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F L Murphy

Publications and source records attributed to F L Murphy.

17 recordsLinked to original sources
PubMed1994

Coaxially stacked RNA helices in the catalytic center of the Tetrahymena ribozyme.

Coaxial stacking of helical elements is a determinant of three-dimensional structure in RNA. In the catalytic center of the Tetrahymena group I intron, helices P4 and P6 are part of a tertiary structural domain that folds independently of the remainder of the intron. When P4 and P6 were fused with a phosphodiester linkage, the resulting RNA retained the detailed tertiary interactions characteristic of the native P4-P6 domain and even required lower magnesium ion concentrations for folding. These results indicate that P4 and P6 are coaxial in the P4-P6 domain and, therefore, in the native ribozyme. Helix fusion could provide a general method for identifying pairs of coaxially stacked helices in biological RNA molecules.

Animals↗
PubMed1994

Two major tertiary folding transitions of the Tetrahymena catalytic RNA.

The L-21 Tetrahymena ribozyme, an RNA molecule with sequence-specific endoribonuclease activity derived from a self-splicing group I intron, provides a model system for studying the RNA folding problem. A 160 nucleotide, independently folding domain of tertiary structure (the P4-P6 domain) comprises about half of the ribozyme. We now apply Fe(II)-EDTA cleavage to mutants of the ribozyme to explore the role of individual structural elements in tertiary folding of the RNA at equilibrium. Deletion of peripheral elements near the 3' end of the ribozyme destabilizes a region of the catalytic core (P3-P7) without altering the folding of the P4-P6 domain. Three different mutations within the P4-P6 domain that destabilize its folding also shift the folding of the P3-P7 region of the catalytic core to higher MgCl2 concentrations. We conclude that the role of the extended P4-P6 domain and of the 3'-terminal peripheral elements is at least in part to stabilize the catalytic core. The organization of RNA into independently folding domains of tertiary structure may be common in large RNAs, including ribosomal RNAs. Furthermore, the observation of domain-domain interactions in a catalytic RNA supports the feasibility of a primitive spliceosome without any proteins.

Animals↗
PubMed1994

GAAA tetraloop and conserved bulge stabilize tertiary structure of a group I intron domain.

The 413 nucleotide self-splicing group I intron from Tetrahymena thermophila pre-rRNA contains a 160 nucleotide independently folding domain of RNA tertiary structure, the P4-P6 domain. This domain consists of sequence elements highly conserved among group I introns (P4 and P6) and peripheral extensions conserved in certain subgroups of these introns (P5abc and P6ab). The effect of mutation of selected bases on the formation of domain structure was analyzed using two probes: solvent-based Fe(II)-EDTA, which monitors backbone accessibility, and dimethyl sulfate, which monitors availability of N(1) of adenine and N(3) of cytosine. A GAAA tetraloop and an adenosine-rich bulge were found to stabilize domain tertiary structure in a sequence-specific manner. A single base change in the GAAA tetraloop disrupted Fe(II)-EDTA protection both locally and in P6a, and a specific base-pair substitution in P6a similarly disrupted protection locally and in the tetraloop; thus remote elements of the secondary structure are linked in tertiary structure. Our model of the domain's tertiary structure is refined to include this long-range tertiary interaction. The interaction requires severe bending of the domain RNA such that sequences separated by approximately 50 bases of largely double-stranded RNA are in proximity in the tertiary structure. The bending causes or allows for contact between sequences of the conserved core and sequences of the P5 extension. Thus the P5 extension may serve to stabilize the structure of the intron core in vivo.

Animals↗
PubMed1994

Visualization of a tertiary structural domain of the Tetrahymena group I intron by electron microscopy.

The P4-P6 domain RNA of the group I intron of Tetrahymena thermophila has previously been shown by chemical probing to be an independently folding domain of the intron's tertiary structure. To directly visualize this tertiary structure, the P4-P6 domain and two folding defective mutants were prepared for high-resolution electron microscopy using tungsten shadowcasting. In the presence of Mg2+, the P4-P6 domain predominantly consists of compact molecules, while the two mutant RNAs are nearly all rod-like molecules. The measured length of the rod-like molecules is 64 (+/- 6) bp, which agrees closely with the length expected for molecules containing secondary structure only. In the absence of Mg2+, the P4-P6 domain contains threefold or tenfold fewer compact structures (depending on the mounting procedures) than in the presence of Mg2+. These results provide direct evidence for the overall shape of the tertiary structure proposed on the basis of biochemical experiment, and they confirm the Mg2+ dependence of tertiary folding. An equilibrium between the extended (rod-like) and the compact structures is suggested, with the concentration of bound Mg2+ and different mounting methods influencing the direction of the equilibrium. The entire group I ribozyme (L-21 Sca I RNA) was also examined by electron microscopy in the presence of Mg2+, and was revealed to have a compact shape. These studies present a direct demonstration of long-range interactions in a catalytic RNA molecule.

Animals↗
PubMed1993

An independently folding domain of RNA tertiary structure within the Tetrahymena ribozyme.

The Tetrahymena thermophila pre-rRNA contains a 413-nucleotide self-splicing group I intron. This intron has been converted into a sequence-specific endonuclease or ribozyme. A 160-nucleotide portion of the ribozyme consisting of both highly conserved sequence elements (P4 and P6) and nonconserved peripheral extensions (P5abc and P6ab) was synthesized as a separate molecule. Solvent-based Fe(II)-EDTA, a probe that monitors higher-order RNA structure, revealed a protection pattern that was a large subset of that observed in the whole ribozyme. Data from dimethyl sulfate modification and partial digestion with nucleases were also consistent with maintenance of the proper secondary and tertiary structure in the shortened RNA molecule. Thus, this 160-nucleotide molecule (P4-P6 RNA) is an independently folding domain of RNA tertiary structure. A series of mutations and deletions were made within the P4-P6 domain to further dissect its tertiary structure. Fe(II)-EDTA and dimethyl sulfate analysis of these mutants revealed that the domain consists of two substructures, a localized subdomain involving the characteristic adenosine-rich bulge in P5a, and a subdomain-stabilized structure involving long-range interactions. Therefore, like some proteins, the intron RNA is modular, containing a separable domain and subdomain of tertiary structure.

Animals↗
PubMed1993

Reconstructive management of patients with greater than 80 per cent TBSA burns.

With improving acute burn care, greater numbers of patients are surviving large burns. Meshed skin grafts or cultured epithelial autografts are often required to achieve rapid wound closure, even in areas such as the hands or face. This, plus the lack of suitable donor tissue for reconstruction, is mandating a change in reconstructive principles. Twenty-eight patients surviving > or = 80 per cent TBSA full skin thickness burns were evaluated using two specially devised instruments (Inventory of Potential Reconstructive Needs; Donor Tissue Surveillance). A total of 564 reconstructive needs were identified in the 28 patients, an average of 20.1 per patient. There were 265 defects in the head and neck, 143 in the upper extremities, and 156 in the torso/lower extremities. The injured anatomical units most frequently identified were the hand (74), trunk (60), nose/nasolabial fold (48), mouth (46), ankle/foot (42), neck (31) and check (28). The Donor Tissue Surveillance form revealed that the necessary donor tissue was frequently not available, and when available, was often of poor quality. These facts require a different set of priorities for reconstruction of the massively burned patient. No longer can a simple stepwise plan of active function, passive function, and aesthetic needs be followed. The patient and family desires must be combined with a realistic outlook by the entire burn team to determine the most judicious and efficient use of available donor tissue to meet the reconstructive needs.

Burns↗
PubMed1992

RNA substrate binding site in the catalytic core of the Tetrahymena ribozyme.

In catalysis by group I introns, the helix (P1) containing the RNA cleavage site must be positioned next to the guanosine binding site. We have identified a conserved adenine in the catalytic core that contributes to the stability of this arrangement and propose that it accepts a hydrogen bond from a specific 2'-OH in P1. Such base-backbone tertiary interactions may be generally important to the organization of RNA tertiary structure.

Adenine↗
PubMed1992

Alkalinization of mepivacaine for axillary block.

We examined the onset and distribution of sensory blockade, the onset of motor blockade, and venous mepivacaine concentrations after axillary block with 1.25% mepivacaine with and without bicarbonate. There were no statistically significant differences between the alkalinized and placebo groups with respect to distribution of analgesia or anesthesia, time to onset of analgesia, or time to onset of paresis. However, alkalinization significantly decreased the time to onset of anesthesia in the medial cutaneous nerve of the forearm, the median nerve, and the ulnar nerve, as well as the time to onset of paralysis. Concentrations of mepivacaine in venous blood did not differ significantly. We conclude that alkalinized mepivacaine offers the advantage of quicker onset of more profound blockade in several terminal nerve distributions.

Adult↗
PubMed1990

Low-dose lovastatin safely lowers cholesterol after cardiac transplantation.

Hypercholesterolemia occurs in many cardiac transplant patients and may aggravate graft coronary arteriopathy as well as contributing to peripheral vascular disease. Lovastatin, which inhibits 3-hydroxy-3-methylglutaryl coenzyme A reductase, in doses of 40-80 mg/day effectively lowers cholesterol in the general cardiac population but has been associated with rhabdomyolysis in cardiac transplant recipients. To determine whether lower doses of lovastatin would be effective and safe for lowering cholesterol after cardiac transplantation, 44 patients with blood cholesterol greater than 200 mg/dl at least 6 months after cardiac transplantation received 10-20 mg lovastatin daily. In addition, lovastatin enzyme inhibitor level was assayed in six patients to determine whether metabolism of the drug was abnormal. Lovastatin decreased total cholesterol by 28% from 282 +/- 54 to 208 +/- 62 mg/dl (p less than 0.005), primarily because of reduction in the low-density lipoprotein fractions, and was well-tolerated without any symptoms or abnormal creatine phosphokinase levels in 43 of 44 patients. One patient developed rhabdomyolysis and reversible renal failure when lovastatin was increased to 40 mg daily. Enzyme inhibitor levels in the six transplant patients were 4.2-7.8 times higher than those measured in normal volunteers. Low-dose lovastatin effectively lowers cholesterol in patients after transplantation, but metabolism is altered, perhaps by cyclosporine. Monitoring of enzyme inhibitor levels may be required to allow safe administration of this drug to cardiac transplant recipients.

Cholesterol↗
PubMed1989

Alteration of substrate specificity for the endoribonucleolytic cleavage of RNA by the Tetrahymena ribozyme.

A shortened form of the intervening sequence of the self-splicing RNA from Tetrahymena thermophila catalyzes sequence-specific cleavage of RNA. Cleavage site selection involves a base-pairing interaction between the substrate RNA and a binding site within the intervening sequence. Single-base changes in this binding site were previously shown to alter substrate specificity in a predictable manner. To examine the generality with which substrate specificity can be altered, six variant catalytic RNAs (ribozymes) have been produced with two- or three-base changes in the active site. Each ribozyme cleaves its predicted substrate. The conditions required for good reactivity and for discrimination against cleavage at mismatched sites vary and were independently determined for each ribozyme.

Animals↗
PubMed1984

Diffuse slow washout of myocardial thallium-201: a new scintigraphic indicator of extensive coronary artery disease.

When coronary artery disease is extensive and of relatively uniform severity, regional myocardial hypoperfusion may be balanced during stress, precluding development of spatially relative perfusion defects. Assessment of the washout of thallium-201 from myocardial regions may provide diagnostic assistance in these cases because washout analysis is spatially nonrelative and hypoperfused myocardial regions manifest a slow thallium-201 washout rate. In 1,265 consecutive patients having quantitatively analyzed stress-redistribution scintigraphy, 46 had a diffuse slow washout pattern with no or a maximum of one regional perfusion defect. Thirty-two underwent clinically indicated coronary angiography, and 23 (72%) of these were found to have three vessel or left main disease. Of 30 similar patients without a diffuse slow washout pattern and with no or a maximum of one perfusion defect, only 5 (17%) had extensive coronary disease. An independent relation between diffuse slow washout and extensive coronary disease was demonstrated by a Mantel- Haentzel chi-square analysis of a wide variety of other indexes of extensive disease. A diffuse washout abnormality, even in the absence of other scintigraphic, clinical or electrocardiographic indicators, carries a high predictive value for three vessel or left main coronary artery disease. The predictive value is maintained when the exercise level achieved is submaximal. Although an infrequent occurrence (3.6% of tested patients), a diffuse slow washout pattern without other scintigraphic indications of extensive coronary disease should lead to further diagnostic testing.

Coronary Angiography↗