Bone Marrow Transplant, 1999 Jul, 24(2), 173 - 7 Spleen sizing by ultrasound scan and risk of pneumococcal infection in patients with chronic GVHD: preliminary observations; Picardi M et al.; Encapsulated bacteria infections (EBI) can cause severe complications after BMT, usually occurring in patients with chronic GVHD (cGVHD) and attributed to functional hyposplenism . Using ultrasound (US) scan, we measured spleen size in 22 patients transplanted from HLA identical siblings, with or without cGVHD . No patient had received TBI, spleen irradiation or penicillin prophylaxis . Results were correlated with occurrence of EBI during a mean follow-up of 55 months (range 7-93) . In the group without cGVHD, the difference between pre- and post-BMT spleen longitudinal diameters was not significant, and no patient developed EBI . In the cGVHD group, post-BMT spleen longitudinal diameters were significantly smaller than those pre-BMT (9.1+/-1.6 vs . 12.3+/-2.2; P = 0.0005) . Out of four patients with cGVHD who showed a major spleen size reduction, two developed a severe infection (an overwhelming sepsis and a pneumococcal meningitis) . In our small series, we found a borderline relationship between spleen size reduction and duration of cGVHD (P = 0.06), as well as an increased risk of life-threatening infection in patients with extensive cGVHD and hyposplenism as detected by US scan . We conclude that US scan may be useful to detect spleen size reduction following allogeneic BMT and that penicillin prophylaxis is to be strongly recommended in patients with extensive cGVHD and spleen size reduction, even in those who have not received total body or spleen irradiation. Medicina (B Aires), 1999, 59(3), 277 - 8 {Acute nephrotic syndrome associated with secondary syphilis}; Baudagna GB et al.; We present a case of a 38 year old woman with nephrotic syndrome and skin rash . She had been previously healthy . At the beginning she showed nonpruritic macular papular lesion in trunk and upper and lower extremities with twenty days of evolution . The week previous to hospitalization, she presented eyelid and pretibial edema . The laboratory informed normal kidney function with proteinuria of 10 g/day, plasma proteins of 4.20 g/dl., cholesterol 334 mg/l, VDRL (+), 1/32 FTA abs (+) HIV non reactive, normal collagenogram . She was diagnosed as secondary syphilitic nephrotic syndrome . She was prescribed penicillin, rest and salt restriction . She presented good evolution before ending treatment and the syndrome was completely solved at the third week . The incidence of kidney condition associated with early staging of syphilis is lower than 0.3% . We call attention to this association, for such an uncommon case may not be detected. Bioorg Med Chem Lett, 1999 Jul 19, 9(14), 1997 - 2002 The synthesis and evaluation of 6-alkylidene-2'beta-substituted penam sulfones as beta-lactamase inhibitors; Buynak JD et al.; Penicillin sulfones, which structurally incorporate both a 6-position alkylidene substituent and a 2'beta substituent, have been synthesized and evaluated as inhibitors of class C and class A serine beta-lactamases . Incorporation of the 2'beta-substituent generally improves inhibitory activity . Substituents that improve transport across the bacterial cell membrane have also been incorporated. Biochim Biophys Acta, 1999 Aug 17, 1433(1-2), 327 - 34 pH dependence of penicillin amidase enantioselectivity for charged substrates; Lummer K et al.; The pH dependence of E (enantiomeric ratio or enantioselectivity, a quantitative measure for enzyme stereospecificity) was studied for penicillin amidase catalysed hydrolysis of charged enantiomeric substrates . Theoretical analysis shows that a pH dependence can only be observed around the pK values of groups in the active site whose ionisation control the enzyme activity . For charged substrates that may perturb these pK values, a pH dependence of E is also expected . This was experimentally verified around these pK values . The S'(1)-stereospecificity of penicillin amidase was studied for the hydrolysis of the enantiomeric phenylacetyl-S/R-Phe and for the racemic phenylacetyl-S,R-PhG . The S(1)-stereospecificity was investigated for the hydrolysis of the enantiomeric S/R-PhG-NH(2) . The observed pH modulation of E (more than 3-fold for the studied substrates in the pH range 4.5-9) was found to be a result of compensatory effects for binding and catalysis . The ratios k(cat, S)/k(cat,R) and K(m,S)/K(m,R) for the hydrolysis of the enantiomeric phenylacetyl-Phe were found to decrease from 1000 to 10 and from 0.1 to 0.01, respectively in the pH range 5-8 . The dependence was stronger for the S'(1)- than for the S(1)-subsite . This is probably due to the stronger influence of the substrate carboxyl group in the S'(1)-subsite than that of the substrate amino group in the S(1)-subsite on the pK of the N-terminal Ser B1 that is essential for the activity . The observed pH dependence of E was used to discuss the importance of ground-state interactions for discrimination between enantiomers and for enzyme catalysis in general . The experimental results conform to the split site model according to which a better binding must not be fundamentally inhibitory. Biochim Biophys Acta, 1999 Aug 17, 1433(1-2), 76 - 86 Intramolecular autoproteolysis initiates the maturation of penicillin amidase from Escherichia coli; Kasche V et al.; The penicillin amidase (PA) from Escherichia coli belongs to a group of proteolytically processed bacterial enzymes . The mechanism of the maturation of the single polypeptide proenzyme has been studied for the PA from E . coli using a slowly processing mutant proenzyme . The mutant proenzyme was constructed by replacing Thr with Gly in the Thr(263)-Ser(264) bond that must be hydrolysed in active PA . The mutant proenzyme was purified by biospecific affinity chromatography using an immobilized monoclonal antibody against PA . The maturation of the free and covalently immobilized purified proenzyme was studied in vitro . For the free proenzyme the same products with PA activity as observed in homogenates of wild-type PA-producing E . coli cells were found to be formed during this process . A kinetic analysis of the possible inter- and intramolecular processes involved in the maturation demonstrated that unambiguous evidence for the existence of intramolecular processes can only be obtained in systems where intermolecular processes are excluded . The Gly(263)-Ser(264) bond was found to be hydrolysed first in the free and immobilized mutant proenzyme, based on determinations of mass spectra, N-terminal sequences and active site concentrations . In the system with immobilized proenzyme intermolecular processes are excluded, demonstrating that this bond is hydrolysed by intramolecular autoproteolysis . Based on the known three-dimensional structure of the PA from E . coli the same maturation mechanism should apply for the wild-type proenzyme. J Med Assoc Thai, 1999 Jun, 82(6), 531 - 5 Pulmonary actinomycosis: a study of 16 cases from Central Chest Hospital; Dujneungkunakorn T et al.; Actinomycosis is a relatively rare infection . This is a report of 16 patients with pulmonary actinomycosis diagnosed from 1990 to 1997 at the Central Chest Hospital, Thailand . Twelve patients were male and 4 were female, with a mean age of 59 years and a mean duration of symptoms of 9 months . Common symptoms were cough and hemoptysis . Mass-like shadowing was the most common radiographic finding (37%) . The diagnosis, based on findings of typical sulfur granules, was reached by bronchoscopy (10 cases), surgery (5 cases) and fine needle aspiration (1 case) . Endobronchial mass with luminal occlusion was the most frequent bronchoscopic finding (56%) . Coexistent bronchial carcinoma was present in one specimen . Penicillin was given in 10 patients, 2 of whom (20%) were cured, 5 (50%) are currently on treatment and have achieved clinical response, whereas, the other 2 patients (20%) did not respond . Surgical resection was performed in 8 patients, all of whom recovered . An awareness of the full spectrum of actinomycosis manifestations will expedite diagnosis and optimize treatment. Fungal Genet Biol, 1999 Jul-Aug, 27(2-3), 243 - 52 HAP-Like CCAAT-binding complexes in filamentous fungi: implications for biotechnology; Brakhage AA et al.; Regulatory CCAAT boxes are found frequently in eukaryotic promoter regions . They are bound by different CCAAT-binding factors . Until now, a single CCAAT-binding complex has been reported in fungi . It is also found in higher eukaryotes and is highly conserved among eukaryotic organisms . This multimeric protein complex is designated HAP, AnCF, CBF, or NF-Y . The complex consists of at least three subunits . In fungi, only the HAP complex of Saccharomyces cerevisiae had been known for a long time . The recent cloning of genes encoding the components of the corresponding complex (AnCF/PENR1) of Aspergillus nidulans and characterization of CCAAT-regulated genes in A . nidulans, as well as other filamentous fungi, led to a deeper insight into the role of this transcription complex, in particular in aerobically growing fungi . An overview of the function of HAP-like complexes in gene regulation in filamentous fungi is presented . Some of the genes that have been found to be regulated by HAP-like complexes encode enzymes of biotechnological interest, like taka-amylase, xylanases, cellobiohydrolase, and penicillin biosynthesis enzymes . The importance of HAP-like complexes in controlling the expression of biotechnologically important genes is discussed . J Bacteriol, 1999 Aug, 181(16), 5107 - 10 Identification of a novel penicillin-binding protein from Helicobacter pylori; Krishnamurthy P et al.; The Helicobacter pylori genome encodes four penicillin-binding proteins (PBPs) . PBPs 1, 2, and 3 exhibit similarities to known PBPs . The sequence of PBP 4 is unique in that it displays a novel combination of two highly conserved PBP motifs and an absence of a third motif . Expression of PBP 4, but not PBP 1, 2, or 3, is significantly increased during mid- to late-log-phase growth. FEMS Microbiol Lett, 1999 Aug 1, 177(1), 7 - 14 New insights into the regulation of the pac gene from Escherichia coli W ATCC 11105; Roa A et al.; The regulation of the pac gene encoding the penicillin G acylase of Escherichia coli W ATCC 11015 has been investigated by a molecular approach using lacZ as a reporter gene . This analysis revealed that a region of 170 bp located upstream of the pac structural gene contains the regulatory sequences that control its expression . The cAMP receptor protein is involved not only in the catabolite repression of penicillin G acylase production caused by glucose but also in the induction of pac gene expression by phenylacetic acid . Primer extension analyses have demonstrated that the transcription of the pac gene can be initiated from at least three different promoters . Although all these promoters are functional, their relative activity depends on the transcribed gene, the P1 and P3 promoters being more active in the presence of the pac gene, whereas the P2 promoter was stronger when the upstream region of the pac gene was fused to the lacZ reporter . A deletion of the region surrounding the -10 box of the P3 promoter produced a constitutive expression of the fused gene indicating that this sequence is required for phenylacetic acid induction and suggesting that the expression of the pac gene is regulated by a repression mechanism . This work reveals that the regulation of the pac gene is more complex than previously envisioned and provides new clues to investigate further this interesting regulatory system. Analyst, 1998 Dec, 123(12), 2779 - 83 Multi-residue analysis of penicillin residues in porcine tissue using matrix solid phase dispersion; McGrane M et al.; The aim of this study was to develop a multi-residue method for the analysis of penicillins in animal tissue . Matrix solid phase dispersion (MSPD) was employed to extract the residues and the extracts were then cleaned-up by C18 solid phase extraction (SPE) . Pre-column derivatisation using acetic anhydride and 1,2,4-triazole in the presence of mercuric chloride was employed to allow detection in 325 nm . Gradient elution was required to elute amoxicillin, ampicillin, penicillin G, cloxacillin and dicloxacillin derivatives from a C18 reversed phase column using phosphate buffer-acetonitrile mobile phase . The developed method had a limit of detection of 20 ng g-1 and had recoveries in the range 40-90% for the 5 drugs in samples fortified at 40 and 200 ng g-1; the maximum residue limits (MRLs) for these drugs were in the range of 50-300 ng g-1 (ppb). Analyst, 1998 Dec, 123(12), 2749 - 54 The potential of monoclonal antibodies against ampicillin for the preparation of a multi-immunoaffinity chromatography for penicillins; Dietrich R et al.; Monoclonal antibodies (Mab) against ampicillin were prepared by immunization of mice with an ampicillin-keyhole limpet hemocyanin conjugate coupled by a glutaraldehyde method . Sensitivity and specificity of these antibodies were tested in a direct competitive enzyme immunoassay, in which an ampicillin-horseradish peroxidase conjugate prepared by a carbodiimide method served as the labelled antigen . According to their cross-reactivities with the other beta-lactam antibiotics, the Mabs could be divided into two groups, which are represented by the clones designated 1D1 and 3B5 . While Mab 3B5 (IgG1) showed no major cross-reactions with the other penicillins frequently used in veterinary medicine except for amoxicillin (108%), Mab 1D1 (IgG2a) had marked cross-reactivities with most of the 17 tested beta-lactam antibiotics (e.g., amoxicillin 187%, penicillin G 31%, cloxacillin 30%, dicloxacillin 44%, and oxacillin 14%) . The detection limits for ampicillin, calculated from the antibiotic concentration giving 30% binding inhibition, were 11.7 (Mab 3B5) and 16.6 ng ml-1 (Mab 1D1) . To prepare multi-immunoaffinity chromatography columns, Mab 1D1 and a previously described antibody against cloxacillin (Mab 1F7) were each coupled to CNBr activated sepharose . The capacity of the resulting immunosorbents was approximately 6.6 and 5.4 micrograms ml-1 gel for ampicillin and cloxacillin, respectively . Recoveries of amoxicillin, ampicillin, cloxacillin, dicloxacillin, penicillin G and oxacillin (in buffer solutions) from the produced immunoaffinity columns were in the range from 67 to 100%. Acad Emerg Med, 1999 Jul, 6(7), 682 - 7 Glottal patency during experimental cortical seizures in piglets; Leaming JM et al.; OBJECTIVE: Systemically-induced seizures produce glottal airflow obstruction in anesthetized pigs, resulting in hypercapnia and respiratory acidosis . Cortically-induced seizures may be more representative of human seizure disorders . The purpose of this study was to describe glottal area patency (GAP) in piglets during cortically-induced seizures . METHODS: Nineteen spontaneously breathing, lightly anesthetized (alphaxalone-alphadolone IV) piglets (aged 10 +/- 2 days) were instrumented for recording nasal airflow, subglottic pressure, and electrocorticogram . Glottal visualization was achieved supraglottically using a 1.2-mm fiberoptic scope inserted through the thyrohyoid membrane . Following baseline-control, hypoxic-rebreathing, and new baseline recordings, seizures were induced using subcortical injections of crystalline penicillin G (100,000 units/ injection) . Five consecutive-breath representative epochs were digitized from baseline-control, hypoxic-rebreathing, and seizure conditions . For each breath, GAP was measured at the onset of inspiratory pressure, peak of inspiratory effort (Ip), and onset of expiration . RESULTS: The piglets were physiologic at baseline-control and new baseline conditions, and showed expected increases in ventilation and GAP during rebreathing experiments . GAP was maximum at Ip under baseline and rebreathing conditions, but was significantly decreased and airway resistances were increased during seizure conditions (p < 0.05, ANOVA) . CONCLUSIONS: Generalized seizure activity results in reduced GAP at the peak of inspiratory effort . Increased work of breathing during seizures is created by direct mechanical obstruction at the level of the larynx. Rev Alerg Mex, 1999 May-Jun, 46(3), 78 - 82 {Effect of naproxen sodium on serum concentrations of IL-1, IL-6, and TNF in patients with acute, purulent pharyngo-tonsillitis}; Salgado Legorreta C et al.; BACKGROUND: Cytokines produced by macrophages and neutrophils participate in the host's defense mechanism; in infectious processes . Interleukins such as LI-1, IL-6, and tumor necrosis factor (TNF) also have a role in these acute phase mechanisms . OBJECTIVE: Assess the effect of sodium naproxen on the serum concentration of IL-1, IL-6 and TNF in acute infectious process . MATERIAL A METHODS: Eighteen patients with clinical diagnosis of acute purulent pharyngitis and/or tonsillitis were randomly assigned to one of two groups of 9 patients each . RESULTS: The patients in the first group received sodium naproxen and those in the other group received placebo . Patients of both groups received procaine G penicillin . Patients in the group receiving sodium naproxen showed improvement of the infectious and febrile syndromes within 72 hours . Patients receiving placebo showed signs and symptoms; of the infections and febrile syndromes for more than three days . DISCUSSION: Patients receiving treatment with sodium naproxen had a statistically significant reduction of the serum concentration of IL-1b as compared to basal and 72 h measurements; there were also statistically significant differences with respect to patients receiving placebo . IL-6 and TNF did not change in any of the groups . These results show that serum IL-1b levels dropped in both groups with a more striking reduction in the group receiving sodium naproxen , that also showed a faster improvement of the symptoms. Rinsho Shinkeigaku, 1999 May, 39(5), 570 - 2 {A case of Lyme disease with the triad of neurologic manifestations (meningitis, radiculoneuritis, facial nerve palsy) and dermatitis of the nail roots}; Murakami I et al.; A 23-year-old man experienced dysesthesia of the distal parts of four limbs and spilling of drinking water from the right corner of his mouth . He also experienced low grade fever, headache, and nausea . Neurological examination showed weakness of the right frontal, orbicularis oculi and orbicularis oris muscles, atrophy and weakness of the interosseous muscles in both upper limbs and dysesthesia in the distal parts of four limbs . Lasegue's sign was also positive on the left side . Examination of CSF showed lymphocytic pleocytosis and an increase of total protein . Serum antibody against Borrelia garinii was positive as determined by ELISA . The patient was diagnosed as a case of Lyme disease . His symptoms were not alleviated by administration of ceftriaxone (2 g/day) for 15 days, and dermatitis of nail roots appeared . Following administration of penicillin G at a high dose (12 million units/day) for 20 days, both neurologic symptoms and dermatitis were alleviated . Since B . garinii was reported to move preferentially toward the sites of low temperature, dermatitis of nail roots may be one of the characteristic features of Lyme disease caused by B . garinii. Biochemistry, 1999 Jul 27, 38(30), 9570 - 6 X-ray structure of the Asn276Asp variant of the Escherichia coli TEM-1 beta-lactamase: direct observation of electrostatic modulation in resistance to inactivation by clavulanic acid; Swaren P et al.; The clinical use of beta-lactam antibiotics combined with beta-lactamase inactivators, such as clavulanate, has resulted in selection of beta-lactamases that are insensitive to inactivation by these molecules . Therefore, therapeutic combinations of an enzyme inactivator and a penicillin are harmless for bacteria harboring such an enzyme . The TEM beta-lactamase variants are the most frequently encountered enzymes of this type, and presently, 20 variants are designated as inhibitor-resistant TEM ("IRT") enzymes . Three mutations appear to account for the phenotype of the majority of IRT enzymes, one of them being the Asn276Asp substitution . In this study, we have characterized the kinetic properties of the inhibition process of the wild-type TEM-1 beta-lactamase and of its Asn276Asp variant with the three clinically used inactivators, clavulanic acid (clavulanate), sulbactam, and tazobactam, and we report the X-ray structure for the mutant variant at 2.3 A resolution . The changes in kinetic parameters for the interactions of the inhibitors with the wild-type and the mutant enzymes were more pronounced for clavulanate, and relatively inconsequential for sulbactam and tazobactam . The structure of the Asn276Asp mutant enzyme revealed a significant movement of Asp276 and the formation of a salt bridge of its side chain with the guanidinium group of Arg244, the counterion of the inhibitor carboxylate . A water molecule critical for the inactivation chemistry by clavulanate, which is observed in the wild-type enzyme structure, is not present in the crystal structure of the mutant variant . Such structural changes favor the turnover process over the inactivation chemistry for clavulanate, with profound phenotypic consequences . The report herein represents the best studied example of inhibitor-resistant beta-lactamases. Antonie Van Leeuwenhoek, 1999 Jan-Feb, 75(1-2), 145 - 54 Metabolic control analysis of the penicillin biosynthetic pathway: the influence of the LLD-ACV:bisACV ratio on the flux control; Theilgaard HA et al.; An extended kinetic model for the first two steps of the penicillin biosynthetic pathway in Penicillium chrysogenum is set up . It includes the formation and reduction of the dimer bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV) from the first pathway intermediate LLD-ACV and their parallel inhibition of the enzyme ACV synthetase (ACVS) . The kinetic model is based on Michaelis-Menten type kinetics, with non-competitive inhibition of the ACVS by both LLD-ACV and bisACV, and competitive inhibition of the isopenicillin N synthetase (IPNS) by glutathione . The inhibition constant of LLD-ACV, KACV is determined to be 0.54 mm . With the kinetic model metabolic control analysis is performed to identify the distribution of rate-control in the pathway at all ratios of LLD-ACV:bisACV . It is concluded that the flux control totally resides at the IPNS . This is a result of the regulation of the ACVS by both the LLD-ACV and bisACV demanding a higher flux through the IPNS enzyme to alleviate their inhibition . The measurement of an intracellular ratio of LLD-ACV:bisACV to be in the range of 1-2 moles per moles emphasises the importance of a fast conversion of LLD-ACV to IPN, and accumulation of LLD-ACV above the K(m)-value of the IPNS should therefore be avoided. Med Mycol, 1999 Jun, 37(3), 163 - 8 The in vitro isolation of Blastomyces dermatitidis from a woodpile in north central Wisconsin, USA; Baumgardner DJ et al.; Environmental isolations of the dimorphic fungus, Blastomyces dermatitidis, the aetiologic agent of blastomycosis are rare, and have usually involved animal inoculation techniques . We report the in vitro isolation of B . dermatitidis from a woodpile in November 1997, from a private property in a highly endemic area of north central Wisconsin, USA . The woodpile was 73 m from the Wisconsin River and 5 m from a kennel which had housed nine dogs over the past 14 years, four of which had been diagnosed with blastomycosis . One of 19 samples from the property yielded B . dermatitidis after 37 degrees C incubation in a neutral aqueous solution of allantoin, Tween-80, potassium phosphate, magnesium sulphate, penicillin and streptomycin followed by plating on yeast-extract phosphate agar at 20 degrees C . Refinements of this technique may help further elucidate the ecological niche of B . dermatitidis. J Colloid Interface Sci, 1999 Aug 15, 216(2), 270 - 275 Influence of Molecular Structure on the Ideality of Mixing in Micelles Formed in Binary Mixtures of Surface-Active Drugs; Taboada P et al.; The influence of the structure of the hydrophobic group on the ideality of mixing in binary mixtures of surface active molecules has been investigated using combinations of amphiphilic penicillins . Critical concentrations (cc) of the binary mixtures of these anionic surfactants were determined by conductivity measurements as a function of the composition . The nonideality of mixing was evaluated using a regular solution approximation and expressed in terms of the interaction parameter, beta . Mixing in micelles formed in binary mixtures of the structurally similar penicillins cloxacillin, dicloxacillin, and flucloxacillin was ideal (beta = 0) . In contrast, the combination of either cloxacillin or dicloxacillin with the penicillin nafcillin produced mixed micelles in which the mixing deviated from ideality (beta = +0.1 to +0.2) . The positive values of beta for these systems indicated negative synergism between components of the mixtures that may be a consequence of the marked structural differences between the hydrophobic groups of these drugs . The composition of the mixed micelles was derived from the cc data by application of a theoretical treatment based on excess thermodynamic quantities . Zh Vyssh Nerv Deiat Im I P Pavlova, 1999 May-Jun, 49(3), 471 - 82 {The behavioral changes and the EEG of the rat brain induced by hyperactivation of the amygdaloid body under noradrenergic deficiency}; Pankova NB et al.; The brain NA deficit was produced by bilateral injection of 6-OHDA into the locus coeruleus . Three weeks later amygdala hyperactivation was initiated by local penicillin injection (1% solution, 0.5 mcl) . Saline in the same volume was used in control groups in both cases . It was shown that the decrease in NA level facilitated the development of epileptiform activity in rat brain and appearance of immobility-related high-voltage spindles during waking . The amygdala hyperactivation after NA deprivation resulted in a decrease in exploratory activity and disruption of the reaction to novelty . The delta component of the EEG power spectrum increased . The alterations appeared 1-2 weeks after the experimental procedure and became more pronounced towards the end of the third week. J R Army Med Corps, 1999 Jun, 145(2), 107 - 8 The history of colonic surgery in war; Edwards DP; The history of laparotomy for penetrating abdominal wounds is reviewed from its origins in the sixteenth century to the large series from World War II . It is shown that the mass casualties of World War I allowed the management of organ specific injuries to be studied . This review concentrates upon one of the most contentious topics in war surgery-the management of colonic wounds, and suggests that post-war surgical practice may have been influenced too strongly by experiences from the pre-Penicillin era. J Biol Chem, 1999 Jul 30, 274(31), 21853 - 60 The crystal structure of a penicilloyl-serine transferase of intermediate penicillin sensitivity . The DD-transpeptidase of streptomyces K15; Fonze E et al.; The serine DD-transpeptidase/penicillin-binding protein of Streptomyces K15 catalyzes peptide bond formation in a way that mimics the penicillin-sensitive peptide cross-linking reaction involved in bacterial cell wall peptidoglycan assembly . The Streptomyces K15 enzyme is peculiar in that it can be considered as an intermediate between classical penicillin-binding proteins, for which benzylpenicillin is a very efficient inactivator, and the resistant penicillin-binding proteins that have a low penicillin affinity . With its moderate penicillin sensitivity, the Streptomyces K15 DD-transpeptidase would be helpful in the understanding of the structure-activity relationship of this penicillin-recognizing protein superfamily . The structure of the Streptomyces K15 enzyme has been determined by x-ray crystallography at 2.0-A resolution and refined to an R-factor of 18.6% . The fold adopted by this 262-amino acid polypeptide generates a two-domain structure that is close to those of class A beta-lactamases . However, the Streptomyces K15 enzyme has two particular structural features . It lacks the amino-terminal alpha-helix found in the other penicilloyl-serine transferases, and it exhibits, at its surface, an additional four-stranded beta-sheet . These two characteristics might serve to anchor the enzyme in the plasma membrane . The overall topology of the catalytic pocket of the Streptomyces K15 enzyme is also comparable to that of the class A beta-lactamases, except that the Omega-loop, which bears the essential catalytic Glu(166) residue in the class A beta-lactamases, is entirely modified . This loop adopts a conformation similar to those found in the Streptomyces R61 DD-carboxypeptidase and class C beta-lactamases, with no equivalent acidic residue. Pathol Biol (Paris), 1999 May, 47(5), 469 - 73 {The Pneumococcus Observatory of the Central Region, from June 1, 1997 to May 31, 1998}; Cattier B et al.; 714 pneumococcus were listed from 14 laboratories between the 1 June 1997 and the 31 May 1998 . Data capture was done on Epi info software and concerned age, file number, consultation/hospitalization, sample type, susceptibility to oxacilline (5 micrograms), the results of the E-test for penicillin G, amoxicillin, cefotaxime and the results of the routine disk diffusion susceptibility method . Strains with reduced susceptibility to penicillin G (PRSP) were collected by the coordinating center to perform MICs by the reference method of agar dilution and serotyping . Over 714 strains, 45.7% of the samples originated from lungs, followed by 22% for blood samples, 14% for ear pus and 2.3% for CSF . 34% of the patients were female . 36.7% were children under 16 (57.8% PRSP) and 63.3% were adults (41% PRSP) . 338 strains (47.3%) were determined as PRSP and 293 of them were studied by the coordinating center . 81 of the 293 PRSP (27.7%) were resistant et 212 (72.3%) were intermediate to penicillin G . 81% of the PRSP studied had a CMI value for penicillin G within +/- 1 log2 dilution . 20 strains of PRSP were resistant for amoxicillin (6.8% of the PRSP) and two (0.7% of the PRSP) for cefotaxime . 289 serotyping were done, most met serotypes were 23 (25%), 14 (23%) . The least met was 15 (2.4%) . These results let assess the epidemiology of pneumococcus in our region. Brain Res, 1999 Jul 31, 836(1-2), 156 - 63 Reduced signal complexity of intracellular recordings: a precursor for epileptiform activity? Widman G, Bingmann D, Lehnertz K, Elger CE. Recent studies have shown that time windowed extraction of nonlinear parameters like an effective correlation dimension from intracranially recorded EEG of epileptic patients often allows to detect and identify an unequivocal "pre-ictal phase" preceding an epileptic seizure . In another study, however, such an anticipation could not be made . These conflicting findings may indicate that observed changes in nonlinear parameters probably depend on the type of elementary mechanisms underlying epileptic processes and/or the spatial distribution of neurons primarily involved in generation of epileptiform discharges . To test the existence of such dependencies, the transition from normal to epileptiform activity (EA) of CA3-neurons in hippocampal slices was analyzed in four epilepsy models, using a time windowed computation of an effective correlation dimension . Indeed, in xanthine and penicillin models, signal complexity in intracellular recordings was reduced before manifestation of paroxysmal depolarization shifts (PDS), whereas a preceding loss of complexity was missing in low-magnesium and veratridine models . These findings indicate that interictal-like EA is predictable only in some epilepsy models . Eur Respir J, 1999 May, 13(5), 1189 - 91 Occupational asthma induced by cephalosporins; Sastre J et al.; A 20-yr-old pharmaceutical worker who developed attacks of shortness of breath and wheezing 9 months after beginning work on a process in which cefadroxil powder was bottled or encapsulated will be described . Skin test with cefaxodril was negative . Baseline spirometry and methacholine inhalation test were normal . A controlled bronchial challenge test was carried out in a closed-circuit system with assessment of respirable dust concentration . Exposure to cefadroxil powder at a mean concentration of 10 mg x m(-3) for 10 min elicited an isolated immediate asthmatic response, but no response was observed to control challenge with lactose . Single-blind oral challenge test with amoxicillin up to 500 mg was well tolerated, whereas the oral challenge with cephalexin (25 mg) elicited an immediate asthmatic response . This patient had developed occupational asthma caused by inhalation of cefadroxil as confirmed by specific inhalation test . Since she tolerated oral amoxicillin, a synthetic penicillin with the side-chain identical to that of cefadroxil, it seems that she may be sensitized to the dihydrothiazine ring of cephalosporins. Wien Klin Wochenschr, 1999 May 21, 111(10), 410 - 3 {Congenital syphilis: late diagnosis in spite of screening}; Vecsei AK et al.; We report the case of an infant in whom congenital syphilis was diagnosed at the age of 5 weeks . The case is remarkable because of (a) the negative venereal disease laboratory test from the cord blood, (b) the incidental diagnosis of the disease in the fifth week of life, (c) pneumonia alba being one of the symptoms, (d) the occurrence of a mild Jarisch-Herxheimer reaction after initiation of penicillin therapy and (e) the successful treatment of infection related anaemia with recombinant human erythropoietin. Laryngorhinootologie, 1999 May, 78(5), 254 - 8 {Stage 3 syphilis of the mouth cavity}; Kirwald H et al.; BACKGROUND: Untreated syphilis or lues is a chronic infectious disease . It is caused by treponema pallidum, which is most commonly transmitted by sexual contact and occasionally by blood transfusion or by intrauterine infection . If the disorder is not treated, its clinical course can be chronic, persisting for decades . During this time, a variety of morphological signs occur depending on the stage of the disease . CASE REPORT: We describe a case of tertiary syphilis in the oropharynx with a defect of the soft palate . In a 37-year-old woman, the first symptom was a dryness of the throat followed by a feeling of foreign body in the palate area . The patient had a history of sexual contact with a man who had had syphilis ten years ago, and our initial suspicion was confirmed by a final diagnosis of tertiary syphilis . Signs of primary or secondary syphilis were not observed . RESULTS: In the course of diagnostic procedures both further manifestatons of syphilis and other infectious or malignant causes were excluded . The serological results showed a typical constellation of Treponema and non-Treponema serum reactions . The histopathological examination of an exploratory excision from the soft palate showed granulomatous changes with peripheral participation of plasma cells . We initiated appropriate antibiotic therapy, using clemizole penicillin G over a period of 21 days, which induced healing of the soft palate . CONCLUSIONS: A defect of the soft palate was diagnosed as a very rare sign of tertiary syphilis. J Korean Med Sci, 1999 Jun, 14(3), 315 - 8 Endobronchial actinomycosis simulating endobronchial tuberculosis: a case report; Lee SH et al.; We report a case of a 70-year-old woman who presented with mild exertional dyspnea and cough . Fiberoptic bronchoscopic findings revealed an endobronchial polypoid lesion with stenotic bronchus . The lesion was very similar to endobronchial tuberculosis . Histologic examination of the biopsy specimen demonstrated Actinomyces infection . There was a clinical response to intravenous penicillin therapy . Primary endobronchial actinomycosis must be considered in the differential diagnosis of an endobronchial lesion, especially endobronchial tuberculosis in Korea. Biotechnol Bioeng, 1999 Aug 20, 64(4), 383 - 91 Electrically immobilized enzyme reactors: bioconversion of a charged substrate . Hydrolysis Of penicillin G by penicillin G acylase; Bossi A et al.; The possibility of using the multicompartment immobilized enzyme reactor (MIER) in presence of a charged substrate is here explored . Penicillin G acylase is used to convert penicillin G (a free acid, with a pK of 2.6) into two charged products: phenyl acetic acid (PAA, with a pK of 4.2) and 6-aminopenicillanic acid (6-APA, a zwitterion with a pI of 3.6) . The enzyme is trapped by an isoelectric mechanism in a chamber of the electrolyzer delimited by a pI 5.0 and a pI 9.0 amphoteric, isoelectric membranes . Under normal operating conditions (continuous substrate feeding in the presence of an electric field), only a low substrate conversion can be achieved, due to rapid electrophoretic transport of unreacted penicillin G out of the reaction chamber towards the anode . Excellent conversion rates (>96%) are obtained under a "doubly-discontinuous" operation mode: a time-lapse substrate feeding, accompanied by short times (4-8 min) of electric field interruption . The product of interest (6-APA, a precursor of semisynthetic penicillins), by virtue of its amphoteric nature, is trapped in a chamber delimited by a pI 3.5 membrane and a pI 5.5 membrane, adjacent to the reaction chamber on its anodic side . The other contaminant product (PAA) first accumulates in the same chamber and then progressively vacates it to collect in the anodic reservoir, leaving behind a pure 6-APA solution . In this operation mode, vanishing amounts of unreacted substrate (penicillin G) leave the reaction chamber to contaminate the adjacent, anodic chambers . A novel class of zwitterionic buffers is additionally reported, able to cover very thoroughly any pH value along the pH 3-10 interval: polymeric, zwitterionic buffers, synthesized with the principle of the Immobiline (acrylamido weak acids and bases) chemicals . Enhanced enzyme reactivity is found in this macromolecular buffers as compared to conventional ones . J Immunol, 1999 Jul 15, 163(2), 1053 - 9 Highly Th2-skewed cytokine profile of beta-lactam-specific T cells from nonatopic subjects with adverse drug reactions; Brugnolo F et al.; A positive lymphocyte transformation test to beta-lactams (beta-L) was found in 12 of 29 subjects with adverse drug reaction (ADR) to beta-L, irrespective of either the type of clinical manifestation or the presence of specific serum IgE . Short-term T cell lines specific for penicillin G, amoxicillin, and ampicillin could be generated only from subjects with ADR (eight with positive and one with negative lymphocyte transformation test), while streptokinase and Dermatophagoides pteronyssinus group 1 (Der p 1)-specific T cells were obtained from all these subjects, from 7 atopic Der p-sensitive donors without history of ADR and 17 healthy nonatopic donors . Streptokinase-specific T cells from all subjects showed intracellular expression of IFN-gamma with poor or no IL-4, whereas Der p 1-specific T cells exhibited IFN-gamma but low or no IL-4 expression in nonatopics, and remarkable IL-4 expression in atopic donors . By contrast, all penicillin G-, ampicillin-, and amoxicillin-specific short-term T cell lines showed high intracellular expression of IL-4, IL-5, and IL-13, but poor or no expression of IFN-gamma, thus exhibiting a clear-cut Th2 profile . Accordingly, most penicillin G-specific T cell clones derived from two subjects with ADR released high concentrations of IL-4 alone or IL-4 and IFN-gamma . These data suggest that cytokines produced by Th2 cells play an important role in all beta-L-induced ADR, even when late clinical manifestations occur and an IgE-mediated mechanism is apparently indemonstrable. Pediatrics . 1999 Jul;104(1):e4. Pituitary gland gumma in congenital syphilis after failed maternal treatment: a case report; Benzick AE et al.; A preterm, very low birth weight infant was born to a mother with early latent syphilis who was treated 10 days and 3 days before delivery with 2.4 mU of benzathine penicillin . The infant had clinical, laboratory, and radiographic abnormalities consistent with congenital syphilis, ie, a Venereal Disease Research Laboratory test titer that was fourfold greater than was the maternal titer, hepatosplenomegaly, abnormal liver function tests, pneumonitis, osteochondritis of the long bones, and cerebrospinal fluid (CSF) examination showing a reactive Venereal Disease Research Laboratory test, pleocytosis, and elevated protein content . The infant died on the third day of life, and an autopsy revealed an evolving gumma of the anterior pituitary . Immunoglobulin M immunoblotting of serum and CSF was positive, and polymerase chain reaction detected Treponema pallidum DNA in endotracheal aspirate and CSF . This case highlights the pathologic abnormalities observed in congenital syphilis and focuses on the rare finding of an evolving anterior pituitary gumma . Furthermore, it documents the failure of maternal syphilis treatment during the last 4 weeks of pregnancy to cure fetal infection and supports the recommendation that all infants born to mothers with syphilis treated during the last 4 weeks of pregnancy should receive penicillin therapy. Antimicrob Agents Chemother, 1999 Jul, 43(7), 1803 - 4 Sensitivity of amoxicillin-resistant Helicobacter pylori to other penicillins; Dore MP et al.; The sensitivities to penicillins and to a penicillin and beta-lactamase inhibitor combination agent were determined for Helicobacter pylori strains that were sensitive, moderately resistant, or highly resistant to amoxicillin . All strains were resistant to nafcillin and oxacillin . Moderately resistant strains showed an intermediate zone of inhibition to ticarcillin, mezlocillin, piperacillin, and amoxicillin-clavulanic acid . High-level resistance was associated with the smallest zone size for all penicillins tested. Kekkaku, 1999 May, 74(5), 447 - 52 {A study of beta-lactamase activity of mycobacteria and clinical trial of penicillin/beta-lactamase inhibitor combinations in the treatment of drug-resistant Mycobacterium tuberculosis}; Nakagawa Y et al.; Beta-lactamase activity was determined using a nitrocefin disc method on 34 Mycobacterium tuberculosis (M . tuberculosis) strains and 13 nontuberculous mycobacteria strains . In the 34 M . tuberculosis strains, 23 strains showed beta-lactamase activity . In 10 Mycobacterium avium complex strains, no beta-lactamase activity was detected . In the Mycobacterium chelonae strains, all three strains examined showed strong beta-lactamase activity . No correlation was found between beta-lactamase activity and resistance to anti-tuberculous chemotherapeutic agents . Four patients who were persistently positive for multi-drug-resistant M . tuberculosis (MDR-TB) on sputum and positive in beta-lactamase activity, were treated with penicillin/beta-lactamase inhibitor combinations . In two cases, the trials were discontinued because of diarrhea; the trials were continued in the remaining two for four months, but the MDR-TB was positive during the course of the therapy . Effectiveness of the therapy with penicillin/beta-lactamase inhibitor combinations against M . tuberculosis was obscure, although many of M . tuberculosis examined showed beta-lactamase activity. J Bacteriol, 1999 Jul, 181(13), 3981 - 93 Escherichia coli mutants lacking all possible combinations of eight penicillin binding proteins: viability, characteristics, and implications for peptidoglycan synthesis; Denome SA et al.; The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall . Much is known about the biochemistry of these proteins, but little is known about their biological roles . To better understand the contributions these proteins make to the physiology of Escherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination . The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two res sites from plasmid RP4 . Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E . coli via lambda phage transduction and selecting for kanamycin-resistant recombinants . Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bp res site . These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs . In addition, the dacD gene was deleted from two septuple mutants, creating strains lacking eight genes . The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal . Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated . Furthermore, when both PBPs 2 and 3 were inactivated by the beta-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active . These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects . In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism. Aliment Pharmacol Ther, 1999 Jul, 13(7), 869 - 73 Comparison of two 3-day Helicobacter pylori eradication regimens with a standard 1-week regimen; Grimley CE et al.; BACKGROUND: The duration of Helicobacter pylori eradication regimens has decreased to 1 week with cure rates of over 90% . This can be attributed to the use of triple drug regimens including potent inhibitors of gastric acid secretion and clarithromycin . There is no theoretical reason why shorter regimens should not be possible . AIM: To compare two 3-day, low-dose, twice daily regimens with 1 week of omeprazole 20 mg b.d., clarithromycin 250 mg b.d., and metronidazole 400 mg b.d . (OCM) METHODS: Outpatients referred for gastroscopy were screened by biopsy urease test . H . pylori-positive patients were randomized to receive either lansoprazole 30 mg b.d., tri-potassium dicitrato bismuthate one tablet b.d., clarithromycin 250 mg b.d., and amoxycillin 1 g b.d . for 3 days (LTdbCA), or ranitidine bismuth citrate 400 mg b.d., clarithromycin 250 mg b.d . and amoxycillin 1 g b.d . for 3 days (RbcCA) or omeprazole 20 mg b.d., clarithromycin 250 mg b.d . and metronidazole 400 mg b.d . for 1 week (OCM) . They were not pre-treated with a gastric acid inhibitor . After 8 weeks, H . pylori status was assessed by 13C urea breath test . RESULTS: 974 out of 1114 patients referred for gastroscopy were screened by biopsy urease test . 140 patients were not screened either because they were anticoagulated or for technical reasons . 334 patients were H . pylori-positive: 154 were excluded mostly because of allergy to penicillin and personal reasons but 180 were randomized to treatment All regimens were well tolerated . For LTdbCA (n=60), RbcCA (n=59), and OCM (n=61) the H . pylori cure rates (95% CI) were 23% (12-34), 14% (5-23) and 87% (79-95), respectively, using intention-to-treat analysis and 25% (14-36), 15% (6-24) and 88% (80-96), respectively, if analysed per protocol . OCM was significantly superior to LTdbCA and RbcCA (P < 0.001) but there was no significant difference between regimens LTdbCA and RbcCA . CONCLUSIONS: OCM is an extremely effective H . pylori eradication regimen . The 3-day regimens tested both have poor cure rates . Pre-treatment with a proton pump inhibitor, higher doses or more frequent dosing may be necessary to increase the cure rate of short duration regimens . However, this could make them less acceptable than the H . pylori eradication regimens currently available. Aliment Pharmacol Ther, 1999 Jun, 13(6), 769 - 74 A new quadruple therapy for Helicobacter pylori: influence of resistant strains on treatment outcome; Okada M et al.; BACKGROUND: There have been no reports concerning the efficacy and safety of a 1-week quadruple therapy regimen of omeprazole, amoxycillin, roxithromycin and metronidazole for Helicobacter pylori infections and the impact of primary resistance on the eradication rate . METHODS: One hundred and sixty-nine consecutive patients with peptic ulcer disease as well as gastritis with biopsy-proven H . pylori infection were entered into an open study of omeprazole 20 mg o.m., amoxycillin 500 mg t.d.s., roxithromycin 150 mg b.d., and metronidazole 250 mg t.d.s . Helicobacter pylori status was determined by urease test, histology and culture . Susceptibility to amoxycillin, metronidazole and roxithromycin was determined by the E-test . RESULTS: H . pylori was eradicated in 155 out of 169 (92%; 95% CI 88-96%) by intention-to-treat analysis, and in 155 out of 163 (95%; 95% CI 92-98%) by per protocol analysis . The prevalence of primary resistance against amoxycillin, roxithromycin and metronidazole was 2 out of 166 (1%), 16 out of 166 (10%) and 27 out of 166 (16%), respectively . H . pylori was eradicated in 25 out of 27 (93%) patients with metronidazole-resistant strains compared with 130 out of 136 (96%) in patients with metronidazole-sensitive strains of H . pylori . It was eradicated in 15 out of 16 (94%) patients with roxithromycin-resistant strains while in 140 out of 147 (95%) patients with roxithromycin-sensitive strains of H . pylori, and in two out of two (100%) patients with amoxycillin-resistant stains compared with 153 out of 161 (95%) in patients with amoxycillin-sensitive strains . H . pylori was eradicated in three out of four (75%) patients with double resistance against metronidazole and roxithromycin compared with 152 out of 159 (96%) patients with sensitive strains to metronidazole and or roxithromycin . None of these differences were statistically significant . Severe side-effects were found in only one out of 169 patients-anaphylaxis due to penicillin . CONCLUSIONS: The 1-week quadruple therapy with omeprazole, amoxycillin, metronidazole and roxithromycin was found to eradicate H . pylori in over 90% of all patients . This regimen was also found to be beneficial for patients with pre-treatment resistant strains to metronidazole, roxithromycin or amoxycillin, and was observed to be safe and well-tolerated. J Antimicrob Chemother, 1999 Jan, 43(1), 23 - 9 Aspartic acid for asparagine substitution at position 276 reduces susceptibility to mechanism-based inhibitors in SHV-1 and SHV-5 beta-lactamases; Giakkoupi P et al.; In SHV-type beta-actamases, position 276 (in Ambler's numbering scheme) is occupied by an asparagine (Asn) residue . The effect on SHV-1 beta-lactamase and its extended-spectrum derivative SHV-5 of substituting an aspartic acid (Asp) residue for Asn276 was studied . Mutations were introduced by a PCR-based site-directed mutagenesis procedure . Wild-type SHV-1 and -5 beta-lactamases and their respective Asn276-->Asp mutants were expressed under isogenic conditions by cloning the respective bla genes into the pBCSK(+) plasmid and transforming Escherichia coli DH5alpha . Determination of IC50 showed that SHV-1(Asn276-->Asp), compared with SHV-1, was inhibited by 8- and 8.8-fold higher concentrations of clavulanate and tazobactam respectively . Replacement of Asn276 by Asp in SHV-5 beta-lactamase caused a ten-fold increase in the IC50 of clavulanate; the increases in the IC50s of tazobactam and sulbactam were 10- and 5.5-fold, respectively . Beta-lactam susceptibility testing showed that both Asn276-->Asp mutant enzymes, compared with the parental beta-lactamases, conferred slightly lower levels of resistance to penicillins (amoxycillin, ticarcillin and piperacillin), cephalosporins (cephalothin and cefprozil) and some of the expanded-spectrum oxyimino beta-lactams tested (cefotaxime, ceftriaxone and aztreonam) . The MICs of ceftazidime remained unaltered, while those of cefepime and cefpirome were slightly elevated in the clones producing the mutant beta-lactamases . The latter clones were also less susceptible to penicillin-inhibitor combinations . Asn276-->Asp mutation was associated with changes in the substrate profiles of SHV-1 and SHV-5 enzymes . Based on the structure of TEM-1 beta-lactamase, the potential effects of the introduced mutation on SHV-1 and SHV-5 are discussed. Infect Immun, 1999 Jul, 67(7), 3631 - 6 Correlation of immunity in experimental syphilis with serum-mediated aggregation of Treponema pallidum rare outer membrane proteins; Lewinski MA et al.; We have previously shown by freeze-fracture electron microscopy that serum from infection-immune syphilitic rabbits aggregates the low-density membrane-spanning Treponema pallidum rare outer membrane proteins (TROMPs) . The purpose of this study was to determine if a relationship could be demonstrated between acquired immunity in experimental rabbit syphilis, serum complement-dependent treponemicidal antibody, and antibody directed against TROMPs as measured by the aggregation of TROMP particles . Three groups of T . pallidum-infected rabbits were treated curatively with penicillin at 9 days, 30 days, and 6 months postinfection to generate various degrees of immunity to challenge reinfection . Sera from rabbits completely susceptible to localized and disseminated reinfection possessed a low titer of treponemicidal antibody (</=1:1 in killing >/=50% of a treponemal suspension) and showed a correspondingly low level of TROMP aggregation (16.5% of the total number of outer membrane particles counted) similar to normal serum controls (13 . 4%); the number of particles within these aggregates never exceeded three . Sera from partially immune rabbits, which were susceptible to local reinfection but had no evidence of dissemination, showed an increase in the titer of treponemicidal antibody (1:16) compared to the completely susceptible group (</=1:1) . Although no significant increase was observed in the total number of TROMP particles aggregated (18.9%) compared to the number in controls (13.4%), approximately 15% of these aggregates did exhibit a significant increase in the number of particles per aggregate (4 to 5 particles) compared to controls (</=3 particles), indicating a measurable increase in anti-TROMP antibody . Finally, sera from rabbits completely immune to both local and disseminated reinfection possessed both high titers of treponemicidal antibody (1:128) and significant aggregation of TROMP (88.6%); approximately 50% of these aggregates contained four to six particles . The results indicate that complete immunity in experimental rabbit syphilis correlates with antibody that kills T . pallidum and aggregates TROMPs, suggesting that TROMPs are molecules which contribute to the development of acquired immunity. Microbiology, 1999 May, 145 ( Pt 5), 1015 - 22 Varying division planes of secondary constrictions in spheroidal Escherichia coli cells; Zaritsky A et al.; Planes of successive divisions in Escherichia coli have been proposed to be either parallel or perpendicular to each other, restricted to one or two dimensions . To test the hypothesis that divisions can occur in planes alternating in three dimensions, a method was developed to generate cells with secondary constrictions during growth in suspension . The method involves a combination of thymine limitation (to manipulate chromosome replication rate) and mecillinam treatment (to inhibit penicillin-binding protein 2) . The former modifies timing of terminations, the latter results in spheroidal cells . Such cells displayed secondary constrictions after adding deoxyguanosine (accelerating replication rate), thus temporarily enhancing division signals . The successive constrictions were seen to develop in planes that were tilted relative to each other, and in positions related to those of the nucleoids, visualized by staining with DAPI (4',6-diamidino-2-phenylindole dihydrochloride hydrate) . Visualizing cell envelopes with FM 4-64 by confocal scanning laser microscopy supported the conclusion that planes of successive divisions can alternate in three dimensions. J Appl Physiol, 1999 Jun, 86(6), 2052 - 8 Effects of experimental cortical seizures on respiratory motor nerve activities in piglets; Terndrup TE et al.; Airway obstruction at the level of the larynx causes respiratory insufficiency during experimental seizures in spontaneously breathing, anesthetized piglets (T . E . Terndrup and W . E . Fordyce, Pediatr . Res., 38: 61-66, 1995) . To investigate further the neural mechanisms of this obstruction, the activities of the phrenic nerve (PH) and the recurrent laryngeal motor branches to the thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles were analyzed in 11 anesthetized, vagotomized, paralyzed, and ventilated piglets . After a control recording period, seizures were induced by subcortical penicillin G injections . Compared with baseline conditions, nerve activities became irregular during seizures . Extraneous TA bursts during PH activation were evident in all piglets during seizures . During ictal phases of seizures, the peak integrated activities of the PH and the expiratory component of the PCA, but not TA or inspiratory PCA activities, were significantly decreased compared with interictal phases . During seizures, a significant delay in the onset of the inspiratory component of PCA activation with respect to the onset of the PH was observed . This study helps to explain respiratory impairment during cortical seizures by providing evidence of impaired timing of activation of laryngeal dilator mechanisms and coordination with those activating the diaphragm . Cyclical PH inhibition during high-intensity cortical discharges may provide a secondary mechanism producing respiratory insufficiency during seizures. J Allergy Clin Immunol, 1999 Jun, 103(6), 1186 - 90 A diagnostic protocol for evaluating nonimmediate reactions to aminopenicillins; Romano A et al.; BACKGROUND: Maculopapular and urticarial rashes are nonimmediate manifestations common during aminopenicillin (AP) treatment, and the former often represent cell-mediated hypersensitivity . OBJECTIVES: We sought to determine the significance and incidence of skin test reactions to APs in adults reporting adverse reactions during therapy with these beta-lactams and, particularly, to evaluate the potential of patch tests, delayed-reading skin tests, and challenges in the diagnosis of nonimmediate reactions . METHODS: We used skin tests with penicilloylpolylysine, minor determinant mixture, benzylpenicillin, ampicillin, and amoxicillin, as well as patch tests with the last 3 drugs . We also performed in vitro assays for specific IgE and challenges with the suspect penicillin in subjects with nonimmediate reactions . RESULTS: Among the 144 patients reporting nonimmediate manifestations (mostly maculopapular rashes), delayed hypersensitivity was diagnosed in 62 on the basis of positive patch test and/or delayed intradermal test results and responses to challenges; negative reactions to challenges allowed us to reasonably exclude the possibility of allergy in 66 subjects, and the challenge confirmed that 1 patient had linear IgA bullous dermatosis . Definitive diagnoses could not be provided for the remaining 15 subjects, who had negative allergologic test results, because they did not consent to challenges . In 40 of 49 immediate reactors, a diagnosis of IgE-mediated hypersensitivity was made . CONCLUSIONS: Both patch and intradermal tests are useful in evaluating nonimmediate reactions to APs . Positive patch test and delayed intradermal responses together indicate delayed hypersensitivity . Intradermal testing appears to be more sensitive than patch testing, but the pattern of positive delayed intradermal test responses and negative patch test responses needs further investigation because of false-positive cases. Biotechnol Prog, 1999 May-Jun, 15(3), 439 - 45 Effect of SecB chaperone on production of periplasmic penicillin acylase in Escherichia coli; Chou CP et al.; The effect of SecB chaperone on production of periplasmic penicillin acylase (PAC) in Escherichia coli was investigated . It appears that formation of PAC required the function of SecB chaperone and the amount of SecB required was at a basal level . The secB mutant was defective in production of PAC, and the impairment could be complemented by extrachromosomally supplementing SecB in trans . The function of SecB might be primarily stabilizing the cytoplasmic PAC precursors . Overproduction of SecB chaperone usually resulted in an increase in the amount of PAC precursors without enhancing PAC activity . In addition, most of the PAC precursors were located in the periplasm, suggesting that formation of active PAC was likely limited by periplasmic processing steps. Br J Dermatol, 1999 May, 140(5), 925 - 30 Isolation of Borrelia afzelii from circumscribed scleroderma; Breier FH et al.; A 45-year-old man presented with circumscribed scleroderma (CS) on the extremities . Histology of lesional skin showed the typical manifestations of scleroderma including a perivascular and interstitial infiltrate of lymphocytes and plasma cells; in one of the biopsies spirochaetes could be detected . Despite treatment with penicillin, progression of CS was observed and spirochaetes were isolated from skin cultures obtained from active scleroderma lesions . These spirochaetes were identified as Borrelia afzelii by sodium dodecyl sulphate-polyacrylamide gel electrophoresis of outer surface proteins and polymerase chain reaction (PCR) analysis of their chromosome . After two courses with ceftriaxone the lesions stopped expanding and sclerosis of the skin was diminished . At this time cultures for spirochaetes and PCR of lesional skin for Borrelia afzelii DNA remained negative . The pathogenetic role of Borrelia afzelii in the development of CS is discussed. Allergol Immunopathol (Madr), 1999 Mar-Apr, 27(2), 104 - 11 {Round Table: Immunological urticaria mediated by IgE}; Eseverri JL et al.; Urticaria is characterized by the appearance of hives and pruritus . Those hives are formed by oedema and vasodilatation and they disappear when they are pressed on . The acute presentation is extremely common and affects between 10 and 20% of the population at a determined moment . In its simplest form, urticaria is envisioned to represent the same sort of wheal-and-flare reaction observed when histamine is injected into the skin . It produces erythema because of capillary vasodilatation, oedema because of increased permeability in capillary and pruritus secondary at local specific receptors stimulation . Angioedema is caused by the same pathologic alterations that occur in the deep dermis and subcutaneous tissue . Thus, an area involved with angioedema has swelling as the prominent manifestation and appearance of the skin itself may be normal . Due to reduced nerve supply in dermis, angioedema is associated with oppression and not pruritus . Immunoallergological study of urticaria and/or angioedema was requested in 133 cases from 648 from the first patient's visits to the surgery . It supposes a 20.52% . The family suspicion of etiology was food in 62 cases, chemical products in 39 cases, other factors (physical, stings, balloons and other manufactured products.) in 7 cases and 25 cases without a direct relation . Out of 100 children diagnosed of allergic urticaria-angioedema 67 was by food; the foods implicated in frequency order were: eggs and nuts, fruit, milk, vegetables, fish and shellfish . In second place, chemical products were responsible of urticaria in 12 children; five of them were positive in diagnosed proof (prick, oral challenge) for penicillin and amoxicillin, both from beta-lactamic group; two of them had and adverse reaction to anesthetic agents; other two cases were after administration of vaccination and due to tetanus toxin; and three cases were due to aspirin, confirmed by oral provocation test . In 10 children the etiological agent was latex . Other etiologies were: three cutaneous reactions after stings (two by wasps and one by mosquito) three reactions due to spices (paprika, cumin, anise, mustard) and two reactions caused by manufactures products containing additives as yellow-orange. Int J Health Plann Manage, 1999 Jan-Mar, 14(1), 41 - 56 A description of outpatient drug use in rural China: evidence of differences due to insurance coverage; Dong H et al.; This paper describes the effects of health financing systems (insurance) on outpatient drug use in rural China . 1320 outpatients were interviewed (exit interview) in the randomly selected county, township and village health care facilities in five counties in three provinces of central China . The interview was face to face . Questions were asked by a trained interviewer and were answered by patient him/herself . The main finding was that health insurance appeared to influence drug use in outpatient services . The average number of drugs per visit was 2.56 and drug expenditures per visit was 16.9 yuan . Between insured and uninsured (out-of-pocket) groups, there were significant differences in the number of drugs and drug expenditures per visit . The insured had a lower number of drugs and a higher drug expenditure per visit than the uninsured, implying the use of more expensive drugs per visit than the uninsured . There were also significant differences in the number of drugs and drug expenditures per visit between the types of insurance . One third of the drugs were anti-infectives, most of which were penicillin, gentamycin, and sulfonamides . The results imply that uninsured patients do not receive the same care as the insured do even if they have the same needs . The fee-for-service financing for hospitals and health insurance have changed health providers' and consumers' behaviour and resulted in the increase of medical expenditure. J Toxicol Sci, 1999 May, 24(2), 95 - 102 Mouse popliteal lymph node assay for assessment of allergic and autoimmunity-inducing potentials of low-molecular-weight drugs; Shinkai K et al.; In the present collaborative study, popliteal lymph node (PLN) responses to penicillin G (an allergenic chemical), D-penicillamine (an autoimmunity-inducing chemical), and barbital (a negative reference chemical) were investigated in three different mouse strains by ten pharmaceutical companies . Two inbred mouse strains (BALB/c and A/J) and one outbred strain (ICR) were subcutaneously injected with saline solutions containing penicillin G (1.25, 2.5 and 5 mg/mouse), D-penicillamine (0.5, 1 and 2 mg/mouse), or barbital (2 mg/mouse) into one hind footpad and saline only was injected into the contralateral footpad . PLN cellularity indices were determined on day 7 . In the three strains tested, the penicillin G and D-penicillamine injections resulted in approximately dose-dependent responses . In contrast, barbital failed to generate a significant PLN reaction . In the typical data from one of the participating laboratories, the PLN responses of A/J, BALB/c, and ICR to penicillin G were high, intermediate and low, respectively, while their PLN responses to D-penicillamine were all high . Some variation in PLN cellularity indices was observed among the participating laboratories, but reproducibility of the popliteal lymph node assay (PLNA) evaluation was partly confirmed . Although the appropriate selection of mouse strains and drug dosage levels has to be considered, these results suggest that the PLNA may be an appropriate screening system for prediction of the allergic or autoimmunity-inducing potentials of low-molecular-weight drugs. Bioconjug Chem, 1999 May-Jun, 10(3), 332 - 7 Toward an in vitro test for the diagnosis of allergy to penicillins . Synthesis, characterization, and use of beta-lactam and beta-lactam metabolite poly-L-lysines which recognize human IgE antibodies; Bolzacchini E et al.; Conjugates of poly-L-lysine (PLL) containing a penicillin or a penicilloyl residue were prepared and characterized by 1H NMR and by size-exclusion (SE) HPLC . These conjugates were used in a radio allergo sorbent tests (RAST) test for the determination of allergy toward beta-lactams . The chemiometric evaluation of the data indicates that allergy to amoxycillin is different from allergy to the other beta-lactams tested . Furthermore, careful chemical characterization of the conjugates appears to be crucial to obtain meaningful information from the RAST data. Am J Prev Med, 1999 Feb, 16(2), 116 - 21 Preventing morbidity and mortality from sickle cell disease . A public health perspective; Olney RS; CONTEXT: Sickle cell disease is a group of conditions characterized by production of abnormal hemoglobin, with clinical manifestations that vary by genotype and age . OBJECTIVE: To discuss current public health issues associated with sickle cell disease, and approaches to preventing complications from these conditions in the United States . DESIGN: Literature review . RESULTS: Most clinical interventions for people with sickle cell disease discussed in the medical literature can be classified as tertiary prevention: for example, therapy to ameliorate anemia, reduce the frequency of pain crises, or prevent stroke recurrences . A form of secondary prevention, newborn screening, has emerged as an important public health approach to identifying affected children before they develop complications . Newborn screening is the starting point for simple public health strategies such as parental education, immunization, and penicillin prophylaxis . Identification of affected families by newborn or community screening programs has also been an entry point for genetic counseling, although utilization of prenatal testing has varied by factors such as geographic location . Public health agencies have had significant involvement with funding, policy making, and formulation of laboratory and clinical guidelines for sickle cell disease . Since the introduction of penicillin prophylaxis policies, newborn screening, new immunizations, and comprehensive medical care centers, the survival of young children with sickle cell disease has improved . CONCLUSIONS: Although the efforts of preventive medicine providers in public health programs are not solely responsible for the improved survival of children with sickle cell disease, such programs remain an important component in preventing sickle cell complications. Aten Primaria, 1999 Mar 31, 23(5), 296 - 300 {Etiology and treatment of choice of respiratory infections in primary care . Opinion of the physicians of the URANO Group}; Ripoll MA et al.; OBJECTIVE: To find the opinion of primary care doctors on the aetiology of respiratory infections and their treatment of choice . DESIGN: Crossover study, through interview with structured questionnaire . SETTING: Primary care . PARTICIPANTS: 400 PC doctors distributed by area in direct proportion to the number of doctors in each area . MEASUREMENTS AND MAIN RESULTS: Most doctors thought that S . pyogenes was the main cause of pharyngitis and tonsillitis; and H . influenzae or S . pneumoniae, of otitis media, sinusitis and exacerbation of acute bronchitis . 78% thought that pneumococci were the main causal agent of pneumonia . 20.5% and 19.8% thought that viruses were the main cause of pharyngitis and acute bronchitis . The treatment of choice depended on the clinical profile and what was thought the most likely cause . For pharyngitis, tonsillitis and acute bronchitis, first choice was aminopenicillins and second was amoxicillin/clavulanic acid . For otitis media, sinusitis and exacerbation of chronic bronchitis, cephalosporins were first choice, followed by amoxicillin/clavulanic acid . For pneumonia, it was aminopenicillins or penicillin, followed by macrolides . CONCLUSIONS: Spanish primary care doctors underestimate the viral aetiology of pharyngo-tonsillitis and acute bronchitis, which may lead to frequent and unnecessary use of antibiotics in these clinical cases . The cause of other infections is well-known, although there are some discrepancies as to the treatment of choice with what is recommended in several medical hand-books. J Biol Chem, 1999 May 28, 274(22), 15706 - 11 Kinetic properties and metal content of the metallo-beta-lactamase CcrA harboring selective amino acid substitutions; Yang Y et al.; The crystal structure of the metallo-beta-lactamase CcrA3 indicates that the active site of this enzyme contains a binuclear zinc center . To aid in assessing the involvement of specific residues in beta-lactam hydrolysis and susceptibility to inhibitors, individual substitutions of selected amino acids were generated . Substitution of the zinc-ligating residue Cys181 with Ser (C181S) resulted in a significant reduction in hydrolytic activity; kcat values decreased 2-4 orders of magnitude for all substrates . Replacement of His99 with Asn (H99N) significantly reduced the hydrolytic activity for penicillin and imipenem . Replacement of Asp103 with Asn (D103N) showed reduced hydrolytic activity for cephaloridine and imipenem . Deletion of amino acids 46-51 dramatically reduced both the hydrolytic activity and affinity for all beta-lactams . The metal binding capacity of each mutant enzyme was examined using nondenaturing electrospray ionization mass spectrometry . Two zinc ions were observed for the wild-type enzyme and most of the mutant enzymes . However, for the H99N, C181S, and D103N enzymes, three different zinc content patterns were observed . These enzymes contained two zinc molecules, one zinc molecule, and a mixture of one or two zinc molecules/enzyme molecule, respectively . Two enzymes with substitutions of Cys104 or Cys104 and Cys155 were also composed of mixed enzyme populations. Am J Perinatol, 1998, 15(12), 649 - 52 Prophylactic amnioinfusion in preganancies complicated by chorioamnionitis: a prospective randomized trial; Parilla BV et al.; The objective of this article is to prospectively investigate the efficacy of amnioinfusion as a means to reduce febrile morbidity in pregnancies complicated by chorioamnionitis . All laboring patients with a temperature > or =100.1 degrees F were approached for study participation . Exclusion criteria included amnionitis diagnosed at greater than 8 cm dilation, multiple gestation, placental abruption, or a nonreassuring fetal heart rate tracing . Consenting patients were randomized to receive antibiotics (ampicillin or penicillin with gentamicin) and acetaminophen with or without amnioinfusion . All patients received intrauterine pressure catheter placement . For study patients, normal saline at room temperature was infused at 10 mL/min for 60 min, then 3 mL/min until delivery . Postpartum endometritis was defined as a temperature = 100.4 degrees F accompanied by uterine tenderness more than 12 hr after delivery . Statistical analysis was performed using the Student's t-test for continuous data and Chi-square for discrete variables . Thirty-six patients were enrolled, and complete data were available for 34 patients (17 in each group) . There were no differences between groups with respect to maternal age, gravidity, race, or gestational age . There were also no differences between groups in duration of rupture of membranes, temperature at randomization, interval from randomization to delivery, cesarean section rate, or umbilical cord arterial pH . The mean temperature at the time of delivery was 99.8+/-0.9 degrees F for the amnioinfusion group versus 100.5+/-1.0 degrees F for the control group (p=0.046) . Three of 17 amnioinfusion patients and 3 of 17 control patients had postpartum endometritis . There was 1 neonatal infection in the treatment group and no neonatal infections among the control patients . Prophylactic amnioinfusion was associated with a decline in temperature at the time of delivery . No untoward effects from the amnioinfusion were identified. J Biol Chem, 1999 May 21, 274(21), 14545 - 50 Disruption of phacA, an Aspergillus nidulans gene encoding a novel cytochrome P450 monooxygenase catalyzing phenylacetate 2-hydroxylation, results in penicillin overproduction; Mingot JM et al.; Aspergillus nidulans utilizes phenylacetate as a carbon source via homogentisate, which is degraded to fumarate and acetoacetate . Mutational evidence strongly suggested that phenylacetate is converted to homogentisate through two sequential hydroxylating reactions in positions 2 and 5 of the aromatic ring . Using cDNA substraction techniques, we have characterized a gene, denoted phacA, whose transcription is strongly induced by phenylacetate and which putatively encodes a cytochrome P450 protein . A disrupted phacA strain does not grow on phenylacetate but grows on 2-hydroxy- or 2, 5-dihydroxyphenylacetate . Microsomal extracts of the disrupted strain are deficient in the NADPH-dependent conversion of phenylacetate to 2-hydroxyphenylacetate . We conclude that PhacA catalyzes the ortho-hydroxylation of phenylacetate, the first step of A . nidulans phenylacetate catabolism . The involvement of a P450 enzyme in the ortho-hydroxylation of a monoaromatic compound has no precedent . In addition, PhacA shows substantial sequence divergence with known cytochromes P450 and defines a new family of these enzymes, suggesting that saprophytic fungi may represent a source of novel cytochromes P450 . Phenylacetate is a precursor for benzylpenicillin production . phacA disruption increases penicillin production 3-5-fold, indicating that catabolism competes with antibiotic biosynthesis for phenylacetate and strongly suggesting strategies for Penicillium chrysogenum strain improvement by reverse genetics. Sex Transm Dis, 1982 Jan-Mar, 9(1), 43 - 4 Unusual location of syphilitic alopecia: a case report; Pareek SS; A case of syphilitic alopecia on the legs of young man is described . Both positive serologic tests and the clinical response to treatment with penicillin suggest that this alopecia was due to syphilis . This report suggests that the physician should be aware of the possible unusual location of syphilitic alopecia at sites other than the scalp, eyebrows, and beard. J Clin Microbiol, 1999 Jun, 37(6), 1958 - 63 Quantitative detection of Borrelia burgdorferi by real-time PCR; Pahl A et al.; Currently, no easy and reliable methods allowing for the quantification of Borrelia burgdorferi in tissues of infected humans or animals are available . Due to the lack of suitable assays to detect B . burgdorferi CFU and the qualitative nature of the currently performed PCR assays, we decided to exploit the recently developed real-time PCR . This technology measures the release of fluorescent oligonucleotides during the PCR . Flagellin of B . burgdorferi was chosen as the target sequence . A linear quantitative detection range of 5 logs with a calculated detection limit of one to three spirochetes per assay reaction mixture was observed . The fact that no signals were obtained with closely related organisms such as Borrelia hermsii argues for a high specificity of this newly developed method . A similar method was developed to quantify mouse actin genomic sequences to allow for the standardization of spirochete load . The specificity and sensitivity of the B . burgdorferi and the actin real-time PCR were not altered when samples were spiked with mouse cells or spirochetes, respectively . To evaluate the applicability of the real-time PCR, we used the mouse model of Lyme disease . The fate of B . burgdorferi was monitored in different tissues from inbred mice and from mice treated with antibiotics . Susceptible C3H/HeJ mice had markedly higher burdens of bacterial DNA than resistant BALB/c mice, and penicillin G treatment significantly reduced the numbers of spirochetes . Since these results show a close correlation between clinical symptoms and bacterial burden of tissues, we are currently analyzing human biopsy specimens to evaluate the real-time PCR in a diagnostic setting. Crit Care Med, 1999 Apr, 27(4), 836 - 42 Prevention and management of overwhelming postsplenectomy infection--an update; Brigden ML et al.; OBJECTIVES: To review the diagnosis and management of overwhelming postsplenectomy infection and to discuss various preventative measures . DATA SOURCES: Data used to prepare this article were drawn from published articles and work in progress . STUDY SELECTION: Articles were selected for relevance to the subject after location by a MEDLINE key word search . DATA EXTRACTION: The literature was reviewed to summarize the etiology and pathophysiology of postsplenectomy sepsis . Preventative strategies were outlined with a particular emphasis on education, immunoprophylaxis, and chemoprophylaxis . DATA SYNTHESIS: Although physicians have become increasingly aware of overwhelming postsplenectomy infection in children, many remain unaware of the risk to asplenic or hyposplenic adults who may have no underlying medical problems . Recent studies have shown that many patients who have had splenectomies have had neither appropriate vaccinations nor teaching that would explain the lifelong nature of their risk . The increasing incidence of penicillin-resistant pneumococci represents a major area of therapeutic and prophylactic concern . The identification of Howell-Jolly bodies on a peripheral blood smear should alert physicians to the need for follow-up to document possible hyposplenism . Attention has focused on a three-pronged attack to this problem, including education, immunoprophylaxis, and chemoprophylaxis . CONCLUSIONS: Overwhelming postsplenectomy infection should be largely preventable if appropriate precautions are taken . Physicians need to know of the spectrum of diseases associated with hyposplenism and how patients noted to have Howell-Jolly bodies should be investigated . They should also be aware of appropriate guidelines for management of patients with asplenia or hyposplenism. Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 583 - 9 Methanococcus vulcanius sp . nov., a novel hyperthermophilic methanogen isolated from East Pacific Rise, and identification of Methanococcus sp . DSM 4213T as Methanococcus fervens sp . nov; Jeanthon C et al.; An autotrophic, hyperthermophilic methanogen (M7T) was isolated from a deep-sea hydrothermal chimney sample collected on the East Pacific Rise at a depth of 2600 m . The coccoid-shaped cells are flagellated and exhibit a slight tumbling motility . The temperature range for growth at pH 6.5 was 49-89 degrees C, with optimum growth at 80 degrees C . The optimum pH for growth was 6.5, and the optimum NaCl concentration for growth was around 25 g l-1 . The new isolate used H2 and CO2 as the only substrates for growth and methane production . Tungsten, selenium and yeast extract stimulated growth significantly . In the presence of CO2 and H2, the organism reduced elemental sulphur to hydrogen sulphide . Growth was inhibited by chloramphenicol and rifampicin, but not by ampicillin, kanamycin, penicillin and streptomycin . The G + C content of the genomic DNA was 31 mol% . As determined by 16S rDNA gene sequence analysis, this organism was closely related to Methanococcus jannaschii strain JAL-1T . However, despite the high percentage of similarity between their 16S rDNA sequences (97.1%), the DNA-DNA hybridization levels between these strains were less than 5% . On the basis of these observations and physiological traits, it is proposed that this organism should be placed in a new species, Methanococcus vulcanius . The type strain is M7T (= DSM 12094T) . During the course of this study, the 16S rDNA sequence analysis placed Methanococcus sp . strain AG86T (= DSM 4213T) as a close relative of M . jannaschii strain JAL-1T . However, the weak level of DNA-DNA hybridization with this strain (< 10%) allowed the proposal that strain AG86T also constitutes a new species, Methanococcus fervens. Wkly Epidemiol Rec, 1999 Apr 23, 74(16), 125 - 7 Resistance monitoring in Denmark, 1997--DANMAP. {Lymphadenosis benigna cutis . A rare variation of Borrelia infection} Danielsen AG, Weismann K. H:S Bispebjerg Hospital, dermato-venerologisk afdeling DWe report three cases of lymphadenosis benigna cutis in children . The lesions were situated on the earlobe and the nipple . The diagnosis was verified by determination of Borrelia antibodies in serum and a positive response to oral penicillin treatment. Hautarzt, 1999 Mar, 50(3), 194 - 7 {Induction of penicillin tolerance in therapy of neurosyphilis in penicillin allergy}; Regele D et al.; Pharmacologically or immunologically induced hypersensitivity to penicillin is frequent . Immunological, i.e., allergic, hypersensitivity reactions are among the most important side effects and can lead to severe and life-threatening reactions . All types of allergic reactions have been described secondary to penicillin therapy . Immediate type hypersensitivity reactions, the symptoms of which range from urticaria and angioedema to respiratory and cardiovascular symptoms, and finally to life threatening full anaphylactic shock are particularly dangerous . This case-report demonstrates that in a patient allergic to penicillin, in whom there is a vital indication to administer this compound, the drug can be given safely after tolerance induction. Acta Derm Venereol, 1999 Mar, 79(2), 139 - 42 Evaluation of systemic provocation tests in patients with suspected allergic and pseudoallergic drug reactions; Hein UR et al.; In order to examine the diagnostic value of systemic provocation tests, we studied 56 inpatients hospitalized for identification of the agent eliciting previous severe allergic or pseudoallergic reactions to non-steroidal anti-inflammatory drugs, local anaesthetics or antibiotics . Skin tests were positive in only 4 patients reacting to antibiotics and propyphenazone and were always negative for local anaesthetics (n = 32) . Only 4 of 26 patients reacted to oral or subcutaneous provocation, 3 times to penicillin and once each to mepivacain, propyphenazone and cyanocobalamine when the suspected drug was tested . In the remaining 30 patients, who for safety reasons were tested only with alternative drugs, none had positive reactions, but 11 patients reported non-specific symptoms, as did 9 of 21 patients given placebo . Systemic provocation tests for drug allergy thus gave few positive results . However, these tests should always be done together with placebo testing for validation of results, and they remain indispensable for identification of alternative, well-tolerated drugs. Postgrad Med, 1999 Apr, 105(4), 106 - 14 Managing community-acquired pneumonia . Factors to consider in outpatient care; Farber MO; Most patients with community-acquired pneumonia are treated as outpatients, and choice of therapy is usually empirical because the etiologic agent is unknown . Therapy should include coverage for both typical and atypical organisms . In geographic areas with highly resistant S pneumoniae, one of the newer fluoroquinolones should be considered, since resistance to penicillin is associated with cross-resistance to macrolides and tetracyclines . Once-daily dosing should be given strong preference because more frequent dosing results in poor compliance, which may lead to inadequate therapy and increased resistance . At present, the duration of therapy should probably be no less than 7 days . Patients should be categorized for mortality risk with objective scoring methods, and the need for hospitalization should be decided accordingly . Greater use of observational and intermediate-care beds is encouraged, as is improved utilization of pneumococcal vaccine. J Mol Biol, 1999 Apr 16, 287(5), 943 - 60 Studies on the active site of deacetoxycephalosporin C synthase; Lloyd MD et al.; The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25 % of total soluble protein in Escherichia coli and purified by an efficient large-scale procedure . Purified protein catalysed the conversions of penicillins N and G to deacetoxycephems . Gel filtration and light scattering studies showed that in solution monomeric apo-DAOCS is in equilibrium with a trimeric form from which it crystallizes . DAOCS was crystallized +/-Fe(II) and/or 2-oxoglutarate using the hanging drop method . Crystals diffracted to beyond 1.3 A resolution and belonged to the R3 space group (unit cell dimensions: a=b=106.4 A, c=71.2 A; alpha=beta=90 degrees, gamma=120 degrees (in the hexagonal setting)) . Despite the structure revealing that Met180 is located close to the reactive oxidizing centre of DAOCS, there was no functional difference between the wild-type and selenomethionine derivatives . X-ray absorption spectroscopic studies in solution generally supported the iron co-ordination chemistry defined by the crystal structures . The Fe K-edge positions of 7121.2 and 7121.4 eV for DAOCS alone and with 2-oxoglutarate were both consistent with the presence of Fe(II) . For Fe(II) in DAOCS the best fit to the Extended X-ray Absorption Fine Structure (EXAFS) associated with the Fe K-edge was found with two His imidazolate groups at 1.96 A, three nitrogen or oxygen atoms at 2.11 A and one other light atom at 2.04 A . For the Fe(II) in the DAOCS-2-oxoglutarate complex the EXAFS spectrum was successfully interpreted by backscattering from two His residues (Fe-N at 1.99 A), a bidentate O,O-co-ordinated 2-oxoglutarate with Fe-O distances of 2.08 A, another O atom at 2.08 A and one at 2.03 A . Analysis of the X-ray crystal structural data suggests a binding mode for the penicillin N substrate and possible roles for the C terminus in stabilising the enzyme and ordering the reaction mechanism . Am Fam Physician, 1999 Apr 15, 59(8), 2233 - 40, 2245-6 Resolving the common clinical dilemmas of syphilis; Birnbaum NR et al.; The diagnosis and treatment of syphilis can present difficult dilemmas . Serologic tests can be negative if they are performed at the stage when lesions are present, and the VDRL test can be negative in patients with late syphilis . Cerebrospinal fluid examination is not required in patients with primary or secondary disease and no neurologic signs or symptoms, but it may be warranted in patients with late latent syphilis or in whom the duration of infection is unknown . Patients with penicillin allergy can be treated with alternative regimens if they have primary or secondary syphilis . Penicillin is the only effective drug for neurosyphilis; oral desensitization should be accomplished before treatment of penicillin-allergic patients . Other dilemmas may be encountered in the treatment of patients who have concurrent human immunodeficiency virus infection. J Am Board Fam Pract, 1999 Mar-Apr, 12(2), 158 - 61 Actinomycosis--an unusual case of an uncommon disease; Petrone LR et al.; Actinomycosis is an uncommon disease caused by organisms of the Actinomyces genus . These organisms are commonly found in the mucous membranes but do not cause infection unless there is disruption of the membranes, as occurs, for example, during dental trauma or abdominal surgery . Use of an IUD is also a risk factor for pelvic actinomycosis . The disease is usually insidious and is often mistaken for other conditions . Treatment of the infection, once diagnosed, is a regimen of long-term antibiotics such as penicillin, clindamycin, and others . Our patient had pelvic and sacral actinomycosis without any of the traditional risk factors for infection. Infection, 1999 Mar-Apr, 27(2), 122 - 4 Endocarditis due to Aerococcus urinae: diagnostic tests, fatty acid composition and killing kinetics; Zbinden R et al.; Two cases of Aerococcus urinae endocarditis are reported . The organism is not included in any database of commercial identification systems at this time . Formation of tetrades and positive reactions for leucine arylamidase and beta-glucuronidase pointed strongly to A . urinae . The cellular fatty acid pattern was similar to that of Aerococcus viridans, with predominantly C16:0, C18:1 omega 9c and C18:0; the presence of C18:1 omega 7t differentiated our isolates from A . viridans and can support the diagnosis of A . urinae . Furthermore, susceptibility to penicillin but resistance to cotrimoxazole represents a pattern opposite to that of A . viridans . Minimal inhibition concentrations of gentamicin and netilmicin were < or = 64 mg/l but those of tobramycin were > or = 256 mg/l . Penicillin combined with either gentamicin or netilmicin showed distinct synergy in killing kinetics . These combinations seem to be the appropriate regimen to treat A . urinae endocarditis. J Bacteriol, 1999 May, 181(9), 2979 - 83 tRNA synthetase mutants of Escherichia coli K-12 are resistant to the gyrase inhibitor novobiocin; Jovanovic M et al.; In previous studies we demonstrated that mutations in the genes cysB, cysE, and cls (nov) affect resistance of Escherichia coli to novobiocin (J . Rakonjac, M . Milic, and D . J . Savic, Mol . Gen . Genet . 228:307-311, 1991; R . Ivanisevic, M . Milic, D . Ajdic, J . Rakonjac, and D . J . Savic, J . Bacteriol . 177:1766-1771, 1995) . In this work we expand this list with mutations in rpoN (the gene for RNA polymerase subunit sigma54) and the tRNA synthetase genes alaS, argS, ileS, and leuS . Similarly to resistance to the penicillin antibiotic mecillinam, resistance to novobiocin of tRNA synthetase mutants appears to depend upon the RelA-mediated stringent response . However, at this point the overlapping pathways of mecillinam and novobiocin resistance diverge . Under conditions of stringent response induction, either by the presence of tRNA synthetase mutations or by constitutive production of RelA protein, inactivation of the cls gene diminishes resistance to novobiocin but not to mecillinam. J Bacteriol, 1999 May, 181(9), 2970 - 2 Disulfide bridges are not involved in penicillin-binding protein 1b dimerization in Escherichia coli; Chalut C et al.; PBP1b can be found as a dimer in Escherichia coli . Previous results suggested that dimerization involved the cysteine(s) in an intermolecular disulfide bond . We show that either deletion mutants or a mutant without cysteines is fully active and still binds penicillin and that the latter can also form dimers. Acta Crystallogr D Biol Crystallogr, 1999 May, 55(5), 1052 - 4 Crystallization of a precursor penicillin acylase from Escherichia coli; Hewitt L et al.; The crystallization of a mutant precursor penicillin acylase {penicillin amidohydrolase (amidase) E.C . 3.5.1.11} from Escherichia coli W (ATCC 11105) using the hanging-drop method is reported . The crystals are in space group P1 with unit-cell parameters a = 51.04, b = 63.58, c = 71.17 A, alpha = 103.0, beta = 110.6, gamma = 105.3 degrees, with one molecule in the asymmetric unit, and diffract to 1 . 8 A using synchrotron radiation. Bull Soc Pathol Exot, 1999 Feb, 92(1), 51 - 5 {Epidemiologic and clinical study of leptospirosis in Bourail (New Caledonia)}; Bouree P et al.; Leptospirosis is a frequent zoonosis in New Caledonia, mostly in the Bourail area, with an incidence of 9.5/1000 inhabitants . This town is an important cross-roads between the main town, Noumea, and the bush . An agricultural show takes place every year in this town, concentrating a great number of domestic animals and several thousand people coming from all over the island . A study was carried out on 78 patients with leptospirosis . The patients were mostly men (70%) of all ages of Melanesian and European origin . People who were living in the bush were more often attacked by leptospirosis than people living in towns . There were two annual peak values (March-April and July-August) . Three symptoms (fever, headache, myalgia) were frequent (56%) . Leucocytosis increased in 45% of cases . The serogroup icterohaemorrhagiae was the more common (38%) . After treatment by penicillin, patients recovered in 83% of cases, but a fatal outcome occurred in 4 patients . An information campaign was necessary in order that people seek medical care as soon as the first symptoms appeared . At present, vaccination does not seem to be possible in such a context. Gut, 1999 May, 44(5), 620 - 4 Characterisation of penicillin G uptake in human small intestinal brush border membrane vesicles; Poschet JF et al.; BACKGROUND: Many beta lactams are well absorbed by the small intestine, although the reasons for this are poorly understood . AIMS: To characterise the uptake of penicillin G into human small intestinal brush border membrane vesicles (BBMV) and to compare the uptake characteristics to those of rabbit BBMV . METHODS AND RESULTS: Uptake of penicillin G was studied in human BBMV . Penicillin G was actively transported into the lumen of BBMV via an H+ dependent, Na+ independent uptake system . The carrier mediated process was saturable and adhered to Michaelis-Menten kinetics (Vmax 52 nmol penicillin G per mg protein per 30 seconds, Km 13.9 mM) . These results are similar to those previously reported in rabbit BBMV . CONCLUSIONS: It is suggested that penicillin G can be used as a model molecule for peptide and beta lactam transport studies as it is cheap and readily available in isotopically labelled form . Furthermore, rabbit BBMV may be used as an acceptable substitute for human BBMV for the study of penicillin transport. Eur J Epidemiol, 1999 Feb, 15(2), 171 - 4 Neonatal tetanus: a continuing challenge in the southeast of Turkey: risk factors, clinical features and prognostic factors; Gurkan F et al.; Neonatal tetanus (NT) still causes significant mortality in developing countries, although in 1989 WHO adopted the goal of eliminating the disease by 1995-2000 . To characterize the regional characteristics, clinical charts of 55 neonates (42 males and 13 females) admitted to the Pediatric Infectious Diseases Ward of Dicle University Hospital, Diyarbakir, Turkey with the diagnosis of NT from 1991 to 1997 were reviewed . Mean age at admittance was 8.9+/-4.3 days with a range of 3-25 days . Mean period for the appearance of first symptoms was 5.8 days ranging between 1 and 21 days . Mean birth weight of the patients was 3369+/-560g . All patients were from rural areas and were delivered at home by untrained traditional birth attendants with no prior antenatal healthcare services . Razor blade (55%), scissors (27%), and knife (18%) were the instruments used to cut the cord in non-hygienic conditions . No mothers had prior vaccination with tetanus toxoid during their pregnancy . Spasticity (76%), lack of sucking (71%), trismus (60%), fever (49%), omphalitis (44%), irritability (24%), risus sardonicus (22%), and opithotonus (15%) were the most common presenting signs and symptoms . Age at admission < 7.5 days and symptoms of onset <4.9 days, risus sardonicus and opisthotonus were associated with fatal outcome . All patients were treated with human tetanus immunglobulin or equine tetanus antitoxin where available, antibiotic therapy by penicillin G (100.000 U/kg/day) and intravenous high dose diazepam (40 mg/kg/day) . Overall mortality rate was 40% (22 cases), without any equipment for mechanical ventilation . Health education of mothers and birth attendants, promotion of hospital delivery and prenatal tetanus immunization of all pregnant women particularly in rural areas are recommended, if NT is to be prevented. J Biotechnol, 1999 Mar 26, 69(1), 27 - 38 Manipulation of carbon assimilation with respect to expression of the pac gene for improving production of penicillin acylase in Escherichia coli; Chou CP et al.; A strategy of genetically manipulating carbon assimilation with respect to expression of the pac gene was employed for overproduction of recombinant penicillin acylase (PAC) . Two expression plasmids of pCLL2902 and pCLL3201, which contain the pac coding region but differ in the pac regulatory region, were constructed for the production experiments . Expression of the pac gene was subjected to phenyl acetic acid (PAA-) induction and glucose catabolite repression for pCLL3201, whereas it was subjected to neither of the two transcriptional regulations for pCLL2902 . The specific PAC activity for strains harboring pCLL2902 was significantly higher than that for strains harboring pCLL3201 due to an improved transcription efficiency . In addition, no inclusion bodies were observed upon production of PAC using the current expression systems . The results suggest that using the native pac promoter instead of a strong promoter such as tac for regulation is a feasible approach for production of PAC . The impact of the current expression systems is also significant from a process viewpoint since, using strains harboring pCLL2902, not only could glucose replace PAA as a carbon source of Escherichia coli cultures for production of PAC but also the volumetric PAC activity was highly improved. Endocr Regul, 1999 Mar, 33(1), 17 - 23 Thymidine kinase activity in homogenates of rat thyroid lobes from euthyroid, hypothyroid and/or hyperthyroid rats incubated with EGF: studies ex vivo in vitro; Karbownik M et al.; OBJECTIVE: To examine thymidine kinase (TK - ATP: thymidine 5'-phosphotransferase, EC 2.7.1.21) activity in homogenates of rat thyroid lobes incubated in vitro with epidermal growth factor (EGF) . METHODS: The thyroid lobes were collected from euthyroid, hypothyroid and/or hyperthyroid animals . Hypothyroidism was developed in the experimental rats by an administration of 0.1 % solution of propylthiouracil (PTU) in drinking water for 2 weeks, while hyperthyroidism was obtained by daily i.p . injections of L-thyroxine (50 microg/kg, B.W.), also for 2 weeks . After collecting, the thyroids were incubated for 4 hours in RPMI 1640 medium with an addition of 20 mM of Hepes buffer, 15% FCS, penicillin (200 U/ml), streptomycin (10 ug/ml) and with EGF (Sigma) (0.1 ng/ml, 10 ng/ml, 1000 ng/ml) . The control lobes were incubated without any addition of EGF to the medium . TK activity was expressed as the amount of reaction products, measured by ascending chromatography . RESULTS: 1 . in the absence of EGF, TK activity in the homogenates of thyroid lobes from hypothyroid rats was lower, while it was higher in the lobes from hyperthyroid animals, when compared to these obtained from euthyroid controls; 2 . EGF in the concentration of 0.1 ng/ml or 1000 ng/ml decreased, while that in the concentration of 10 ng/ml increased TK activity in lobes collected from euthyroid or hyperthyroid rats; 3 . in the tissue collected from hypothyroid rats, the addition of EGF (0.1 ng/ml or 10 ng/ml) caused a slight increase in TK activity versus hypothyroid controls - a tendency towards diminishing TK activity could be observed as parallel to increasing EGF concentration . CONCLUSIONS: TK activity in the homogenates of rat thyroid lobes depends on the functional thyroid status and on applied EGF concentration in vitro. Clin Microbiol Rev, 1999 Apr, 12(2), 187 - 209 Syphilis: review with emphasis on clinical, epidemiologic, and some biologic features; Singh AE et al.; Syphilis is a chronic disease with a waxing and waning course, the manifestations of which have been described for centuries . It occurs worldwide, and the incidence varies significantly with geographic location . Transmission is mainly by sexual contact . The causative organism, Treponema pallidum, was first described in 1905, but because of the inability to culture the organism and the limitations of direct microscopy, serologic testing is the mainstay of laboratory diagnosis . The disease has been arbitrarily divided into several stages . The primary stage is defined by a chancre at the site of inoculation . The secondary stage is characterized by a polymorphic rash, lymphadenopathy, and other systemic manifestations . A variable asymptomatic latent period follows, which for epidemiologic purposes is divided into early (<1 year) and late (>1 year) stages . The early stages (primary, secondary, and early latent) are potentially infectious . The tertiary stage is the most destructive and is marked by cardiovascular and neurologic sequelae and gummatous involvement of any organ system . Congenital infection may result in protean early or late manifestations . Unlike many other bacteria causing infectious diseases, the organism remains sensitive to penicillin, and this remains the mainstay of therapy. J Cutan Pathol, 1999 Jan, 26(1), 51 - 4 Syphilis in an HIV infected patient misdiagnosed as leprosy; Fonseca E et al.; A 42-year-old man with uveitis and a widespread cutaneous eruption, histopathologically characterized by dermal granulomatous infiltrates with perineurial invasion, was incorrectly diagnosed and treated as having borderline leprosy . Further studies demonstrated secondary syphilis and human immunodeficiency virus (HIV) infection . Standard penicillin therapy resolved his cutaneous and ocular lesions . Reports on clinical and pathological findings of active syphilis in HIV infected patients are scarce but this case and isolated previous case reports suggest that granulomatous infiltrates might be a common feature in secondary syphilis with short evolution in HIV infected patients. Can J Hosp Pharm, 1991 Apr, 44(2), 83 - 7 A targeted review of vancomycin use; Piquette RK; In recent years vancomycin usage at the Ottawa Civic Hospital has been steadily increasing . In an effort to determine the reason for this resurgence, and whether or not it is justified, a prospective assessment of vancomycin utilization was performed . All new orders for vancomycin received in the pharmacy in a two-month period were evaluated against predetermined criteria for appropriate use, which were developed in conjunction with Infectious Diseases and Cardiac Surgery . Of the 55 orders evaluated during the study period, 32 (58.2%) were considered inappropriate, translating to a cost of approximately $5,500.00 for the seven-week period . Use of vancomycin in penicillin-allergic patients without a confirmed history of IgE-mediated reaction, was responsible for the majority of vancomycin prescribed unnecessarily (-66%) . As a result of the review's findings, the following actions were taken by the Pharmacy and Therapeutics Committee: (1) vancomycin was restricted to specific indications; (2) vancomycin will be prospectively monitored by the Pharmacy Department; (3) physician education on approved indications and dosing of vancomycin; and (4) development of guidelines for assessment and prescribing in penicillin-allergic patients. Biotechnol Bioeng, 1999 May 5, 63(3), 263 - 72 Genetic manipulation to identify limiting steps and develop strategies for high-level expression of penicillin acylase in Escherichia coli; Chou CP et al.; We have identified the bottleneck steps limiting expression of penicillin acylase (PAC) through comparison of the expression performance for various PAC-expression vectors constructed by genetically modulating the efficiencies of transcription and/or translation of the pac gene . To our knowledge, this is the first report demonstrating that expression of PAC could be limited by various steps, such as transcription, translation, and post-translational steps (i.e . translocation and periplasmic processing), depending on the host/vector systems . Results also indicate that the structure of the wild-type pac gene might not be optimal for direct use in production of PAC using recombinant DNA technology . To improve the gene expression, transcription was enhanced by manipulating certain DNA bases in the pac regulatory region, whereas translation was enhanced by enlarging the spacing between the ribosome binding site and the ATG initiation codon to increase the initiation efficiency . The information is useful in terms of developing genetic strategies for overproduction of recombinant PAC in Escherichia coli . Biotechnol Bioeng, 1999 Feb 5, 62(3), 267 - 77 pH gradients in immobilized amidases and their influence on rates and yields of beta-lactam hydrolysis; Spiess A et al.; The pH gradients developing within immobilized biocatalysts during hydrolysis of penicillin G and glutaryl-7-aminocephalosporanic acid have been estimated both theoretically and experimentally . For the latter a fluorimetric method for the direct measurement of the average pH value within the carrier during reaction has been developed using the pH-dependent fluorescence intensity of an enzyme-bound fluorophore determined with a fiber bundle . The theoretical calculations were based on a model for the hydrolysis with immobilized enzymes using a kinetic expression with five pH-dependent, measurable kinetic and equilibrium constants . The transport reaction differential equation which considers the laminar boundary layer has been solved numerically for the key component . The calculated values agreed well with the experimental data . Under the typical reaction conditions of penicillin G hydrolysis the average pH value in the carrier was 1 and 2.5 pH units below the bulk pH (=8) with and without buffer, respectively . The corresponding changes for the hydrolysis of glutaryl-7-aminocephalosporanic acid at bulk pH 8 in the presence of buffer was 0.5 . This demonstrates the existence of considerable pH gradients in carriers during hydrolytic reactions, even in buffered systems with negligible mass transfer resistance . The low pH value causes suboptimal reaction rates, reduced equilibrium conversion, and reduced enzyme stability . These pH gradients can be minimised by using buffers with pK values approximately equal to the bulk pH used for the hydrolysis . The prediction quality of the model has been tested applying it to fixed bed reactor design . The reduction in rate and yield due to concentration and pH gradients can be overcome with simple measures such as high initial pH value and pH adjustments in segmented or recycling fixed bed reactors . Thus, enzymatic conversions with high yield and high operational effectiveness are achieved . Biotechnol Bioeng, 1998 Dec 20, 60(6), 761 - 7 Modelling of the protonophoric uncoupling by phenoxyacetic acid of the plasma membrane potential of Penicillium chrysogenum; Henriksen CM et al.; Physiological effects of phenoxyacetic acid, the penicillin V side-chain precursor, on steady-state continuous cultures of Penicillium chrysogenum have been studied both theoretically and experimentally . Theoretical calculations show that at an extracellular pH of 6.50, phenoxyacetic acid has negligible influence on the growth energetics due to protonophoric uncoupling of membrane potentials by passive diffusive uptake . On the other hand, when the extracellular pH is lowered to 5.00, a severe maintenance-related uncoupling effect of phenoxyacetic acid is calculated . These findings were confirmed experimentally by steady-state continuous cu