Intermediate species possessing bent DNA are present along the pathway to formation of a final TBP-TATA complex.

Binding of the TATA-binding protein (TBP) to the "TATA" sequences present in the promoters of eukaryotic class II genes is the first step in the sequential assembly of transcription pre-initiation complexes. Myriad structural changes, including severe bending of the DNA, accompany TBP-TATA complex formation. A detailed kinetic study has been conducted to elucidate the mechanistic details of TBP binding and DNA bending. The binding of Saccharomyces cerevisiae TBP to the adenovirus major late promoter (AdMLP) was followed in real-time through a range of temperatures and TBP concentrations using fluorescence resonance energy transfer (FRET) and stopped-flow mixing. The results of association and relaxation kinetics and equilibrium binding experiments were analyzed globally to obtain the complete kinetic and energetic profile of the reaction. This analysis reveals a complex mechanism with two intermediate species, with the DNA in the intermediates apparently bent similarly to the DNA in the final complex. TBP binding and DNA bending occur simultaneously through the multiple steps of the reaction. The first and third steps in this sequential process show nearly identical large increases in both enthalpy and entropy, whereas the middle step is highly exothermic and proceeds with a large decrease in entropy. The first intermediate is significantly populated at equilibrium and resembles the final complex both structurally and energetically. It is postulated that both this intermediate and the final complex bind transcription factor IIB in the second step of pol II pre-initiation complex assembly. A consequence of such a reactive intermediate is that the rate of assembly of transcriptionally competent pre-initiation complexes from bi-directionally bound TBP is greatly increased.

Detection of stroke during cardiac operations with somatosensory evoked responses.

OBJECTIVES: The objectives of this study were to determine if monitoring of intraoperative somatosensory evoked potentials could be used to detect stroke during cardiac operations and to establish indicators of cerebral ischemia based on changes in these potentials. METHODS: Twenty-five patients undergoing cardiac operations underwent preoperative and postoperative neurologic examinations as well as intraoperative recording of somatosensory evoked potentials. Detailed analysis of the waveforms of these potentials was performed. RESULTS: Two of the 25 patients had intraoperative strokes. These patients and only these patients had changes in their somatosensory evoked potentials during the operation suggesting cerebral ischemia. The unilateral disappearance of the cortical somatosensory evoked potential waves correlated significantly with the clinical outcome of stroke (p < 0.004). Ischemic changes were detected in real time and were related to the removal of the aortic crossclamp in one patient and to the initiation of cardiopulmonary bypass in the other. CONCLUSIONS: Somatosensory evoked potentials can detect intraoperative stroke during cardiac operations. Acute, unilateral decreases in amplitude of the cortical potential are more useful than changes in latency in detecting intraoperative stroke.

An electronmicroscope study of the effect of sulphadiazine and trimethoprim on Enterobacter cloacae.

Electronmicroscopy of thin sections of log phase cells of Enterobacter cloacae NCTC 10005 grown for 4 h in the presence of sulphadiazine 250 micrograms/ml, trimethoprim 12.5 microliters/ml or the combination of sulphadiazine 250 micrograms/ml plus trimethoprim 12.5 micrograms/ml indicated that both agents caused marked morphological damage even though the MIC of sulphadiazine for the E. cloacae strain was > 3000 micrograms/ml. The damage took the form of electron-transparent areas devoid of ribosomes in the cytoplasm and detachment of the outer membrane. The latter was most marked with trimethoprim, which also caused damage to the cytoplasmic membrane. It is postulated that the synthesis of the peptidoglycan layer was affected by the antimetabolites since the morphological effects were strikingly similar to those caused by treatment of E. cloacae with disodium edetate plus lysozyme. Viable counts of cultures undergoing the same treatments as those prepared for electronmicroscopy indicated that although sulphadiazine merely partially inhibited growth it nevertheless enhanced the bactericidal action of trimethoprim over a 5-h period.

A triterpene from Rubus pinfaensis.

A new pentacyclic triterpene, pinfaensin, was isolated from the roots of Rubus pinfaensis and the structure elucidated by 13C NMR methods. This triterpene, a glycoside of pinfaensic acid (glucosyl 23-formyl-2 alpha,3 beta,19 alpha-trihydroxyurs-12-en-28-oate), in order to achieve purification and characterization, was hydrolysed to previously unreported pinfaensic acid which on acetylation and methylation gave methyl diacetoxypinfaensate (methyl 2 alpha,3 beta-diacetoxy-23-formyl-19 alpha-hydroxyurs-12-en-28-oate).

Determination of peptidoglycan-associated protein in Escherichia coli NCIB 8545 by capillary zone electrophoresis.

An efficient reliable and sensitive capillary zone electrophoresis assay for the six major bacterial peptidoglycan-associated proteins of Escherichia coli NCIB 8545 is described. The method provides the facility to determine quantitatively the effect of antibacterials on bacterial peptidoglycan-associated protein synthesis and thus to further elucidate the mechanism of antibacterial action of such drugs as the antifolates which recently have been shown to adversely affect peptidoglycan synthesis.

Pseudomonas cepacia resistance to antibacterials.

Reproducible growth rates of Pseudomonas cepacia were obtained. P. cepacia had a markedly different resistance pattern to single and combined antibacterials from that characteristic of Pseudomonas aeruginosa. Benzalkonium and chlorhexidine were more active against log phase P. cepacia than against log phase P. aeruginosa, but polymyxin B sulfate was inactive against log phase P. cepacia at all concentrations tested (smaller than or equal to 16 units/ml). Antagonism of antibacterial activity between edetate disodium-benzalkonium and edetate disodium-chlorhexidine combinations was marked with 16-hr P. cepacia and with 16-hr Staphylococcus aureus. Phenylethanol-benzalkonium and phenylethanolchlorhexidine combinations had no more than additive activity against log phase P. cepacia. These results have relevance to hospital disinfection and preservation of pharmaceutical solutions.

In vitro evaluation of the antimicrobial activities of selected lozenges.

The in vitro antimicrobial activities of 10 lozenges (Merothol, Merocets, Merocaine, Strepsils (two varieties), Dequacaine, Dequacets, Zensyls, Tyrozets, and Labosept) were determined by use of a microtiter counting method with Streptococcus pyogenes, Staphylococcus aureus, and Candida albicans as the test organisms. Merothol, Merocets, Merocaine, and both Strepsils formulations all reduced the counts of both S. aureus and S. pyogenes suspensions by approximately 6 log cycles within 5 and 20 min, respectively. Merothol, Merocets, and Merocaine also caused a reduction in the counts of the C. albicans suspension approximately 5 log cycles within 40 min, but no other lozenge formulation showed rapid and marked activity against C. albicans. Dequacaine and Dequacets showed marked but much slower activities against this yeast. Zensyls caused an approximately 6-log-cycle reduction in bacterial counts within 40 min, and Dequacaine, Dequacets, and Tyrozets showed marked but slower antibacterial activities. This work confirmed by a statistically sound in vitro method the in vivo antibacterial activities reported for Merothol, Merocets, and Merocaine, demonstrated equivalent antibacterial activities for Strepsils, and indicated that Merothol, Merocets, and Merocaine also showed marked activities against C. albicans.

Electron microscope study of effect of benzalkonium chloride and edetate disodium on cell envelope of Pseudomonas aeruginosa.

Electron micrographs of Pseudomonas aeruginosa grown in nutrient broth or broth containing subinhibitory concentrations of benzalkonium chloride indicate that benzalkonium chloride at 50 and 100 mug/ml strips off the outer cell membrane. Cells grown in the presence of edetate disodium, 50-100 mug/ml had convoluted surfaces (wavy in cross section). Cells damaged by growing on nutrient agar containing benzalkonium (200 mug/ml), when subsequently grown for 16 hr in nutrient broth, produced cells with apparently normal outer lavers. Similar cells grown on nutrient agar plus benzalkonium (500 mug/ml) when grown for 16 hr in nutrient broth had normal resistance to edetate disodium lysis, but cells grown overnight in broth plus benzalkonium (500 mug/ml) showed increased sensitivity to edetate disodium lysis. Cells grown on nutrient agar in the presence of benzalkonium (800 mug/ml) grew in broth plus benzalkonium (10 mug/ml) without stripping of their external membrane, but replicate inocula into broth plus benzalkonium (10 mug/ml) and edetate disodium (50 mug/ml) produced cells with structural damage to the outer lavers of the cell envelope.

Differences in antibacterial activity of benzalkonium chloride.

Benzalkonium solutions obtained from different manufacturers were shown to have different activities. This difference in activity was related to the composition of the benzalkonium chloride. The potential seriousness of this situation is emphasized, and a recommendation is made that the official monographs on benzalkonium chloride be amended appropriately, noting the apparently superior antibacterial activity of the tetradecyl (C14) homolog.

An evaluation of the antibacterial activities of combinations of sulfonamides, trimethoprim, dibromopropamidine, and silver nitrate compared with their uptakes by selected bacteria.

Modifications of antibacterial activity have been demonstrated using combinations of two antibacterials from trimethoprim, sulfonamides (sulfadiazine, sulfamerazine, and silver sulfadiazine), silver nitrate, and dibromopropamidine isethionate, either formulated in a cream base or dissolved in peptone water. The creams were evaluated using the agar cup diffusion method in isosensitest agar. The peptone water solutions provided fractional inhibitory concentrations for combinations of the antibacterial substances. The test organisms were Pseudomonas aeruginosa, Enterobacter cloacae, and Staphylococcus aureus. Bacterial uptakes of antibacterial combinations, determined by either an HPLC assay method or an atomic absorption method, combined with dry cell weight determinations, indicated that enhancement of activity of the antibacterial combinations against P. aeruginosa (two strains) and E. cloacae were related to marked increases in the bacterial uptake of the chemical agents. Decreases in activity were related to decreased uptake of either dibromopropamidine and/or silver ions. The effect of the trimethoprim and the sulfonamides was shown to depend on their effect on bacterial folate synthesis. It is suggested that partial blockade of the folate synthetic pathway leads to an effect on cell permeability which results in increased uptake of antibacterials. Dibromopropamidine isethionate also has an effect on cell permeability which produces an increased bacterial uptake of a second antibacterial present in the medium. These findings provide further explanation of how subinhibitory concentrations of trimethoprim and sulfonamide combinations are synergistic against a wide range of bacteria even when certain bacteria are resistant to either member of the combination.

Investigation of cell envelope damage to Pseudomonas aeruginosa and Enterobacter cloacae by dibromopropamidine isethionate.

Electron micrographs of log-phase Pseudomonas aeruginosa and Enterobacter cloacae cultured for 4 h in the presence of subinhibitory concentrations of dibromopropamidine isethionate indicate that this antibacterial agent can cause marked damage to the cell envelope structures of both species. This result provides an explanation of how dibromopropamidine can enhance the uptake and thus the activity of a second antibacterial agent used in combination with it.

Determination of thymidine, uracil and p-aminobenzoic acid in bacteriological cultures by capillary zone electrophoresis.

A capillary zone electrophoresis assay has been developed which simultaneously separates and quantifies the bacterial metabolites thymidine, uracil and p-aminobenzoic acid present at bacteriologically relevant concentrations in a supplemented minimal bacteriological medium. Sulphadiazine was used as the internal standard.

Capillary zone electrophoresis assay of the uridine diphosphate N-acetylmuramyl peptide precursors and the disaccharide pentapeptide derivative of bacterial cell wall peptidoglycan.

Uridine diphosphate N-acetylmuramyl peptide (EDP-MurNac) precursors and disaccharide pentapeptide of bacterial cell wall peptidoglycan were extracted from Enterobacter colacae cells and examined by capillary zone electrophoresis. Five UDP-MurNac derivatives with dibromopropamidine isethionate as the internal standard, and disaccharide pentapeptide with pyrimethamine as the internal standard, were successfully and rapidly analysed by using a fused-silica capillary and sodium phosphate buffer in methanol as the organic modifier at appropriate pH. Accurate quantitation was also achieved. The method provides the potential to investigate quantitatively the effect of antibacterials on the biosynthesis of peptidoglycan and to determine the relative cellular concentrations of the murein precursors within the cell cycle.

Investigation of synergism between combinations of ciprofloxacin, polymyxin, sulphadiazine and p-aminobenzoic acid.

Subinhibitory concentrations of combinations of any two of ciprofloxacin, colistin (or polymyxin B), sodium sulphadiazine and p-aminobenzoic acid were shown by checkerboard minimum inhibitory concentration determinations to have synergistic inhibitory activity against Pseudomonas aeruginosa and to have either synergistic or additive activity against Staphylococcus aureus. In addition, sulphadiazine plus either ciprofloxacin or polymyxin showed markedly enhanced killing activity against both P. aeruginosa and S. aureus. p-Aminobenzoic acid plus either ciprofloxacin or polymyxin also demonstrated enhanced killing activity against P. aeruginosa but these combinations were less effective in enhancing activity against S. aureus. Ciprofloxacin in combination with polymyxin had a marked synergistic effect against P. aeruginosa but only a slight synergistic effect against S. aureus. These findings indicate a potential usefulness for the synergistic combinations against P. aeruginosa and S. aureus in the clinical situation; that is, they indicate an extended role for sulphonamides and support a potential role for p-aminobenzoic acid as enhancers of the activity of primary antibacterial agents such as ciprofloxacin and polymyxin. We suggest that for a second antibacterial to enhance the activity of ciprofloxacin, it may be necessary for the second antibacterial to increase cell permeability so increasing bacterial uptake of ciprofloxacin.

Quantification of uptake of liposomal carboxyfluorescein by professional phagocytes in-vitro. A flow microfluorimetric study on the J774 murine macrophage cell line.

Unilamellar egg phosphatidylcholine/cholesterol liposomes containing carboxyfluorescein were prepared by an ether injection method. The ability of cells of the J774.2 murine macrophage cell line to incorporate the liposomal fluorophore during incubation at 37 degrees C was measured by flow microfluorimetry. Liposomes incorporating additional phosphatidylserine or phosphatidic acid were taken up much more avidly than those lacking these phospholipids and the greatest uptake of carboxyfluorescein was observed with the phosphatidylserine species. Calculation of the number of liposomes taken up, greater than or equal to 0.2% of the number given, showed that this was an inefficient process. However these uptake data support previous findings based on the intracellular bactericidal activity of liposomal antibiotics determined in an identical in-vitro system.

An in-vitro model of intracellular bacterial infection using the murine macrophage cell line J774.2.

A simple model of intracellular bacterial infection based on the ability of the macrophage cell line J774.2 to phagocytose Escherichia coli in a reproducible manner is described. Viable counting of intracellular bacteria and microscopic examination of infected macrophage cultures after treatment with fluorescent E. coli antibody showed that the bacteria multiplied within the J774.2 cells. Viable intracellular bacteria may be used to study the activity of bactericidal and bacteriostatic drugs within the macrophage. The low apparent intracellular bactericidal activity of streptomycin, which was time- and concentration-dependent, accords with a low permeability of the J774.2 macrophages to this antibiotic. An exclusively intracellular infection could be achieved by inactivation of non-phagocytosed extracellular bacteria by streptomycin treatment of infected macrophage cultures. Under appropriate conditions intracellular bacterial viability was unaffected.

Vancomycin resistance reversal in enterococci by flavonoids.

The development of clinical vancomycin-resistant strains of enterococci (VRE) is a major cause for concern. Here we show that a combination of galangin or 3,7-dihydroxyflavone with vancomycin may be used to sensitize resistant strains of Enterococcus faecalis and Enterococcus faecium to the level of vancomycin-sensitive strains. Minimum inhibitory concentrations (MICs) and viable counts were determined in Iso-sensitest broth using a microtitre method. MICs of vancomycin against 67% of resistant clinical isolates and a type strain of enterococci were lowered from > 250 microg mL(-1) to < 4 microg mL(-1) in the presence of galangin (12.5 microg mL(-1)) or 3,7-dihydroxyflavone (6.25 microg mL(-1)). Viable counts for type culture E. faecalis ATCC 51299 showed the flavonoids alone significantly lowered numbers of colony forming units (CFUs). CFUs were maintained at low levels (< 10(3) CFU mL(-1)) for 24 h by vancomycin/flavone combinations. This combinational action in reversing vancomycin resistance of enterococci highlights novel drug targets and has importance in the design of new therapeutic regimes against resistant pathogens.

Mechanism for synergism between sulphonamides and trimethoprim clarified.

Pseudomonas aeruginosa, Escherichia coli, Pseudomonas cepacia and Moraxella catarrhalis were selected for their markedly different resistance patterns to sulphonamides and trimethoprim. In addition, strains of E. coli and P. cepacia were selected having different resistance profiles to the inhibition of dihydropteroate synthetase and dihydrofolate reductase. All inhibitors of dihydropteroate synthetase combined in any combination with inhibitors of dihydrofolate reductase resulted in mutual enhancement of bacterial uptakes of the inhibitors and corresponding increased antibacterial activity of the combinations. High concentrations of sulphonamides or p-aminobenzoic acid plus trimethoprim caused a decrease in overall activity of the combination and indicated that both sulphonamides and p-aminobenzoic acid at high concentrations can interact with dihydrofolate reductase. The antibacterial activity of p-aminobenzoic acid at high concentrations is considered to be a blocking effect on dihydrofolate reductase even though p-aminobenzoic acid at low concentrations is an essential part of the synthesis of dihydrofolic acid. These findings support an alternative hypothesis for the mechanism of antibacterial action of individual antifolates and their mechanism of synergism in combination.

Baicalin synergy with beta-lactam antibiotics against methicillin-resistant Staphylococcus aureus and other beta-lactam-resistant strains of S. aureus.

Bacterial resistance to antibiotics is a serious global problem and includes strains of beta-lactam-resistant Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). Novel antimicrobials and/or new approaches to combat the problem are urgently needed. The Chinese herb Xi-nan Huangqin (Scutellaria amoena C.H. Wright) has been used in traditional Chinese medicine to treat a wide range of infectious diseases. In this study we have examined the antibacterial action of baicalin, a flavone isolated from the herb. When combined with 16 microg mL(-1) baicalin, minimum inhibitory concentrations (MICs) of benzylpenicillin against MRSA and penicillin-resistant S. aureus were reduced from 125 and 250 microg mL(-1) to 4 and 16 microg mL(-1), respectively. This activity of baicalin was dose-dependent. Viable counts showed that the killing of MRSA and beta-lactam-resistant S. aureus cells by 10 to 50 microg mL(-1) ampicillin, amoxycillin, benzylpenicillin, methicillin and cefotaxime was potentiated by 25 microg mL(-1) baicalin. From the study it was concluded that baicalin has the potential to restore the effectiveness of beta-lactam antibiotics against MRSA and other strains of beta-lactam-resistant S. aureus. In view of its limited toxicity baicalin offers potential for the development of a valuable adjunct to beta-lactam treatments against otherwise resistant strains of microorganisms.

Study of the mutual effects of sulphadiazine and ciprofloxacin on their uptakes by Pseudomonas aeruginosa.

A high-performance liquid chromatography (HPLC) assay was developed for ciprofloxacin and sulphadiazine in Isosensitest broth. Combining the HPLC assay with cell dry-weight determinations indicated that both compounds were able to enhance the uptake of the other by log phase Pseudomonas aeruginosa cultured in the presence of the compounds. It is hypothesized that the increased bacterial uptakes are the reason for the enhanced antibacterial activity previously reported for combinations of ciprofloxacin and sulphadiazine.