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T Powers

Publications and source records attributed to T Powers.

At least 37 records · Page 2Linked to original sources
PubMed1995

Hydroxyl radical footprinting of ribosomal proteins on 16S rRNA.

Complexes between 16S rRNA and purified ribosomal proteins, either singly or in combination, were assembled in vitro and probed with hydroxyl radicals generated from free Fe(II)-EDTA. The broad specificity of hydroxyl radicals for attack at the ribose moiety in both single- and double-stranded contexts permitted probing of nearly all of the nucleotides in the 16S rRNA chain. Specific protection of localized regions of the RNA was observed in response to assembly of most of the ribosomal proteins. The locations of the protected regions were in good general agreement with the footprints previously reported for base-specific chemical probes, and with sites of RNA-protein crosslinking. New information was obtained about interaction of ribosomal proteins with 16S rRNA, especially with helical elements of the RNA. In some cases, 5' or 3' stagger in the protection pattern on complementary strands suggests interaction of proteins with the major or minor groove, respectively, of the RNA. These results reinforce and extend previous data on the localization of ribosomal proteins with respect to structural features of 16S rRNA, and offer many new constraints for three-dimensional modeling of the 30S ribosomal subunit.

Base Sequence↗
PubMed1994

Selective perturbation of G530 of 16 S rRNA by translational miscoding agents and a streptomycin-dependence mutation in protein S12.

Previous studies have shown that a concise set of universally conserved bases in 16 S rRNA are strongly protected from attack by chemical probes when tRNA is bound specifically to the ribosomal A site. Two of these bases, A1492 and A1493, are located in the cleft of the 30 S subunit, the site of codon-anticodon interaction. A third residue, G530, is located within the highly conserved 530 stem-loop, a region that is involved in interactions with proteins S4 and S12, mutations in which perturb the translational error frequency. The 530 loop is also thought to be located at or near the site of interaction of elongation factor Tu on the 30 S subunit, a location that is distinct from the decoding site. This study monitors the response of these two A-site-related regions of 16 S rRNA to a variety of translational miscoding agents. Several of these agents, including streptomycin, neomycin and ethanol, selectively potentiate tRNA-dependent protection of residue G530 from kethoxal modification; in contrast, little change in reactivity of residues A1492 and A1493 is observed. These results are consistent with the previously demonstrated importance of G530 for A-site function and, moreover, suggest a common mechanism of action for these miscoding agents, even though they appear to have distinctly different modes of interaction with 16 S rRNA. In contrast to the miscoding agents, we find that a streptomycin-dependence (SmD) mutation in protein S12, which causes ribosomes to be hyperaccurate, antagonizes tRNA-dependent protection of G530. The possibility that 5' or 3' flanking regions of mRNA could be involved in tRNA-dependent protection of G530 was tested by using different lengths of oligo(U) to promote binding of tRNA(Phe) to the A site. The relative levels of protection of G530, A1492 and A1493 were unchanged as the size of the mRNA fragment was decreased from 16 to 6 bases in length. We conclude, therefore, that for protection of G530 to be the result of direct contact with message, it must necessarily be located directly at the decoding site; otherwise, its protection is best explained by allosteric interactions, either with mRNA, or with the codon-anticodon complex. These results are discussed in terms of a model wherein the conformation of the 530 loop is correlated with the affinity of the ribosome for elongation factor Tu.

Base Sequence↗
PubMed1994

The 530 loop of 16S rRNA: a signal to EF-Tu?

The molecular mechanism of tRNA selection has remained elusive. Recent evidence, both biochemical and genetic, suggests the involvement of 'smart' ribosomes, that can signal the elongation factor Tu by way of conformational changes in ribosomal RNA.

Models, Biological↗
PubMed1994

Intratracheal pulmonary ventilation (ITPV): control of positive end-expiratory pressure at the level of the carina through the use of a novel ITPV catheter design.

A new mode of pulmonary ventilation called intratracheal pulmonary ventilation (ITPV) was studied. Briefly, a continuous flow of air/oxygen is introduced through a small catheter, the tip of which is positioned at the carina, with a diffuser mounted at its distal end. A timed expiration valve, when closed, provides for inspiration; when open, it provides for expiration. The system as first described had a potential for significant back pressure at the level of the carina, which was more at rapid gas flows and with smaller endotracheal tubes. We have now mounted a venturi on the tip of the catheter (reverse thrust catheter [RTC]) that avoids back pressure, and which facilitates expiration. At respiratory rates from 10 to 120/min, the ITPV system with the RTC maintained end-expiratory pressure at the level of the carina at, or near 0 cm H2O. Compared to conventional mechanical ventilation, at identical respiratory rates, this system reduced tidal volume by one half at the lowest respiratory rates, and by as much as two thirds at the highest respiratory rates, with a proportional decrease in peak inspiratory pressure. ITPV has the smallest minute volume ventilation of any conventional or nonconventional mode of pulmonary ventilation.

Animals↗
PubMed1994

Comparison of diode and argon laser trabeculoplasty in cadaver eyes.

PURPOSE: To evaluate differences in the effects of diode and argon trabeculoplasty on cadaver eye trabecular meshwork. METHODS: The argon laser was used at a 50 mu spot size, 500 to 100 mW of power, and a 0.1 second time interval, and the diode laser was used at a 0.1 to 0.2 second time interval, 100 mu spot size, and 400 to 1200 mW of power. Analysis was performed using videography, microscopy, and scanning electron microscopy. RESULTS: This study found grossly that burns with the argon laser caused tissue blanching and pigment dispersion, whereas no reaction was observed with the diode laser. The diode laser made no observable histologic alterations, but the argon caused fragmentation and coagulation of trabecular beams. Scanning electron microscopy showed that the diode laser caused an area of trabecular beam coalescence measuring 50 to 100 mu in diameter with energies > or = 0.12 J. A surrounding zone of more superficial trabecular beam fragmentation measuring 200 to 400 mu in diameter also was observed. Similar energy levels from the argon laser caused hole formation 200 to 400 mu in diameter. Lower argon energy levels (0.05 J) caused inconsistent coalescence 50 to 100 mu in diameter similar to the diode laser. CONCLUSIONS: This study suggests that in the trabecular meshwork the diode laser causes a reaction at, but not at less than, energy levels shown previously to produce a clinical ocular hypotensive effect. Also, at similar energy per area, the tissue effects of the diode and argon laser are comparable.

Argon↗
PubMed1993

Anophthalmic socket pain.

We examined and treated four patients with anophthalmic socket pain. Conditions responsible for this problem in this series included scleritis after evisceration, amputation neuroma, pain from a skull-base meningioma, and chemical dependency with drug-seeking behavior. The pain associated with the scleritis after evisceration responded to removal of the scleral remnant. The pain associated with the amputation neuroma responded to removal of the orbital implant and its pseudocapsule in which the amputation neuroma was embedded. The pain associated with the meningioma was intractable. The pain associated with the chemical dependency remained a persistent problem. A careful history and physical examination are critical in the evaluation of anophthalmic socket pain. Computed tomography or magnetic resonance imaging may be helpful in some cases.

Adult↗
PubMed1993

Dynamics of in vitro assembly of 16 S rRNA into 30 S ribosomal subunits.

One of the important unsolved problems in the ribosome field is the molecular basis for the sequential and co-operative nature of ribosome assembly. As an approach to this problem, we have taken advantage of the temperature dependence of in vitro reconstitution and have used chemical probing methods to examine the conformation and reactivity of 16 S rRNA at successive stages during subunit assembly. One class of nucleotides displays reactivities similar to those observed in native 30 S particles when the RNA and protein are incubated in the absence of any heat step (0 degrees C effects). At 30 degrees C, where the assembly process takes 2 hours, other bases can be assigned to one of several additional kinetic classes, determined by the rate at which their chemical reactivities transit from levels observed in naked RNA to levels observed in fully assembled subunits: (1) fast (t1/2 = < 5 min at 30 degrees C); (2) slow (t1/2 = 15 to 30 min at 30 degrees C); (3) delayed slow (t1/2 = 30 to 60 min at 30 degrees C). Finally, several nucleotides display transient kinetics in their reactivities, showing increasing reactivity at early time points and becoming protected later in assembly; most of these effects correspond to residues that were previously shown to display reciprocal enhancement and protection patterns during step-wise in vitro assembly. These findings, together with our previous studies using purified individual proteins lead to the following conclusions: (1) there is a predominant 5' to 3' polarity to in vitro assembly, even though it is uncoupled from transcription; (2) portions of the central and 3' major domains fold into an active conformation only at a very late stage of assembly; (3) bases footprinted by late-assembling proteins, according to the 30 S subunit assembly map, show generally slower kinetics than residues footprinted by proteins that bind early in the assembly map, providing direct evidence for the sequential nature of the in vitro assembly process; (4) most proteins are associated with nucleotides that fall into more than one kinetic class, suggesting that assembly proceeds through multiple pathways, or that individual proteins interact sequentially with different regions of the RNA.

Cold Temperature↗
PubMed1993

Evidence for functional interaction between elongation factor Tu and 16S ribosomal RNA.

Translation of the genetic code requires the accurate selection of elongation factor (EF)-Tu.GTP.tRNA ternary complexes at the ribosomal acceptor site, or A site. Several independent lines of evidence have implicated the universally conserved 530 loop of 16S rRNA in this process; yet its precise role has not been identified. Using an allele-specific chemical probing strategy, we have examined the functional defect caused by a dominant lethal G-->A substitution at position 530. We find that mutant ribosomes are impaired in EF-Tu-dependent binding of aminoacyl-tRNA in vitro; in contrast, nonenzymatic binding of tRNA to the A and P sites is unaffected, indicating that the defect involves an EF-Tu-related function rather than tRNA-ribosome interactions per se. In vivo, the mutant ribosomes are found in polysomes at low levels and contain reduced amounts of A-site-bound tRNA, but normal levels of P-site tRNA, in agreement with the in vitro results; thus the dominant lethal phenotype of mutations at G530 can be explained by impaired interaction of mutant ribosomes with ternary complex. These results provide evidence for a newly defined function of 16S rRNA--namely, modulation of EF-Tu activity during translation.

Alleles↗
PubMed1993

Allele-specific structure probing of plasmid-derived 16S ribosomal RNA from Escherichia coli.

Biochemical analysis of Escherichia coli ribosomes containing mutant 16S or 23S (r)ribosomal RNAs, produced via cloned rDNA genes on multicopy plasmids, has been hindered by the background of wild-type (wt) ribosomes containing host-derived rRNA. Here, we describe a method for the construction of unique priming sites in 16S rRNA that allow allele-specific structure probing of ribosomes containing plasmid-encoded RNA. Phenotypically silent mutations, designed to mimic related eubacterial sequences, have been introduced into four phylogenetically variable regions in the 16S rDNA gene that allow inspection of several 16S rRNA functional sites. When oligodeoxyribonucleotides complementary to these altered sequences are used to prime cDNA synthesis in primer extension reactions using reverse transcriptase, only plasmid-derived 16S rRNA is used as a template, thus rendering the wt background invisible. Unexpectedly, we were unable to introduce silent mutations into one nonconserved helix in 16S rRNA, suggesting that constraints in addition to Watson-Crick pairing are important in this region.

Alleles↗
PubMed1991

A functional pseudoknot in 16S ribosomal RNA.

Several lines of evidence indicate that the universally conserved 530 loop of 16S ribosomal RNA plays a crucial role in translation, related to the binding of tRNA to the ribosomal A site. Based upon limited phylogenetic sequence variation, Woese and Gutell (1989) have proposed that residues 524-526 in the 530 hairpin loop are base paired with residues 505-507 in an adjoining bulge loop, suggesting that this region of 16S rRNA folds into a pseudoknot structure. Here, we demonstrate that Watson-Crick interactions between these nucleotides are essential for ribosomal function. Moreover, we find that certain mild perturbations of the structure, for example, creation of G-U wobble pairs, generate resistance to streptomycin, an antibiotic known to interfere with the decoding process. Chemical probing of mutant ribosomes from streptomycin-resistant cells shows that the mutant ribosomes have a reduced affinity for streptomycin, even though streptomycin is thought to interact with a site on the 30S subunit that is distinct from the 530 region. Data from earlier in vitro assembly studies suggest that the pseudoknot structure is stabilized by ribosomal protein S12, mutations in which have long been known to confer streptomycin resistance and dependence.

Base Sequence↗
PubMed1991

Specific structural probing of plasmid-coded ribosomal RNAs from Escherichia coli.

The preferred method for construction and in vivo expression of mutagenised Escherichia coli ribosomal RNAs (rRNAs) is via high copy number plasmids. Transcription of wild-type rRNA from the seven chromosomal rrn operons in strains harbouring plasmid-coded mutant rRNAs leads to a heterogeneous ribosome population, which consequently hinders direct probing of mutant rRNAs. Here, we describe how nonconserved helical regions of plasmid-coded rRNA have been altered in a manner that preserves their secondary structures while creating new sites for primer extension of mutant rRNAs. This facilitates specific biochemical probing of mutagenised rRNA regions despite the background of wild-type molecules. Four priming sites have been made to investigate the structural effects of mutations in the GTPase centre, helix 1200-1250, the peptidyl transferase region and the alpha-sarcin loop of 23S rRNA.

Anti-Bacterial Agents↗
PubMed1991

A nutrition support team quality assurance plan.

The Nutrition Support Team at the Veterans Affairs Medical Center is responsible for the development of guidelines related to the provision of both total parenteral nutrition and enteral nutrition support. A Quality Assurance plan which was approved by the Joint Commission on Accreditation of HealthCare Organizations was implemented by the Nutrition Support Team. This plan addresses: nutritional assessment and consultation, indications for enteral and parenteral nutrition support, provision of optimal nutrition support including attainment of nutritional goals, and the prevention, detection, and management of complications. The indicators and criteria for each aspect of care are described. This program has provided documentation of the activities of the Nutrition Support Team as well as data defining the patient population requiring specialized nutrition support, and has helped identify areas where improvement is required.

Dietary Services↗
PubMed1990

Dominant lethal mutations in a conserved loop in 16S rRNA.

The 530 stem-loop region in 16S rRNA is among the most phylogenetically conserved structural elements in all rRNAs, yet its role in protein synthesis remains mysterious. G-530 is protected from kethoxal attack when tRNA, or its 15-nucleotide anticodon stem-loop fragment, is bound to the ribosomal A site. Based on presently available evidence, however, this region is believed to be too remote from the decoding site for this protection to be the result of direct contact. In this study, we use a conditional rRNA expression system to demonstrate that plasmid-encoded 16S rRNA genes carrying A, C, and T point mutations at position G-530 confer a dominant lethal phenotype when expressed in Escherichia coli. Analysis of the distribution of plasmid-encoded 16S rRNA in ribosomal particles, following induction of the A-530 mutation, shows that mutant rRNA is present both in 30S subunits and in 70S ribosomes. Little mutant rRNA is found in polyribosomes, however, indicating that the mutant ribosomes are severely impaired at the stage of polysome formation and/or stability. Detailed chemical probing of mutant ribosomal particles reveals no evidence of structural perturbation within the 16S rRNA. Taken together, these results argue for the direct participation of G-530 in ribosomal function and, furthermore, suggest that the dominant lethal phenotype caused by these mutations is due primarily to the mutant ribosomes blocking a crucial step in protein synthesis after translational initiation.

Base Sequence↗
PubMed1990

Statistical analysis of pharmacokinetic data with special applications to bioequivalence studies.

The objectives of this investigation are: 1) to describe techniques for determining the validity of the assumptions; 2) to suggest data transformations which may validate the use of parametric procedures; and 3) to describe a non-parametric alternative to the analysis of variance for crossover designs. Two assumptions common to all parametric procedures include the underlying normal distribution of the observations and equality of variances across treatment groups. Normal probability plots and/or stem and leaf plots are good diagnostic techniques to address the assumption of normality, while Bartlett's test is the most common method of determining equality of variances. To evaluate bioequivalence data, the Food and Drug Administration suggests the use of analysis of variance for crossover designs. If the underlying assumptions are valid, the appropriate statistical models are well known. On the other hand, if the assumptions are not valid, the investigator has one of two choices: 1) transform the data in such a way as to satisfy the assumptions, or 2) use a non-parametric procedure. Square root or logarithmic transformations are commonly used in this situation. However, if a suitable transformation cannot be found, then a non-parametric procedure should be used. Koch (Biometrics (1972) 28, 577-584) developed a non-parametric crossover test, which is relatively easy to apply, but the corresponding power calculations required by the FDA are less obvious.

Animals↗
PubMed1990

Clinical and economic effect of ciprofloxacin as an alternative to injectable antimicrobial therapy.

The effect of the use of oral ciprofloxacin on patient outcome and the cost of antimicrobial therapy was investigated. In 1988 ciprofloxacin was placed on the antimicrobial formulary at a Veterans Affairs medical center. Patients with urinary tract infections, soft tissue infections, osteomyelitis, or pneumonia due to organisms that were documented as being susceptible to ciprofloxacin and either resistant to other oral antimicrobials or susceptible to other oral antimicrobials in patients allergic to such agents were monitored in a prospective open study over 12 months. When a patient was enrolled, the physician was asked to select the i.v. antimicrobial regimen that would have been used if ciprofloxacin were not available. Patient outcome was determined from medical records, and the difference in the costs of the oral and i.v. regimens was calculated. Clinical cure occurred in 96/100 (96%) of patients with urinary tract infection, 19/22 (86%) with soft tissue infection, 14/16 (88%) with osteomyelitis, and 10/12 (83%) with pneumonia. The overall cure rate was 139/150 (93%). The 11 clinical failures occurred in patients infected with methicillin-resistant Staphylococcus aureus (MRSA) alone, group D enterococcus alone, MRSA and Pseudomonas sp., and Pseudomonas sp. alone. The total cost avoidance achieved by using oral ciprofloxacin instead of i.v. antimicrobials was $77,158. Oral ciprofloxacin was an effective and cost-efficient alternative to traditional i.v. antimicrobial therapy in the patients studied.

Anti-Infective Agents↗