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Biomedical subjects

E Wickstrom

Publications and source records attributed to E Wickstrom.

At least 73 records · Page 4Linked to original sources
PubMed1981

Synthesis and intramolecular crosslinking of chlorambucilyl (prolyl)n [3H]phenylalanyl-tRNAPhe (yeast), n = 0, 5, 11 and 15.

Rigid, variable-length oligoproline crosslinking reagents, which we call molecular rulers, are a potentially powerful tool for probing the solution structures of tRNA and other biological macromolecules. We wish to demonstrate the feasibility of molecular rulers on a well-studied model system, yeast phenylalanine tRNA, before applying them to less well understood structures. We have found chlorambucil (4-(4-(bis(2-chloroethyl)amino)phenyl)-butanoic acid) to be suitable for use as an alkylating function attached to the imino end of oligo-L-proline spacers which are peptide bonded at their carboxyl ends to the alpha-amine of [3H]Phe-tRNAPhe (yeast). Chlorambucil and chlorambucilyl oligoprolines may be readily and sensitively assayed by their alkylation kinetics with aqueous pyridine as measured by optical absorbance of the product. The pyridine reaction seemed to be the first order in chlorambucil, k1 = (5.4 +/- 1.0) X 10(-3) min-1, zero order in pyridine, and was strongly inhibited by Me2SO. Filter assays of tRNA alkylation by chlorambucilyl [3H]prolyl proline suggested that this reaction is also first order in alkylation reagent, but somewhat dependent on tRNA concentration, and also strongly inhibited by Me2SO. Full alkylation activity was regained upon removal of Me2SO. Modification of [3H]Phe-tRNAPhe (yeast) with the N-hydroxysuccinimide esters of chlorambucilyl (prolyl)n was accomplished with yields of 100% for n = 0, 92% for n = 5, 94% for n = 11 and 44% for n = 15, in 80% Me2SO/CHCl3 at pH 9, 37 degrees C, conditions under which chlorambucil alkylation of tRNA is strongly inhibited. The rates of intramolecular crosslinking of chlorambucilyl (prolyl)n [3H]Phe-tRNAPhe (yeast) were measured assuming a first-order process, giving K1 = (5.3 +/- 0.2) X 10(-3) min-1 for n = 0, (3.2 +/- 0.4) X 10(-4) min-1 for n = 5, (6.8 +/- 0.8) X 10(-5) min-1 for n = 11 and (1.6 +/- 0.4) X 10(-4) min-1 for n = 15. Yields of intramolecularly crosslinked tRNA were 80% for n = 0 after 4 h in 10 mM NH4OAc (pH 6)/1 mM Mg(OAc)2 at 37 degrees C, 7% for n = 5, 3% for n = 11, and 5% for n = 15.

Chemical Phenomena↗
PubMed1981

Molecular rulers for measuring RNA structure: sites of crosslinking in chlorambucilyl-phenylalanyl-tRNAPhe (yeast) and chlorambucilyl-pentadecaprolyl-phenylalanyl-tRNAPhe (yeast) intramolecularly crosslinked in aqueous solution.

Intramolecular crosslinking of yeast phenylalanine tRNA in aqueous solution with rigid, variable-length crosslinking reagents, which we call "molecular rulers," has given results in reasonable agreement with the crystal structure. Chlorambucilyl-[3H]phenylalanyl-tRNAPhe crosslinked intramolecularly at G-71 and A-73, whereas chlorambucilyl-pentadecaprolyl-[3H]phenylalanyl-tRNAPhe crosslinked at G-20 and Y-37. The pentadecaprolyl reagent was predicted to be 62 A long, including chlorambucil and phenylalanine; the sites that it reached are 60 A distant from the 3' OH (in the case of G-20) or 80 A distant (in the case of Y-37) in the crystal structure of tRNAPhe. The close agreement between the length of the reagent and the distance of G-20 from the 3' OH in the crystal structure illustrates the rigidity of the tRNAPhe molecule in the dihydrouridine loop region at the corner of the molecule. The apparent ability of the 62-A-long reagent to crosslink to a site, Y-37, that is 80 A distant from the 3' OH in the crystal structure appears to illustrate the flexibility of both the 3' A-C-C-A terminus and the anticodon stem and loop, with respect to the tRNA molecule. These observations demonstrate the utility of oligoproline-based crosslinking reagents as rigid, variable-length molecular rulers for biological macromolecules in solution.

Alkylation↗
PubMed1980

Circular dichroism study of Escherichia coli initiation factor 3 binding to nucleic acids.

The circular dichroic spectral features of (A)10-20, (C)10-20, A8UGU6, poly(A), and poly(C), at both neutral and acidic pH values and in the presence and absence of Mg2+, are significantly altered by Escherichia coli initiation factor 3 (IF3), implying the occurrence of protein-induced changes in nucleic acid secondary structure. Similarly, the circular dichroic spectral characteristics of helical poly(U), poly-(A)-poly(U), and poly(I)-poly(C) are modified by IF3. However, no structural perturbation of poly(A)-poly(U) occurs in the absence of Mg2+ by IF3. The oligonucleotides (A)10-20 and (C)10-20 at both pH 7.5 and 5.5 titrate to end point of 26 +/- 4 nucleotide residues per IF3 [except (C) 10-20 at pH 5.5 which titrates to 17 +/- 1 nucleotide residues per IF3], whereas the hairpin A8UGU6 under similar conditions at neutral pH and in the presence of Mg2+ titrates to an end point of 56 +/- 3 nucleotide residues per IF3, thereby suggesting the presence of multiple binding sites on the protein. By contrast, poly(A) and poly(C) at neutral pH and in the absence of Mg2+ titrate to an end point of 13 +/- 1 nucleotide residues per IF3. The occurrence of significant light-scattering artifacts precluded a determination of the end point stoichiometry in most other cases. The circular dichroic spectra of E. coli tRNA, MS2 RNA, phiX174 DNA, and sonicated calf thymus DNA were unaffected by IF3 at physiological concentrations. Addition of an equimolar mixture of IF3 and ribosomal protein S1 titrates the circular dichroism of poly(C) at acid pH as did S1 alone. However, addition of IF3 to mixture of poly(A) and S1 at neutral pH did not result in significant titration of the optical activity until IF3 was in excess over S1, even though filter binding assays indicate normal IF3 binding to the polynucleotide. The possible relation of these observations to the biological function of IF3 is briefly considered.

Circular Dichroism↗
PubMed1980

Chlorambucil inhibition by dimethyl sulfoxide and thiosulfate: implications for chlorambucil chemotherapy.

Chlorambucil alkylation of pyridine, a model for nucleotides, appears to be first order in chlorambucil, but zero order in pyridine, and is strongly inhibited by dimethylsulfoxide. The same results are seen with chlorambucilyl [3H]prolyl proline; full alkylation activity was regained upon removal of dimethylsulfoxide. Chlorambycilyl [3H]prolyl proline alkylation of tRNA also appears to be first order in alkylator, but somewhat dependent on tRNA concentration, and is also strongly inhibited by dimethylsulfoxide. 10 mM Na2S2O3 completely inhibits alkylation by aqueous 1 mM chlorambucil. Up to 1 M chlorambucil in Me2SO retains full alkylation activity for at least a year. We suggest that the risk of carcinogenesis inherent in chlorambucil chemotherapy may be avoided by the topical application or injection of 1 M chlorambucil in Me2SO, in conjunction with oral administration of Na2S2O3.

Alkylation↗
PubMed1980

Modifications of solid phase peptide synthesis to obtain homogeneous oligoprolines in high yield.

When oligoprolines containing a [3H]proline in the second residue from the carboxyl terminus are synthesized by the standard solid phase method using stepwise addition of prolines there appears, in addition to the oligoprolines, a steady increase of ninhydrin-negative, but radioactive, impurities. These impurities were found to be N-trifluoroacetyl oligoprolines, and a substantial amount of diketopiperazine was present at the diproline stage. The trifluoroacetyl blockage of the N-termini was almost complete by the time 14 residues were added. To eliminate these side reactions, a block of Boc[3H]Pro2 was coupled to prolyl-resin in order to avoid the diprolyl-resin stage, and 25% CF3CO2H in CHCl3 was replaced by 4N HCl in dioxane as the deprotecting reagent. These changes eliminated the formation of diketopiperazine and trifluoroacetyl impurities, but still a steady increase of ninhydrin-negative, but radioactive, impurity was seen, reaching 22% after three couplings. These side reactions were traced to the neutralization step, and in order to avoid them it became necessary to eliminate the neutralization step, and replace it by incremental additions of Et3N during the coupling step. As a result of these modifications, we now obtain homogeneous length oligoprolines upon cleavage from the resin, with no further purification, when analyzed by reversed-phase liquid chromatography.

Chromatography↗
PubMed1980

A dose-response study in situational insomnia with zopiclone, a new tranquilizer.

Patients scheduled for next-day elective surgery were included in a randomized double-blind trial that compared the hypnotic and adverse effects of a placebo and four doses (3,5,6,9,13.5 mg) of a new compound, zopliclone. Sleep patterns were evaluated using a questionnaire; side effects were noted, and tests evaluating coordination and alertness were performed. Except for dreams and morning responses, there were significant differences in sleep indices at all dose levels. The 3.5-mg dose produced some significant differences compared with the placebo. All responses to the higher three doses were statistically significant; however, there was only a slight increase in effectiveness between 6 and 9 mg. Alertness, coordination tests, blood pressure, and adverse effects did not differ significantly among doses, except for the incidence of drowsiness and a bitter after-taste in the mouth.

Adolescent↗
PubMed1974

Escherichia coli initiation factor IF3 binding to AUG and AUG-containing single strands and hairpin loops, and nonspecific binding to polymers.

Nitrocellulose filter binding and equilibrium dialysis detected the binding of Escherichia coli initiation factor IF3 to AUG, An UGUm single strands and hairpin loops, poly(A,U,G), poly(U), and f2 RNA. No binding was detected for GUA, A8 U, or the hairpin loop A5 GC5 U5. AUG-specific binding, per nucleotide, is strong; nonspecific binding, per nucleotide, is weak.

Binding, Competitive↗