Preparation of a-GeOx:H alloys: Vibrational, optical, and structural properties.
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Journal Article↗
Biomedical subjects
M Zacharias
Publications and source records attributed to M Zacharias.
The bend in RNA created by the trans-activation response element bulge of human immunodeficiency virus is straightened by arginine and by Tat-derived peptide.
The trans-activation response element (TAR) found near the 5' end of the viral RNA of the human immunodeficiency virus contains a 3-nt bulge that is recognized by the virally encoded trans-activator protein (Tat), an important mediator of transcriptional activation. Insertion of the TAR bulge into double-stranded RNA is known to result in reduced electrophoretic mobility, suggestive of a bulge-induced bend. Furthermore, NMR studies indicate that Arg causes a change in the structure of the TAR bulge, possibly reducing the bulge angle. However, neither of these effects has been quantified, nor have they been compared with the effects of the TAR-Tat interaction. Recently, an approach for the quantification of bulge-induced bends has been described in which hydrodynamic measurements, employing the method of transient electric birefringence, have yielded precise estimates for the angles of a series of RNA bulges, with the angles ranging from 7 degrees to 93 degrees. In the current study, transient electric birefringence measurements indicate that the TAR bulge introduces a bend of 50 degrees +/- 5 degrees in the absence of Mg2+. Addition of Arg leads to essentially complete straightening of the helix (to < 10 degrees) with a transition midpoint in the 1 mM range. This transition demonstrates specificity for the TAR bulge: no comparable transition was observed for U3 or A3 (control) bulges with differing flanking sequences. An essentially identical structural transition is observed for the Tat-derived peptide, although the transition midpoint for the latter is near 1 microM. Finally, low concentrations of Mg2+ alone reduce the bend angle by approximately 50%, consistent with the effects of Mg2+ on other pyrimidine bulges. This last observation is important in view of the fact that most previous structural/binding studies were performed in the absence of Mg2+.
Amino Acid Sequence↗
Bulge-induced bends in RNA: quantification by transient electric birefringence.
Bulges represent one of the most common non-helical elements in RNA, often displaying a strong degree of phylogenetic conservation, both in location and sequence, within larger RNA molecules. Thus, knowledge of the conformation and flexibility of RNA bulges is an important prerequisite for understanding the rules governing the formation of tertiary structure within the larger molecules. In the current investigation, the magnitudes of the bends induced in a 148 base-pair duplex RNA molecule by single, centrally located bulges of varying size (n = 1 to 6) and base composition (An and Un series) have been determined through the use of transient electric birefringence (TEB). The TEB approach is highly sensitive to the changes in the global shape of RNA (or DNA) helices that accompany the introduction of points of bending or flexibility near the center of the helix. In the current instance, bulge angles deduced from TEB measurements ranged from approximately 7 degrees to approximately 93 degrees, with the angle increasing with increasing n for both An and Un series. For both An and Un series in the absence of Mg2+, the angle increment per added nucleotide varied from approximately 20 degrees to approximately 8 degrees as n increased from 1 to 6. These angle increments remained unchanged for the An series in the presence of Mg2+; however, the angle increments for the Un series were reduced by a factor of 2 for all values of n. Thus the current observations have identified structural transitions in one of the simplest non-helical elements in RNA, transitions that are dependent on both sequence and counterion valence. Finally, the measured bend angles are strongly correlated with the degree of reduction in electrophoretic mobility of bulge-containing RNA helices. The observed correlation was used to obtain a semi-empirical relationship between bend angle and mobility in order that additional angles might be assigned, by interpolation, through the use of gel data alone.
Base Sequence↗
Combined conformational search and finite-difference Poisson-Boltzmann approach for flexible docking. Application to an operator mutation in the lambda repressor-operator complex.
The N-terminal domain of the phage lambda repressor binds as a dimer to its palindromic DNA operator sequence. In addition to a helix-turn-helix DNA recognition motif, the first six amino acids of the phage lambda repressor form a flexible peptide segment which wraps around DNA. Site-directed mutagenesis studies have shown that amino acid replacements or partial removal of the arm structure, or changes in the DNA sequence contacting the N-terminal arm, can lower the repressor-operator binding affinity by several orders of magnitude. The finite-difference Poisson-Boltzmann approach in combination with a conformational search procedure was used to study energetic contributions of the lambda arm to repressor-operator recognition based on the high resolution X-ray structure. It allows for the local relaxation of the structure upon changing the DNA sequence in the lambda arm binding region. A simplified potential energy function including torsional, truncated Lennard-Jones and approximate electrostatic terms is used in the initial step to screen out energetically unfavorable structures. The electrostatic energy of selected conformations is subsequently calculated more accurately using the finite-difference Poisson-Boltzmann approach. The method was applied to study the effect of a C-->T mutation at position 6 of the consensus half-site of the operator. This base-pair contacts Lys4 which is part of the arm segment. Keeping only the Lys4 side-chain mobile and with the wild-type DNA operator sequence, several conformations close to the X-ray structure were identified as those with lowest energy. In the case of the DNA mutation, lowest energy conformations differed significantly from those selected for the wild-type sequence. These initial calculations indicate that the approach might be a useful tool to estimate conformational and energetic effects upon mutagenesis of protein-DNA complexes.
Base Sequence↗
Patient-controlled sedation using midazolam.
Midazolam was given for sedation as an initial bolus, followed by either a continuous infusion or a patient controlled infusion during third molar extractions. The results showed that there were no significant changes in blood pressure, pulse rate or oxygen saturation during the procedure. Both methods gave good amnesia to events at the start, (100%), as well as to events during, (70% and 75%), and at the end, (61% and 70%), of surgery. There was high acceptance of both methods of sedation (93% and 98% respectively). There was no patient preference for either method of sedation, nor was the operator able to distinguish between the two methods. Hence it is concluded that patient controlled infusion and continuous infusion of midazolam are both satisfactory methods of sedation for patients undergoing surgery under local anaesthesia.
Adolescent↗
Cardiopulmonary resuscitation in dental practice--an update.
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Cardiopulmonary Resuscitation↗
Effect of flumazenil on the recovery from intravenous midazolam.
Psychomotor recovery characteristics were studied in young, healthy patients undergoing intravenous sedation using midazolam for the extraction of third molars. There were significant changes to Choice Reaction Time and Critical Flicker Fusion Threshold noted half an hour after the administration of midazolam (P < 0.001). The administration of the benzodiazepine antagonist flumazenil significantly improved the Choice Reaction Times, but failed to reverse changes in Critical Flicker Fusion Threshold up to 120 minutes later. However, the use of flumazenil assured that no post-operative changes in oxygen saturation occurred in any of the patients.
Adolescent↗
Inversion of receptor binding preferences by mutagenesis: free energy thermodynamic integration studies on sugar binding to L-arabinose binding proteins.
The Escherichia coli L-arabinose-binding protein (ABP) participates as a specific receptor in the transport of L-arabinose, D-fucose, and D-galactose through the periplasmic space. The wild-type protein binds L-arabinose about 40 times more strongly than D-fucose. A mutation of the protein at position 108 (Met-->Leu) causes a specificity change. The Met108Leu ABP slightly prefers binding of D-fucose over L-arabinose. Molecular dynamics (MD) and thermodynamic integration (TI) computer simulations were performed to study the mechanism of sugar discrimination and specificity change based on the known high-resolution X-ray structures. The specificity change was evaluated by calculating the difference in free energy of L-arabinose versus D-fucose bound to wild-type and Met108Leu ABP. The calculated free energy differences are consistent with the experimentally observed specificity of wild-type and Met108Leu ABP. The simulations indicate that the specificity change of Met108Leu ABP is accomplished mainly by reduced Lennard-Jones interactions of residue 108 with L-arabinose and improved interactions with D-fucose. In addition to MD/TI calculations on sugar binding, finite difference Poisson-Boltzmann calculations were performed to identify the most stable ionization state of buried ionizable residues in ABP.
Arabinose↗
Local anesthesia during surgery: when is the best time to give it?
Patients undergoing third molar extractions while under general anesthesia were given local anesthesia either before (group 1) or after (group 2) surgery. The medication intake and pain scores were studied for 7 days but showed no difference between the two groups. It is suggested that timing of local anesthesia alone may not prevent or reduce the postoperative pain experience.
Adult↗
Analysis of the Fis-dependent and Fis-independent transcription activation mechanisms of the Escherichia coli ribosomal RNA P1 promoter.
The role of the curved DNA sequence upstream to the Escherichia coli ribosomal RNA P1 promoter in transcription activation was studied. This sequence region had been shown to activate transcription from P1 in vivo and in vitro and to harbor binding sites for the trans-activating protein Fis. We have constructed a series of linker scanning mutants spanning the region -104 to -47, relative to the transcription start site. DNA fragments carrying the mutations show altered gel electrophoretic mobilities, consistent with reduced DNA bending angles compared to the wild-type sequence. Using gel retardation assays, qualitative as well as quantitative differences in the binding of the trans-activating protein Fis to the mutant DNA fragments could be observed. The effects of the mutations on rrnB P1 promoter activation were studied in vivo in fis+ and fis- backgrounds. A reduction in the promoter strength for some of the linker mutants correlates with altered Fis binding to two of the known Fis binding sites. Shifting the Fis binding region by half a helical turn, relative to the promoter core sequence, abolishes Fis-mediated activation almost totally, whereas activation is partly restored by a shift of a complete helical turn. For one mutant, which does not show alterations in Fis binding, a decrease in the promoter strength was observed in a fis- strain. From the results, we conclude that two upstream activating mechanisms, one Fis-dependent and one Fis-independent, influence the rrnB P1 promoter strength. Sequence determinants for the Fis-independent mechanism are closer to the promoter core region than the Fis binding sites. In addition, the study demonstrates that both the helical geometry and the absolute distance of the UAS region relative to the promoter are crucial for transcription activation.
Base Sequence↗
Poisson-Boltzmann analysis of the lambda repressor-operator interaction.
A theoretical study of the ion atmosphere contribution to the binding free energy of the lambda repressor-operator complex is presented. The finite-difference form of the Poisson-Boltzmann equation was solved to calculate the electrostatic interaction energy of the amino-terminal domain of the lambda repressor with a 9 or 45 base pair oligonucleotide. Calculations were performed at various distances between repressor and operator as well as at different salt concentrations to determine ion atmosphere contributions to the total electrostatic interaction. Details in the distribution of charges on DNA and protein atoms had a strong influence on the calculated total interaction energies. In contrast, the calculated salt contributions are relatively insensitive to changes in the details of the charge distribution. The results indicate that the ion atmosphere contribution favors association at all protein-DNA distances studied. The theoretical number of ions released upon repressor-operator binding appears to be in reasonable agreement with experimental data.
Bacteriophage lambda↗
Bolus dose with continuous infusion of midazolam as sedation for outpatient surgery.
This double-blind, randomised, cross-over trial in 41 patients for 3rd molar surgery compared the safety, amnesic properties and psychomotor recovery between a bolus injection of midazolam and a bolus injection followed by continuous infusion of midazolam. The latter showed good safety and better amnesia to events during the procedure, but prolonged the recovery time.
Adolescent↗
Oxygen saturation during intravenous sedation using midazolam.
Intravenous sedation with midazolam was given to young, fit, adult patients undergoing third molar surgery; oxygen saturation (SpO2) was continuously measured during the surgery and for 30 minutes after the surgery. There were some instances of a brief fall in the oxygen saturation, during the surgery and in the immediate period following surgery. It is suggested that it may be necessary to observe and selectively monitor some patients in the immediate post-surgical period as well as during the period of operation. Although not addressed by this investigation, this would be particularly true of medically compromised patients and the elderly.
Adult↗
Analysis of sequence elements important for the synthesis and control of ribosomal RNA in E coli.
The regulation of the synthesis of ribosomal RNA is a key problem for the understanding of bacterial growth. Many different regulatory mechanisms involving cis and trans acting components participate in a concerted way to achieve the very efficient, flexible and coordinated production of this class of molecules. We have studied three different sequence regions within a ribosomal RNA transcription unit which are believed to control different stages of ribosomal RNA expression. In the first part of the study the function of AT-rich sequences upstream of the -35 hexamer of rRNA promoter P1 in the activation of rRNA transcription was analyzed. We confirm that a sequence dependent bend upstream of P1 is responsible for the high promoter activity. Experiments employing linker scanning mutations demonstrated that the distance as well as the angular orientation of the bent DNA is crucial for the degree of activation. In addition, the effect of the trans activating protein Fis on the transcription initiation of promoter P1 was investigated. We can show, using the abortive initiation assay, that the predominant effect of Fis is due to an increase in the affinity of RNA polymerase for the promoter (binding constant KB) while the isomerisation rate (kf) from a closed to an open RNA polymerase promoter complex is not altered significantly. We also describe the characterization of sequence determinants important for stringent regulation and growth rate control. Evidence is provided that the discriminator motif GCGC is a necessary but not sufficient element for both types of control. Furthermore we show that not simply a particular DNA primary structure but the higher order conformation of the complete promoter region is recognized and triggers the two regulatory mechanisms, both of which are apparently mediated by the effector molecule guanosine tetraphosphate (ppGpp). Finally, we have carried out a systematic mutational analysis of the rrnB leader region preceding the structural gene for 16S RNA. We could demonstrate that highly conserved sequence elements within the rrnB leader, which were believed to be involved in transcription antitermination have post-transcriptional functions. We present evidence that these sequence elements direct the biogenesis of active ribosomal particles.
Base Sequence↗
The signal for growth rate control and stringent sensitivity in E. coli is not restricted to a particular sequence motif within the promoter region.
Hybrid promoter constructs were used to determine the DNA sequence requirements for stringent and growth rate control within a promoter region. The promoters were obtained by fusing complementing sequence regions located upstream and downstream from the GCGC discriminator motif of the growth rate regulated rRNA P1 promoter and a non-regulated tac promoter variant. The activities and the regulatory response of the hybrid promoters were determined in vivo using a promoter test vector system with the chloramphenicol acetyltransferase (CAT) reporter gene. Measurements were made at different growth rates and after starvation for isoleucine to induce the stringent response. Neither the upstream nor the downstream sequence of P1 relative to the GCGC discriminator motif conferred comparable regulatory features when fused to the complementing sequences of the non-regulated mutant tac promoter. A minor response to amino acid deprivation or changes in the growth rate was noted for the hybrid promoter with the rrnB P1 upstream segment and the tac downstream element, pointing to a slightly different importance of the two sequence elements for regulation. The parallel effects for stringent as well as growth rate regulation of the hybrid promoters supports the view of a common mechanism for both types of control. However, none of the promoter sequence elements on its own was able to restore the complete regulatory behaviour of their 'parent' promoters.
Base Sequence↗
The tL structure within the leader region of Escherichia coli ribosomal RNA operons has post-transcriptional functions.
We have investigated a series of mutations within a plasmid encoded E. coli ribosomal RNA leader region. The mutations are localized within a structure known as tL, which has been shown to mediate RNA polymerase pausing in vitro, and which is assumed to have a control function in rRNA transcription antitermination. The effects of the mutated plasmids were analyzed by in vivo and in vitro experiments. Some of the base change mutations led to severely reduced cell growth. As opposed to previous results obtained with mutants where the tL structure has been deleted in part or totally, the tL base change mutations did not result in polar transcription in vivo, rather they revealed a general reduction in the amount of the promoter proximal 16S versus the distal 23S RNA. The deficiency of the 16S RNA, which was most pronounced for some of the slowly growing transformants, can only be explained by a post-transcriptional degradation. In addition, many mutants showed a defective processing after the initial RNase III cut. In line with these results a quantitative analysis of the ratio of ribosomal subunits and 70S tight couple ribosomes showed a reduced capacity to form stable 70S particles for the slowly growing mutants. Together, these findings indicate an important function of the tL structure in post-transcriptional events like processing of rRNA precursors and correct assembly of 30S subunits.
Escherichia coli↗
Polyacrylamide gradient gel electrophoresis for the detection of bended DNA fragments.
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DNA↗
Comparison of two methods of intravenous administration of morphine for postoperative pain relief.
Morphine sulphate was used for the control of pain following major abdominal surgery for a period of three days either as patient-controlled or continuous infusion. The two groups of patients were comparable with regard to patient and operation details, duration of infusion, pain scores and complications. The only significant difference was a reduced dose requirement of morphine in the patient-controlled analgesia group (P less than 0.005). Some possible explanations for this finding are given. It is suggested that a properly supervised continuous infusion of morphine is as good as patient-controlled administration. There was a negative correlation between the age of the patient and the dose of morphine used.
Aged↗