Biomedical subjects
Publications and source records attributed to P Woolley.
This article describes a current view of the events that initiated the transition from the rich organic and inorganic chemistry of the primitive Earth to the earliest forms of life. It is a personal condensation of the basic ideas developed in the so-called Göttingen school. Most of these will be found in the seminal paper of Eigen and the other sources cited. A detailed exposition is given by Küppers.
We have investigated the use of the pH-sensitive fluorescein label as a probe for electrostatic potential in macromolecules. The practicality of this technique is demonstrated by its application to the 16S RNA molecule. The dependence of the electrostatic potential upon ionic conditions and upon the presence of ribosomal proteins and the state of the RNA was studied. The combination of electrostatic and anisotropy data emphasizes the rôle of the 30S ribosomal proteins, rather than of the renaturation of the 16S RNA or the presence of the 50S subunit, in shaping the environment of the 3' terminus of the 16S RNA in the active ribosome.
The procedures developed earlier (Friedrich and Woolley, preceding paper in this journal) for probing electrostatic potential with the fluorescein label were applied to transfer RNA. By using tRNA species that contain chemically reactive bases we were able to label these bases with fluorescein derivatives and thus to 'map' the electrostatic potential around the molecule. Both the electrostatic potential and the fluorescence emission anisotropy data that were obtained at the same time could be understood in terms of the well-known, paradigmatic crystal structure of tRNA(Phe). However, within the distribution of the various tRNA species, tRNA(Met)f appeared to occupy an extreme position, which suggests a relation between the conformation in solution and the initiation function of this molecule. Comparison with theoretical predictions by others of the electrostatic potential map of tRNA showed agreement in respect of trends, but the values of the potentials measured were orders of magnitude lower than predicted. This we attribute primarily to solvation.
Using the technique of singlet-singlet (Förster-type) resonance energy transfer, we have determined five distances in the programmed ribosome, either with the P site or with both the A and the P sites occupied. Two of the distances are new and two agree with earlier measurements; the fifth showed disagreement in detail with earlier results of others, but a consistent general trend. The distances substantiate a current model for the location of ribosomally bound tRNA, except in regard of the position of the 3' end of P-site tRNA, which seems according to our results to lie too far away from the 3' terminus of the 16 S RNA to be accommodated in the model. We present new evidence for the hypothesis that anomalously charged tRNA does not bind to the cognately programmed A site in the same way as does tRNA charged with an amino acid. Occupation of the A site restricts mobility of the 3' end of tRNA in the P site.
77 patients with a first attack of genital herpes were entered into a double-blind trial to compare the efficacy of acyclovir with that of inosine pranobex. 24 patients received acyclovir with that of inosine pranobex, and 28 both drugs. Patients treated with acyclovir or both drugs healed more quickly and had a shorter duration of viral shedding than those treated with inosine pranobex. The time to first recurrence and frequency of subsequent recurrences were similar in the three treatment groups. Acyclovir is the treatment of choice for patients with a first attack of genital herpes.
The general case of Förster-type energy transfer is that in which energy is exchanged in both directions between two unlike fluorophores. In such cases, energy is transferred from the conventionally defined donor to the conventionally defined acceptor (forward transfer) and at the same time from the acceptor to the donor (reverse transfer). Expressions are derived to describe the fluorescence intensities and lifetimes of fluorophores undergoing simultaneous forward and reverse transfer; these are compared with corresponding quantities for the case more usually considered, in which only forward transfer is significant. It is shown that the presence of reverse transfer removes the distinction between donor and acceptor, and allows such anomalous effects as 'acceptor quenching'. A confirmatory example is described. It is shown that the equations generally used in distance determination by steady-state fluorescence spectroscopy can also be applied in the presence of reverse transfer, if a correction term is included; however, for lifetime spectroscopy the correction is more complex.
Fluorescence anisotropy studies performed on 6-demethylchlortetracycline, binding to the ribosome of E. coli in competition with tRNA at the P site or at both P and A sites, have provided a quantitative assessment in situ of the interaction of this antibiotic with the A site and have demonstrated that there is also an interaction between tetracycline and the P site.
The binding of Phe-tRNAPhe at the programmed ribosomal A site has been investigated using antibiotics that influence this binding in different ways. The adhesion of Phe-tRNAPhe, the consumption of GTP and the extent of the peptidyl transfer reaction were monitored. All of the five known misreading-inducing antibiotics that were tested stabilised the binding of Phe-tRNAPhe after its affixture to the A site by EF-Tu with GTP hydrolysis. The stabilisation was sufficient to overcome a single mismatch in the codon-anticodon interaction. Combinations of stabilising and destabilising influences were found to be additive, thus supporting the concepts: (1) that there is a 'correct' binding energy for aminoacyl tRNA in the A site, whose reduction hampers polypeptide synthesis and whose increase makes it inaccurate by by-passing proofreading; and (2) that the different antibiotics affect the bound aminoacyl tRNA at different points.
Sixteen patients were enrolled in a Phase I study of the combined use of recombinant DNA alpha-2-interferon (IFN) and radiation therapy, conducted at the Georgetown University Hospital (GUH) from February 1, 1984 to September 20, 1985. Escalating IFN doses ranging from 2.0 X 10(6) IU/m2 to 5 X 10(6) IU/m2 were administered to groups of six patients per IFN dose level. Three patients at each dose level were treated on a 5-day-a-week schedule and three patients were treated on a 3-day-a-week schedule. Significant toxicity including dehydration, infection, deep vein thrombosis, and myocardial infarction was noted throughout in patients receiving IFN five times per week, with eight of nine requiring hospitalization during the treatment course. There was one treatment-related death. In the five-times-per-week group, only 22% of patients tolerated the full initially planned IFN dosage and 44% tolerated the full initially planned radiation dosage, compared to 100 and 86%, respectively, in the three-times-per-week group. A tolerance dose and schedule of 5.0 X 10(6) IU/m2 of alpha-2-interferon administered subcutaneously three-times-per-week in conjunction with standard radiotherapy has been identified for use in future combined modality trials.
It is shown on the basis of the excluded-volume effect that inert macromolecules may be expected to suppress the dissociation of double-helical nucleic acids into single helices and thus to raise the melting point of the double helix. The rise in melting temperature of the ribonucleic acid [poly(I).poly(C)] caused by dextran polymers and by poly(ethylene oxide) is described and compared with the theoretical prediction. Good agreement was found in respect of the extent of the rise in melting point and in respect of its dependence upon polymer length. An additional dependence upon the identify of the polymer was attributed to detailed effects of shape in solution.
Ribosomes from Escherichia coli were programmed by being allowed to bind a molecule of tRNAMetf or fMet-tRNAMetf and the hexanucleotide messenger AUGN1N2N3. The interaction of the ternary complex [EF-Tu X GTP X Phe-tRNAPhe] with the A site (containing the codon N1N2N3) was then studied by measuring the extent of (i) the binding of Phe-tRNAPhe to the ribosome, (ii) the hydrolysis of GTP, and (iii) the formation of the dipeptide fMet-Phe. By variation of N1,N2, and N3, a defined degree and position of mismatch could be obtained; the correct A-site codon UUU was compared with the incorrect codons CUU, UCU, GUU, and UUG. Each single-point alteration led to catalytic hydrolysis of GTP and to a strong reduction in the amounts of Phe-tRNAPhe binding and of dipeptide formation. The observations were explicable qualitatively by a hypothesis according to which the behavior of the bound aa-tRNA, after hydrolysis of GTP and before peptidyl transfer, is determined principally by the energy of binding of the aminoacyl-tRNA to the A site. This binding in turn was found to depend upon both the nature and the position of the mismatch. The results further suggest a steric interplay between the 3' (acceptor) end of the A-site tRNA and the second and third positions of the anticodon, so that a mismatch at one of these positions can impair directly the interaction between the aminoacylated 3' end and the ribosome and can thus reduce the rate of peptide bond formation and contribute to the overall fidelity of the elongation cycle.
The binding of demeclocycline (6-demethylchlortetracycline) to ribosomes and ribosomal subunits from Escherichia coli was investigated by using the fluorescence anisotropy of the antibiotic to determine the extent of binding. Binding data obtained from 70S and 30S particles differed fundamentally from those obtained from 50S subunits: the first two showed a strong, specific interaction while the third did not. In addition, all three particles possessed weak, unspecific binding sites. Computer-aided least-squares analysis of the data yielded the following numbers of sites and equilibrium constants: for 30S, n1 = 1, K1 = 2.2 X 10(6) M-1, n2 K2 = 0.029 X 10(6) M-1; for 50S, n1 = 0, n2 K2 = 0.035 X 10(6) M-1; for 70S, n1 = 1, K1 = 3.2 X 10(6) M-1, n2 K2 = 0.082 X 10(6) M-1. These data resolve current disagreement in the literature and are a prerequisite for quantitative studies of the mechanism of inhibition by tetracycline of protein biosynthesis.
Twenty-one patients with head and neck carcinomas relapsing after radiotherapy were treated with a combination of cis-platinum, bleomycin, and methotrexate. Four patients (19%) achieved a partial response. Toxicity was significant in selected cases; three patients developed WBC counts less than 1,000/mm3 and one of these patients died with sepsis. Severe mucositis was present in three of the twenty-one patients. Considering the toxicity of this combination and the limited therapeutic activity with the dose and schedule used in this study, this regimen is not recommended for the treatment of squamous head and neck carcinomas relapsing after radiotherapy.
In spite of considerable effort there is still serious disagreement in the literature about the question of whether epitopes of ribosomal protein S4 are accessible for antibody binding on the intact small ribosomal subunit. We have attempted to resolve this issue using three independent approaches: (i) a re-investigation of the exposure and the location of epitopes of ribosomal protein S4 on the surface of the 30S subunit and 30S core particles of the E. coli ribosome, including rigorous controls of antibody specificity, (ii) a similar investigation of protein S4 from Bacillus stearothermophilus and (iii) the labelling of residue Cys-31 of E. coli S4 with a fluorescein derivative the accessibility of which towards a fluorescein-specific antibody was demonstrated directly by fluorimetry. In each of the three cases the antigen (E. coli S4, B. stearothermophilus S4 or fluorescein) was found to reside on the small lobe.
Ribosomal proteins L6, L11 and the complex [(L12)4 X L10] were labelled specifically at their respective single thiol groups, either with the acetylaminoethyl-dansyl or with the acetamidofluorescein fluorophore. The labelled proteins were then reconstituted, singly or in pairs, into ribosomal 50S subunits; the presence of the label had no observable effect on the composition, shape or activity of the reconstituted subunits. The distances between the labelled thiol groups were measured by a fluorescence energy transfer method detailed elsewhere [Epe, B. et al. (1983) Proc. Natl Acad. Sci. USA, 80, 2579-2583] and were found to be: for L6-L10, 60 A (6.0 nm); for L6-L11, 46 A (4.6 nm); for L10-L11, 56 A (5.6 nm). Reversal of the direction of energy transfer by exchanging labels gave duplicate distances which differed, on average, by about 4%. The distance between the fluorescent labels on L10 and L11 in the [23 S-RNA X L10 X L11 X (L12)4] ribonucleoprotein complex was the same as in the 50S subunit, but all three distances were greater in 50S subunits which had been reconstituted without the final activation step (incubation at 50 degrees C). This suggests a tightening of the L6/L10/L11 domain of the 50S subunit during the activation step.
By coupling fluorescein to a defined amino acid of a single ribosomal protein and incorporating this protein into the ribosome, we have obtained ribosomes labelled at a single, defined position. A fluorescein-specific antibody preparation was used to locate the fluorescein residues bound to the two cysteines at positions 58 and 63 of protein S17 and to the cysteine at position 86 of protein L6. This study demonstrates the advantages which accrue from the combination of electron microscopy and fluorimetry.
Explore the source record for details and available documents.
Intercalation of the ethidium analogue 3,5-diazido-5-ethyl-6-phenylphenanthridinium into double helices followed by irradiation with blue or ultraviolet light results in cross-linking between the two strands with an efficiency around 30% for DNA, RNA, and DNA-RNA hybrids. Details of this reaction and a convenient synthesis of the ethidium analogue are described. Stable tertiary structure in RNA impedes intercalation and thus reduces the efficiency of cross-linking. In contrast to the ethidium derivative, various acridine diazides show little or no cross-linking ability.