first Japanese translation of Brotzu's Italian publication entitled "Ricerche su di un nuovo antibiotico (Research on a new Antibiotic)" and reveals how cephalosporin was developed, together with a cross reference to the first report of penicillin, a similar antibiotic compound, which was discovered by A . Fleming in 1928 . Brotzu's brief academic and social background is also presented. Ann Dermatol Venereol, 2003 Mar, 130(3), 321 - 4 {Immediate hypersensitivity is rarely implicated in drug induced urticaria}; Cousin F et al.; INTRODUCTION: The unexpected appearance of acute urticaria during the course of drug treatment gives rise to the following question: is it an allergic urticaria (due to an immediate hypersensitivity: IgE mediated specific immunity) or is it pseudo-allergic? We report our findings in an immuno-allergological study of patients who were sent for drug intolerance which presented as immediate hypersensivity (urticaria, angiooedema, anaphylactic shock) . METHODS: A prospective study was conducted including all the patients who were sent to the unit for urticaria or angiooedema type drug intolerance . Patients were questioned about previous chronic urticaria and also about urticaria after taking different medicines . The clinical examination looked for a dermographism . All the patients then took skin tests for immediate hypersensitivity, the molecule was contra-indicated and tests for cross-reactivity were conducted . PATIENTS: Three hundred fifty patients were sent to this unit between February 2000 and April 2001 for drug intolerance, mostly with urticaria/angiooedema but in 7 cases with anaphylactic shock . The incriminated drugs were varied: 50 p . 100 were due mainly to penicillins and cephalosporins . Other drug groups were also involved: non steroid anti-inflammatories, aspirin and paracetamol for the most part, along with local anesthetics, morphine-based products, contrast iodine products, corticosteroids . RESULTS: Of the 350 patients tested, only 22 were allergic and had positive tests for the incriminated drug . In these 22 patients, with the exception of 2 of them, the effects were severe (anaphylactic shock in 7 patients) and the urticaria was only a minor manifestation of the reaction . The drugs responsible were cephalosporin (10 patients), the penicillin (6 patients), insulin (2 patients), gonadorelin (1 patient), carboxymethylcellulose (1 patient), lidocain (1 patient), and sulfamethoxazole (1 patient) . The 328 other patients had negative tests and were able to retake the tested molecule without incident . Most of them had antecedents of chronic urticaria or dermographism . DISCUSSION: Only 22 patients of the 350, i.e . 6 p . 100 were genuinely allergic . These patients were those who presented the most severe symptoms . The other patients, i.e . the majority, suffered from pseudo-allergic drug-induced urticaria, which made retaking the medicines possible. Otolaryngol Pol, 2003, 57(1), 99 - 101 {Multicenter, open clinical investigation on using cefprozil in therapy of otitis media in children}; Gryczynska D et al.; We present results of multi center, open clinical trial on using cefprozil (cephalosporin II generation) in acute otitis media in children . Results indicate very good treatments effect (89.3%) and good tolerance of the drug (10.7% side effects). Farmaco, 2003 May, 58(5), 363 - 9 An improved method for preparation of cefpodoxime proxetil; Rodriguez JC et al.; Cefpodoxime proxetil, a third-generation cephalosporin for oral administration, was synthesized by a method based on the following sequence of reactions: acylation of 7-aminocephalosporanic acid (7-ACA) with S-benzothiazol-2-yl(2-amino-4-thiazolyl)(methoxyimino)thioacetate (MAEM), chloroacetylation of the cefotaxime formed with chloroacetyl chloride, esterification of the acid function with 1-iodoethyl isopropyl carbonate and final cleavage of chloroacetamide protective group by treatment with thiourea in N,N-dimethylacetamide . The developed procedure allows us to obtain better yields of cefpodoxime proxetil and to eliminate the final purification step by column chromatography, necessary during the synthesis of this antibiotic by the previously reported methods. Bioorg Med Chem Lett, 2003 May 19, 13(10), 1687 - 90 Synthesis of beta-lactamase activated nitric oxide donors; Tang X et al.; In order to achieve site specific delivery of NO, we designed conjugates of cephalosporin with NO donors . NO donors such as cupferron and SIN-1 were evaluated as potential choices for conjugates . Cephalosporin conjugated with SIN-1 demonstrated promising beta-lactamase dependent NO releasing ability. Electrophoresis, 2003 Apr, 24(7-8), 1215 - 20 Strategies for the determination of cefazolin in plasma and microdialysis samples by short-end capillary zone electrophoresis; Mayer BX et al.; Capillary zone electrophoresis was employed to determine cefazolin, a first-generation cephalosporin antibiotic, in plasma and microdialysis samples from patients . To shorten the analysis, the samples were injected from the short end of the capillary, resulting in a separation time of < 3 min . Due to a high ionic strength of the biological matrices it was necessary to optimize the stacking conditions . For microdialysis samples a 1:10 dilution with water before injection was sufficient to obtain good peak shape . For plasma samples a protein removal step was required to obtain clean electropherograms and a good peak shape . Acetonitrile was used as precipitant resulting in an enhanced sample stacking in comparison to water dilution . The disadvantage of using acetonitrile was severe evaporation loss making quantitation impossible . A self-sealing film was used to seal each individual sample vial to suppress evaporation during long-term sequences . The calibration curves for spiked plasma and cefazolin in Ringer's solutions were linear in the range from 2-500 and 2.5-100 microg/mL, respectively . Limits of detection were 1.0 and 2.0 microg/mL in plasma and microdialysis samples, respectively . The assay was successfully applied to plasma and microdialysis samples obtained in vivo from the interstial space fluid of subcutaneous adipose and muscle tissue of patients undergoing cardiac surgery. Am J Hematol, 2003 May, 73(1), 41 - 3 Serologically documented loracarbef (Lorabid)-induced immune thrombocytopenia; Aljitawi OS et al.; We report here the first case of severe immune thrombocytopenia induced by a second-generation cephalosporin antibiotic, Loracarbef . A 56-year old white female developed acute severe thrombocytopenia associated with acute respiratory symptoms following administration of Loracarbef . She responded to Loracarbef withdrawal and systemic corticosteroid administration . Loracarbef-dependent platelet-reactive antibodies were demonstrable in her serum by flow cytometry . Curr Med Chem, 2003 Jun, 10(12), 1005 - 19 The HAG mechanism: a molecular rationale for the therapeutic application of iron chelators in human diseases involving the 2-oxoacid utilizing dioxygenases; Hanauske-Abel HM et al.; 'Iron chelation' is widely understood as synonymous with non-specificity and viewed as a purely physicochemical mode of action, without any defined biomolecular target, broadly interfering with metalloenzymes . The 2-oxoacid-utilizing dioxygenases challenge this preconception . A family of non-heme iron enzymes that rely on chelation-dependent catalysis, they employ common molecules like Krebs cycle intermediates as endogenous iron chelators and consume atmospheric oxygen, inserting one of its atoms into cellular components . These enzymes control the adaptation of cells to hypoxia; the reversal of mutagenic DNA alkylations, the initiation of DNA replication, the translation of mRNAs; the production of extracellular matrix proteins like collagens and fibrillins; and numerous metabolic pathways: from the synthesis of the gibberellin growth hormones of plants, and the formation of carnitine, atropine, endotoxins, and cephalosporin antibiotics, to the breakdown of amino acids . Their pivotal roles in human pathology encompass oncogenesis and cancer angiogenesis, scarring and organ fibrosis, inherited diseases, and retroviral infections . Their unique catalysis, termed earlier the 'HAG mechanism' and known in subatomic detail, requires at least three different substrates to form three different products, and proceeds as a ligand reaction at the non-heme iron atom inside the active site pocket, without any direct involvement of apoenzyme residues . The apoenzyme sterically controls ligand access to the metal . The HAG mechanism-based concept of catalytic chelation directed by an apoenzyme, not merely by complexation parameters, has enabled knowledge-guided design of systemic and tissue-selective inhibitors, and of clinical trials . The HAG mechanism also lends itself to the development of novel, man-made biocatalysts. Appl Biochem Biotechnol, 2003 Mar, 104(3), 185 - 98 Immobilization of glutaryl-7-aminocephalosporanic acid acylase on silica gel and enhancement of its stability; Park SW et al.; Glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase is an enzyme that converts GL-7-ACA to 7-aminocephalosporanic acid, a starting material for semisynthetic cephalosporin antibiotics . In this study, optimal conditions for the immobilization of GL-7-ACA acylase were determined by experimental observations and statistical methods . The optimal conditions were as follows: 1.1 M phosphate buffer (pH 8.3) as buffer solution, immobilization temperature of 20 degrees C, and immobilization time of 120 min . Unreacted aldehyde groups were quenched by reaction with a low-molecular-weight material such as L-lysine, glycine, and ethanolamine after immobilization in order to enhance the activity of immobilized GL-7-ACA acylase . The activities of immobilized GL-7-ACA acylase obtained by using the low-molecular-weight materials were higher than those obtained by immobilized GL-7-ACA acylase not treated with low-molecular-weight materials . In particular, the highest activity of immobilized GL-7-ACA acylase was obtained using 0.4% (v/v) ethanolamine . We also investigated the effect of sodium cyanoborohydride in order to increase the stability of the linkage between the enzyme and the support . The effect on operational stability was obvious: the activity of immobilized GL-7-ACA acylase treated with 4% (w/w) sodium cyanoborohydride remained almost 100% after 20 times of reuse. Acta Anaesthesiol Scand, 2003 Feb, 47(2), 230 - 2 Complete recovery from prolonged cardio-pulmonary resuscitation following anaphylactic reaction to readministered intravenous cefazolin; Gibbs MW et al.; We describe a patient who developed a type I anaphylactic reaction to intravenous cefazolin . The patient had no known drug allergies and had previously received intraoperative intravenous cefazolin 2 months prior without any problems . Forty-fives after receiving cefazolin 1 g i.v . and while fully awake, the patient experienced shortness of breath, became unconscious, and then suffered a cardiac arrest . Resuscitation included endotracheal intubation, external cardiac compression, electrical defibrillation and multiple large doses of epinephrine, atropine, and sodium bicarbonate over the course of 2.5 h and three cardiac arrests . Nevertheless, the patient fully recovered . The intent of this case report is to address widely held concerns regarding cross-reactivity of cephalosporin, particularly cefazolin, to penicillin, the legitimacy of test dosing as a means to safely identify patients who will have an allergic reaction to cephalosporins and comment on patient-related predictors of survival following cardiopulmonary resuscitation and the good outcome in this case . Copyright Acta Anaesthesiologica Scandinavica 47 (2003) Pol Merkuriusz Lek, 2002 Nov, 13(77), 396 - 8 {Empirical therapy of peritonitis in peritoneal dialysis patients--it is reasonable to use a new therapeutic schedule?}; Baczynski D et al.; The Ad Hoc Advisory Committee on Peritonitis Management recommended in the year 2000 a new protocol of empirical peritonitis therapy in patients on peritoneal dialysis with preserved residual renal function (RRF) . This protocol comprises 1st and 3rd generation cephalosporins . According to these recommendations the old protocol of therapy, comprising 1st generation cephalosporin and aminoglycoside may be used only in patients with diuresis lower than 100 ml/day . The aim of the study was a retrospective assessment of the efficacy and cost of peritonitis therapy using "old" and "new" protocols . The analysed episodes of peritonitis were divided into two groups . Group 1 included 22 episodes of peritonitis in 13 patients treated with the old protocol, in whom RRF was lower than 100 ml/day . Group 2 included 6 episodes of peritonitis in 4 patients with preserved RRF treated with the new protocol . The efficacy of the treatment according to the old protocol was 64% and according to the new protocol--33% . The average cost of 14-day therapy with the old and new schedule was 67.1 and 247.2 Euro, respectively . In our studied population a considerably lower efficacy and higher cost were revealed of the new empirical schedule of peritonitis treatment in comparison to the old schedule . The results of the study indicate the need of further estimation of the usefulness of the new peritonitis empirical treatment protocol. Appl Environ Microbiol, 2003 Feb, 69(2), 1308 - 14 Cloning and expression analysis of the pcbAB-pcbC beta-lactam genes in the marine fungus Kallichroma tethys; Kim CF et al.; Here we report the identification of the beta-lactam biosynthesis genes pcbAB and pcbC from a cosmid genomic DNA library of the marine fungus Kallichroma tethys . A BLAST homology search showed that they share high sequence identity with the delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetases and isopenicillin N synthases, respectively, of various fungal and bacterial beta-lactam producers, while phylogenetic analysis indicated a close relationship with homologous genes of the cephalosporin-producing pyrenomycete Acremonium chrysogenum . Expression analysis by reverse transcription-PCR suggested that both genes are highly regulated and are expressed in the late growth phase of K . tethys cultures . Complementation of an Aspergillus nidulans strain deficient in ACV synthetase suggested that at least pcbAB is functional, although attempts to isolate active antibiotic from K . tethys were unsuccessful. Bioorg Med Chem Lett, 2003 Feb 10, 13(3), 539 - 42 Cephalosporin prodrugs of paclitaxel for immunologically specific activation by L-49-sFv-beta-lactamase fusion protein; Vrudhula VM et al.; Paclitaxel conjugates of 7-phenylacetamidocephalosporanic acid were prepared as prodrugs for site specific activation by targeted beta-lactamase . Immunologically specific activation of the prodrug 5 containing 3,3-dimethyl-4-amino-butyric acid as linker in combination with the fusion protein L-49-sFv-beta-lactamase was demonstrated in vitro on 3677 melanoma cells. Se Pu, 1999 Nov, 17(6), 570 - 2 {Purity analysis of cephalosporins with capillary zone electrophoresis}; Long H et al.; A capillary zone electrophoresis method is proposed for the purity determination of nine cephalosporin drugs . A background electrolyte comprising of either 20 mmol/L pH 9.20 borate buffer or 20 mmol/L pH 6.86 phosphate buffer was used for most drugs studied except for cefaloridine which formed neutral molecules at these pH values . For it 50 mmol/L pH 2.05 phosphate buffer was used instead . Internal normalization method was employed for quantitation . The method is simple, rapid and versatile . Analysis was completed within 8 min . The merits and limitations of the method were also discussed. Antimicrob Agents Chemother, 2003 Feb, 47(2), 689 - 96 Effects of organic anion, organic cation, and dipeptide transport inhibitors on cefdinir in the isolated perfused rat kidney; Lepsy CS et al.; Cefdinir (Omnicef; Abbott Laboratories) is a cephalosporin antibiotic primarily eliminated by the kidney . Nonlinear renal elimination of cefdinir has been previously reported . Cefdinir renal transport mechanisms were studied in the erythrocyte-free isolated perfused rat kidney . Studies were performed with drug-free perfusate and perfusate containing cefdinir alone to establish the baseline physiology and investigate cefdinir renal elimination characteristics . To investigate cefdinir renal transport mechanisms, inhibition studies were conducted by coperfusing cefdinir with inhibitors of the renal organic anion (probenecid), organic cation (tetraethylammonium), or dipeptide (glycylsarcosine) transport system . Cefdinir concentrations in biological samples were determined using reversed-phase high-performance liquid chromatography . Differences between treatments and controls were evaluated using analysis of variance and Dunnett's test . The excretion ratio (ER; the renal clearance corrected for the fraction unbound and glomerular filtration rate) for cefdinir was 5.94, a value indicating net renal tubular secretion . Anionic, cationic, and dipeptide transport inhibitors all significantly affected the cefdinir ER . With probenecid, the ER was reduced to 0.59, clearly demonstrating a significant reabsorptive component to cefdinir renal disposition . This finding was confirmed by glycylsarcosine studies, in which the ER was elevated to 7.95, indicating that reabsorption was mediated, at least in part, by the dipeptide transporter system . The effects of the organic cation tetraethylammonium, in which the ER was elevated to 7.53, were likely secondary in nature . The anionic secretory pathway was found to be the predominant mechanism for cefdinir renal excretion. Cytometry A, 2003 Feb, 51(2), 68 - 78 Detection of beta-lactamase reporter gene expression by flow cytometry; Knapp T et al.; BACKGROUND: Flow cytometry of gene expression in living cells requires accurate, sensitive, nontoxic fluorescent indicators capable of detecting transcription of specific genes . This is typically achieved by using genes that encode fluorescent proteins or enzymes coupled to promoters of interest . The most commonly used reporters are green fluorescent protein and beta-galactosidase (lacZ) . In this study, we characterized the performance of a cell-permeant, ratiometric, beta-lactamase substrate, coumarin cephalosporin fluorescein (CCF2/AM) . We compared its characteristics with that of the beta-galactosidase/fluorescein di-beta-D-galactopyranoside reporter system . METHODS: Jurkat cell lines were generated for beta-lactamase and beta-galactosidase reporters with the use of similar plasmid constructs . Rare event flow cytometric detection for the beta-galactosidase and beta-lactamase reporters were assayed by using mixed populations of negative (WT) and positive (constitutively expressing) cells for each reporter . To determine sensitivity at low reporter copy number, we measured the activity of an unstimulated inducible promoter and detected positive events as a function of substrate incubation time . Technical issues related to data processing and optical configuration are also presented . RESULTS: The low population coefficients of variation afforded by ratiometric detection of the beta-lactamase system improved the statistical performance of the assay in comparison with a single-dye, intensity-based assay, leading to markedly improved detection for low copy number and rare events . At low levels of gene expression, beta-lactamase was detected with approximately 10-fold higher confidence than was beta-galactosidase . In rare event detection experiments, cells expressing high levels of beta-lactamase proteins were reliably detected at frequencies of 1:10(6) compared with about 1:10(4) for beta-galactosidase . CONCLUSION: The ratiometric fluorescence readout of the beta-lactamase system based on fluorescence resonance energy transfer allowed more sensitive and accurate detection of gene expression than the currently available beta-galactosidase substrates . Further, the cell-permeant nature of the substrate improved experimental convenience . These properties facilitated cell engineering and enabled a variety of applications including selection of rare cells from large populations and measurement of low-expressing or downregulated genes . Antimicrob Agents Chemother, 2003 Jan, 47(1), 387 - 9 Site-specific mutagenesis analysis of PBP 1A from a penicillin-cephalosporin-resistant pneumococcal isolate; Smith AM et al.; Our mutagenesis study has investigated all amino acid mutations in the penicillin-binding domain of PBP 1A from Hungarian pneumococcal isolate 3191 to determine the importance of every mutation in the development of penicillin and cefotaxime resistance . Our data reveal that mutations at amino acid positions 574 to 577 and position 539 cause penicillin and cefotaxime resistance. J Perianesth Nurs, 2002 Dec, 17(6), 393 - 8 Allergic reactions to drugs: implications for perioperative care; Golembiewski JA; Clinically, one must be able to differentiate between an allergic reaction and an adverse reaction . Clinical manifestations of allergic reactions range from urticaria and rash to bronchoconstriction, laryngeal edema, hematologic disorders, and other serious reactions . Many drugs administered in the perioperative setting can cause allergic reactions . Antibiotics such as penicillins, beta-lactam antibiotics, and sulfonamides are the most common class of drugs that produce allergic reactions . A detailed allergy history is important when deciding if a patient can receive a drug that may cross-react (eg, a cephalosporin in a patient with a penicillin allergy) . Vancomycin can cause a reaction that ranges from erythema and pruritis to clinically significant hypotension . Proper dilution and rate of administration are essential in minimizing the histamine from vancomycin that is thought to produce this reaction . "Sulfa allergy" describes an allergy to sulfonamide antibiotics; a patient with a "sulfa allergy" is not allergic to drugs containing sulfur, sulfites, or sulfates . Although true allergic reactions to opioids are rare, naturally occurring compounds like morphine and codeine can cause allergic reactions . After stopping the offending drug, mild allergic reactions can be managed with diphenhydramine, with or without a steroid . Significant allergic reactions require more aggressive management with oxygen, intravenous fluids, epinephrine, and histamine blockers . Trends Biotechnol, 2002 Dec, 20(12), 502 - 7 Unraveling the methionine-cephalosporin puzzle in Acremonium chrysogenum; Martin JF et al.; Methionine has long been known as the major stimulant of the formation of cephalosporin C in Acremonium chrysogenum . Enzymatic and genetic studies of methionine have revealed that it induces four of the enzymes of cephalosporin-C biosynthesis at the level of transcription . It is also converted to cysteine, one of three precursors of cephalosporin C, by cystathionine-gamma-lyase . The main effect of methionine on cephalosporin production results from its regulatory role, which can be duplicated by the non-sulfur analog norleucine . Eliminating cystathionine-gamma-lyase prevents the enhancing precursor effect of methionine on cephalosporin-C production, and cystathionine-gamma-lyase overproduction in moderate doses increases cephalosporin-C formation. Antimicrob Agents Chemother, 2002 Dec, 46(12), 3971 - 7 Amino acid substitutions at Ambler position Gly238 in the SHV-1 beta-lactamase: exploring sequence requirements for resistance to penicillins and cephalosporins; Hujer AM et al.; Site saturation mutagenesis of the 238 position in the SHV beta-lactamase was performed to identify the complete sequence requirements needed for the extended spectrum beta-lactamase (ESBL) phenotype . MICs (in micrograms per milliliter) in an isogenic background, Escherichia coli DH10B, demonstrated that the Gly238Ala mutation conferred the most resistance to penicillins and cephalosporins . The absolute increase in resistance was greatest against cefotaxime for the Gly238Ala mutant (0.06 to 8 micro g/ml) . Except for the strain possessing the Gly238Pro beta-lactamase, ceftazidime MICs were also elevated . None of the mutant SHV beta-lactamases were expressed in as great an amount as the wild-type beta-lactamase . Kinetic analysis of the Gly238Ala mutant revealed that penicillin and cephalosporin substrates have a lower K(m) for the enzyme because of this mutation . Ampicillin and piperacillin MICs were inversely proportional to the side chain volume of the amino acid in cases larger than Ser, suggesting that steric considerations may be a primary requirement for penicillin resistance . Secondary structural effects explain increased resistance to oxyiminocephalosporins . Based upon this study, we anticipate that additional mutations of Gly238 in the SHV beta-lactamase will continue to be discovered with an ESBL (ceftazidime or cefotaxime resistant) phenotype. Biochim Biophys Acta, 2002 Nov 19, 1601(1), 93 - 9 Nonribosomal peptide synthetases-evidence for a second ATP-binding site; Kallow W et al.; delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) catalyses, via the protein thiotemplate mechanism, the nonribosomal biosynthesis of the penicillin and cephalosporin precursor tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) . The complete and fully saturated biosynthetic system approaches maximum rate of product generation with increasing ATP concentration . Nonproductive adenylation of ACVS, monitored utilising the ATP-{32P}PP(i) exchange reaction, has revealed substrate inhibition with ATP . The kinetic inhibition pattern provides evidence for the existence of a second nucleotide-binding site with possible implication in the regulatory mechanism . Under suboptimal reaction conditions, in the presence of MgATP(2-), L-Cys and inorganic pyrophosphatase, ACVS forms adenosine(5')tetraphospho(5')adenosine (Ap(4)A) from the reverse reaction of adenylate formation involving a second ATP molecule . The potential location of the second ATP binding site was deduced from sequence comparisons and molecular visualisation in conjunction to data obtained from biochemical analysis. J Biol Chem, 2002 Dec 6, 277(49), 47719 - 23 Epub 2002 Sep 26. Unexpected advanced generation cephalosporinase activity of the M69F variant of SHV beta-lactamase; Helfand MS et al.; Infections with bacteria that contain hydrolytic beta-lactamase enzymes are becoming a serious problem in the United States . Mutations at Met-69, an amino acid proximal to the active site Ser-70 in the TEM-1 and SHV-1 beta-lactamases, have emerged as a puzzling cause of bacterial resistance to inhibitors of beta-lactamases . Site-saturation mutagenesis of the 69 position in SHV beta-lactamase was performed to determine how mutations of this non-catalytic residue play a role in increasing 50% inhibitory concentrations (IC(50) concentrations) for clinically important beta-lactamase enzyme inhibitors . Two distinct phenotypes are evident in the variant beta-lactamases studied: significantly increased minimum inhibitory concentrations (microg/ml) and IC(50) concentrations to clavulanic acid for the Met69Ile, Leu, and Val substitutions, and unanticipated increased minimum inhibitory concentrations and hydrolytic activity toward