Correlations between fluorine-19 nuclear magnetic resonance chemical shift and the secondary and tertiary structure of 5-fluorouracil-substituted tRNA.

To complete assignment of the 19F nuclear magnetic resonance (NMR) spectrum of 5-fluorouracil-substituted Escherichia coli tRNA(Val), resonances from 5-fluorouracil residues involved in tertiary interactions have been identified. Because these assignments could not be made directly by the base-replacement method used to assign 5-fluorouracil residues in loop and stem regions of the tRNA, alternative assignment strategies were employed. FU54 and FU55 were identified by 19F homonuclear Overhauser experiments and were then assigned by comparison of their 19F NMR spectra with those of 5-fluorouracil-labeled yeast tRNA(Phe) mutants having FU54 replaced by adenine and FU55 replaced by cytosine. FU8 and FU12, were assigned from the 19F NMR spectrum of the tRNA(Val) mutant in which the base triple G9-C23-G12 substituted for the wild-type A9-A23-FU12. Although replacement of the conserved U8 (FU8) with A or C disrupts the tertiary structure of tRNA(Val), it has only a small effect on the catalytic turnover number of valyl-tRNA synthetase, while reducing the affinity of the tRNA for enzyme. Analysis of the 19F chemical shift assignments of all 14 resonances in the spectrum of 5-fluorouracil-substituted tRNAVal indicated a strong correlation to tRNA secondary and tertiary structure. 5-Fluorouracil residues in loop regions gave rise to peaks in the central region of the spectrum, 4.4 to 4.9 parts per million (p.p.m.) downfield from free 5-fluorouracil. However, the signal from FU59, in the T-loop of tRNA(Val), was shifted more than 1 p.p.m. downfield, to 5.9 p.p.m., presumably because of the involvement of this fluorouracil in the tertiary interactions between the T and D-loops. The 19F chemical shift moved upfield, to the 2.0 to 2.8 p.p.m. range, when fluorouracil was base-paired with adenine in helical stems. This upfield shift was less pronounced for the fluorine of the FU7.A66 base-pair, located at the base of the acceptor stem, an indication that FU7 is only partially stacked on the adjacent G49 in the continuous acceptor stem/T-stem helix. An unanticipated finding was that the 19F resonances of 5-fluorouracil residues wobble base-paired with guanine were shifted 4 to 5 p.p.m. downfield of those from fluorouracil residues paired with A. In the 19F NMR spectra of all fluorinated tRNAs studied, the farthest downfield peak corresponded to FU55, which replaced the conserved pseudouridine normally found at this position.     

Seasonal changes in the activation of crossbridge motions of isolated thick filament from Limulus striated muscle

Summary In dynamic light scattering, measurements of the intensity-intensity time correlation function from a suspension of rod-like particles of length L could reveal dynamical information related to translational and internal motions of those particles. For a suspension of thick filaments isolated from the myosin-regulated, striated muscles of Limulus at KL>1 (where K is the scattering vector), the average characteristic linewidth ( ) increased with the addition of Ca²⁺ or with the depletion of ATP. The increase in the with the addition of Ca²⁺ could be due to the presence of energy-requiring, high-frequency motions of the crossbridges activated by Ca²⁺. The increase in which occurred with the depletion of ATP was assumed to be mainly due to the thermal motions of the crossbridges after they had moved radially away from the filament backbone. The percentage increase in following the addition of Ca²⁺ was found to be seasonal, i.e., values of obtained from thick filaments isolated between the middle of June and the middle of September were smaller than those obtained during the rest of the year. The effect of temperature on the percentage increase in was also different. The increase showed a maximum at about 35°C during the summer and at about 25°C at other times. However, the percentage increase in developed under ATP-depleted conditions showed no temperature-related maximum. The number of bound Ca²⁺ per myosin molecule was 1 during the summer and 2 at other times.Abbreviations DLS dynamic light scattering - L length - K scattering vector - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - average characteristic line width Deceased     

Plant thioredoxin h: an animal-like thioredoxin occurring in multiple cell compartments

Thioredoxin h has been purified to electrophoretic homogeneity from spinach roots using a procedure devised for leaves. The root thioredoxin (h2 form) differed from chloroplast and animal thioredoxins in showing an atypical active site (Cys-Ala-Pro-Cys) but otherwise resembled animal thioredoxin in structure. Sequence data for a total of 72 residues of spinach root thioredoxin h2 (about 69% of the primary structure) showed 43-44% identity with rabbit and rat thioredoxin. Analysis of cell fractions from the endosperm of germinating castor beans revealed that thioredoxin h occurs in the cytosol, endoplasmic reticulum, and mitochondria. The present findings demonstrate a similarity between plant thioredoxin h and animal thioredoxins in structure and intracellular location and raise the question of whether these proteins have similar functions.     

Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: a fluorine-19 NMR study.

Interactions of 5-fluorouracil-substituted Escherichia coli tRNAVal with its cognate synthetase have been investigated by fluorine-19 nuclear magnetic resonance. Valyl-tRNA synthetase (VRS) (EC 6.1.1.9), purified to homogeneity from an overproducing strain of E. coli, differs somewhat from VRS previously isolated from E. coli K12. Its amino acid composition and N-terminal sequence agree well with results derived from the sequence of the VRS gene [Heck, J.D., & Hatfield, G.W. (1988) J. Biol. Chem. 263, 868-877]. Apparent KM and Vmax values of the purified VRS are the same for both normal and 5-fluorouracil (FUra)-substituted tRNAVal. Binding of VRS to (FUra)tRNAVal induces structural perturbations that are reflected in selective changes in the 19F NMR spectrum of the tRNA. Addition of increasing amounts of VRS results in a gradual loss of intensity at resonances corresponding to FU34, FU7, and FU67, with FU34, at the wobble position of the anticodon, being affected most. At higher VRS/tRNA ratios, a broadening and shifting of FU12 and of FU4 and/or FU8 occur. These results indicate that VRS interacts with tRNAVal along the entire inside of the L-shape molecule, from the acceptor stem to the anticodon. Valyl-tRNA synthetase also causes a splitting of resonances FU55 and FU64 in the T-loop and stem of tRNAVal, suggesting conformational changes in this part of the molecule. No 19F NMR evidence was found for formation of the Michael adduct between VRS and FU8 of 5-fluorouracil-substituted tRNAVal that has been proposed as a common intermediate in the aminoacylation reaction.     

Inhibition of endotoxin-induced monocytic procoagulant activity by n-alcohols and anesthetics.

1. The effect of n-alcohols (methanol and ethanol) and anesthetics (lidocaine, thiopental, methohexital and thiamylal) on procoagulant activity (PCA) in human peripheral-blood monocytes and non-adherent cultured leukemia promonocytic U937 and THP-1 cells was examined herein. 2. Exposure of whole blood to ethanol showed no effect on PCA in human monocytes. However, ethanol dose-dependently inhibited LPS-induced PCA in isolated human monocytes. 3. In THP-1 cells, ethanol had no significant effect on PCA in either non-challenged or LPS-induced status. However, the induction of PCA by LPS was substantially inhibited when cells were pretreated with 1% ethanol (v/v) for 72 hr. 4. In U937 cells, n-alcohols and anesthetics resulted in dose-dependent depressions in PCA. Importantly, the percent reduction in LPS-induced PCA was much more pronounced than that in non-challenged PCA. 5. These data clearly suggest that n-alcohols and anesthetics readily inhibit the LPS-stimulatory action on monocytic PCA.     

The exon organization of the triple-helical coding regions of the human alpha 1(VI) and alpha 2(VI) collagen genes is highly similar.

The alpha 1(VI) and alpha 2(VI) chains, two of the three constituent chains of type VI collagen, are highly similar in size and domain structure. They are encoded by single-copy genes residing in close proximity on human chromosome 21. To study the evolution of the type VI collagen genes, we have isolated and characterized genomic clones coding for the triple-helical domains of the human alpha 1(VI) and alpha 2(VI) chains, which consist of 336 and 335 amino acid residues, respectively. Nucleotide sequencing indicates that, in both genes, the exons are multiples of 9 bp in length (including 27, 36, 45, 54, 63, and 90 bp) except for those encoding for regions with triple-helical interruptions. In addition, the introns are positioned between complete codons. The most predominant exon size is 63 bp, instead of 54 bp as seen in the fibrillar collagen genes. Of particular interest is the finding that the exon structures of the alpha 1(VI) and alpha 2(VI) genes are almost identical. A significant deviation is that a segment of 30 amino acid residues is encoded by two exons of 54 and 36 bp in the alpha 1(VI) gene, but by a single exon of 90 bp in the alpha 2(VI) gene. The exon arrangement therefore provides further evidence that the two genes have evolved from tandem gene duplication. Furthermore, comparison with the previously reported gene structure of the chick alpha 2(VI) chain indicates that the exon structure for the triple-helical domain of the alpha 2(VI) collagen is strictly conserved between human and chicken.     

Coordinated regulation of vascular endothelial cell growth and prostacyclin production by epidermal growth factor: Evidence of clonal variations among rat aortic endothelial cells

Summary Rat aortic endothelial cells were found to exhibit clonal variations in response to EGF stimulation in cell growth and prostacyclin synthesis. EGF-induced growth and prostacyclin synthesis appeared to be regulated in a coordinated manner in that a clone with a higher response to EGF growth stimulation also exhibited a higher response to EGF-stimulated prostacyclin synthesis. This observation implys a possible involvement of prostacyclin synthesis in some of the biological effects of EGF on vascular endothelial cells.     

Segmental flexibility of ribosomal protein S1 bound to ribosomes and Q beta-replicase

The protonization pattern of the endogenous donor component D₁ which feeds electrons directly into chl-a⁺_II has been analyzed in Tris-washed inside-out thylakoids with the aid of appropriate pH-indicators. It was found that under repetitive flash excitation the amount of protons released is proportional to the extent of D₁-oxidation, depending on the time between the flashes. The kinetics of D₁-oxidation (being practically the same as in normal Tris-washed chloroplasts) are faster than the proton release by two orders of magnitude. The results lead to the conclusion that D₁ is protonized in the reduced state with pK(D^ox₁) < 5 and becomes deprotonized in the oxidized state with pK(D^red₁) ? 8. The proton release is kinetically limited by a transport barrier. Implications on the interpretation of the proton release pattern in preparation with intact water oxidation are discussed.     

Modification of cell membrane in variants of chinese hamster cells resistant to abrin

Stable and heritable variants of Chinese hamster ovary (CHO) cells which are resistant to different levels (0.1, 1.0 and 10 μg/ml) of the toxin abrin have been isolated and characterized. The frequency of resistant colonies to abrin was increased with the concentration of a chemical mutagen. There was no effect of cell density or cross-feeding on the recovery of variants. In experiments using fluorescein-labeled abrin and ricin which bind to terminal (non-sialylated) galactose residues of cell-surface oligosaccharides, parental cells exhibited strong binding toward both toxins, whereas no fluorescence was observed in the resistant clones. A fluorescein-conjugated lectin, BS II, which is specific for terminal N-acetyl- -glucosaminyl residues, did not interact with the parental cells, but did with the resistant clones. This suggests that on the surface of resistant cells the number of terminal galactosyl residues of oligosaccharide chains in glycoproteins was reduced, exposing the penultimate N-acetyl- -glucosaminyl residues. The number of available endogenous acceptor sites for galactosyl transferase in the abrin-resistant clones was directly proportional to the degree of resistance. In the presence of great excess of exogenous acceptor, the rates of galactosyl transfer were similar in all the abrin-resistant cell types tested, with levels ranging from 1.4 to 1.7 times parental cell values. Studies with tetraploid cell hybrids reveal that resistance was a recessive trait. Fluctuation analysis showed that abrin resistance occurred in CHO cell populations at a rate of 4−7 × 10⁻⁸/cell/generation. The system may serve as a new marker for quantitative mutagenesis studies.