Deoxyribose conformation in [d(GTATATAC)]2: evaluation of sugar pucker by simulation of double-quantum-filtered COSY cross-peaks

Exchangeable and nonexchangeable proton and phosphorus resonances (11.75 T) of [d(GTATATAC)]2 in aqueous solution were assigned by using proton two-dimensional nuclear Overhauser effect (2D NOE) spectra, homonuclear proton double-quantum-filtered COSY (2QF-COSY) spectra, proton spin-lattice relaxation time measurements, and 31P1H heteronuclear shift correlation spectra. Due to the large line widths, it was not possible to directly extract vicinal proton coupling constant values from any spectrum including ECOSY or 2QF-COSY. However, comparison of quantitative 2QF-COSY spectral simulations with experimental spectra enabled elucidation of coupling constants. The scope and limitations of this approach were explored by computation and by use of experimental data. It was found that proton line widths exhibit some variability from one residue to the next as well as from one proton to the next within a residue and the exact line width is critical to accurate evaluation of coupling constants. Experimental 2QF-COSY spectra were not consistent with a rigid deoxyribose conformation for any of the nucleotide residues. A classical two-state model, with rapid jumps between C2'-endo (pseudorotation angle P = 162 degrees) and C3'-endo (P = 9 degrees) conformations, was able to account for the spectral characteristics of terminal residue sugars: 60% C2'-endo and 40% C3'-endo. However, the 2QF-COSY cross-peaks from the -TATATA- core could be simulated only if the classical two-state model was altered such that the dominant conformer had a pseudorotation angle at 144 degrees instead of 162 degrees. In this case, the major conformer amounted to 80-85%. Alternatively, the spectral data were consistent with a three-state model in which C2'-endo and C3'-endo conformations had the largest and smallest populations, respectively, but a third conformer corresponding to C1'-exo (P = 126 degrees) was present, consistent with recent molecular dynamics calculations. This alternative yielded populations of 50% (P = 162 degrees), 35% (P = 126 degrees), and 15% (P = 9 degrees) for the -TATATA- sugars. The spectral results indicate little variation of sugar pucker between T and A. Small differences in cross-peak component intensities and characteristic spectral distortions, however, do suggest some unquantified variation. 31P1H heteronuclear chemical shift correlation spectra manifested alternating chemical shifts and coupling constants suggestive of phosphodiester backbone conformational differences between TA and AT junctions.     

Aggregation of sponge cells. XX. Self-aggregation of the circular proteid particle.

In the extracellular space of the tissue of the sponge Geodia cydonium, circular proteid particles are found which carry as subunits the aggregation factor and a series of glycosyltransferases. Using the technique of velocity sucrose gradient centrifugation, the sedimentation coefficient (S020,w) of the particle-monosomes was determined to be 90. By means of the Svedberg equation a molecular weight of 1.3 . 10(8) daltons could be estimated. The monosomes aggregate in the presence of Ca2+ to higher complexes via disomes, trisomes, and pentasomes. The complexes can be redissociated by dodecyl sulfate but not by EDTA. During the Ca2+-mediated self-aggregation, the particles lose their biological activity with respect to their aggregation promoting function.     

Production of 3-acetoxyscirpene-4,15-diol from anguidine (4,15-diacetoxyscirpene-3-ol) by Fusarium oxysporum f.sp. vasinfectum.

Growing cells of Fusarium oxysporum f.sp. vasinfectum (ATCC 7808) formed 3-acetoxyscirpene-4,15-diol from anguidine (4,15-diacetoxyscirpene-3-ol) by way of the intermediates triacetoxyscirpene, 3,4-diacetoxyscirpene-15-ol and 3,15-diacetoxyscirpene-4-ol. The new 3-acetoxy analog was found to be less active than anguidine and the other monoacetoxy derivatives when tested against a series of fungal strains and against HeLa cells in vitro.     

Steroidogenesis in isolated adrenocortical cells. Correlation with receptor-bound adenosine 3′:5′-cyclic monophosphate

Because several groups have recently questioned a mediating role for cyclic AMP in adrenocortical steroidogenesis, we analysed the problem in more detail by measuring three different cyclic AMP pools in cells isolated from decapsulated rat adrenals. Extra-cellular, total intracellular and bound intracellular cyclic AMP were determined by radioimmunoassay in comparison with corticosterone production induced by low corticotropin concentrations. The increase in extracellular and total intracellular cyclic AMP with low corticotropin concentrations was dependent on the presence of a phosphodiesterase inhibitor and short incubation times. Bound intracellular cyclic AMP was less dependent on these two parameters. In unstimulated cells cyclic AMP bound to its receptor represents only a small fraction of the total intracellular cyclic AMP. After stimulation by a concentration of corticotropin around the threshold for corticosterone production, an increase in bound cyclic AMP was observed which correlated very well with steroidogenesis both temporally and with respect to corticotropin concentration. This finding was complemented by measuring a concomitant decrease in free receptor sites. Full occupancy of the receptors was not necessary for maximal steroidogenesis. Binding kinetics of cyclic [(3)H]AMP in concentrations equivalent to the intracellular cyclic AMP concentration suggest the presence of at least three different intracellular cyclic AMP pools. These observations are in agreement with a possible role for cyclic AMP as a mediator of acute steroidogenesis induced by low corticotropin concentrations.     

The interaction of RNA polymerase and lac repressor with the lac control region.

We have examined the interactions of lac repressor and RNA polymerase with the DNA of the lac control region, using a method for direct visualization of the regions of DNA protected by proteins from DNAase attack. The repressor protects the operator essentially as reported by Gilbert and Maxam (1) with some small modifications. However, the evidence reported here concerning the binding of RNA polymerase to the DNA of the promoter mutant UV5 indicates that : 1) the RNA polymerase molecule binds asymmetrically to the promoter DNA, 2) RNA polymerase protects DNA sequences to within a few bases of the CAP binding site, suggesting direct interaction between polymerase and the CAP protein at this site, 3) RNA polymerase still binds to the promoter when repressor is bound to the operator, but fails to form the same extensive complex.     

Kinetics of binding of bovine trypsin-killikrein inhibitor (K unitz) in which the reactive-site peptide bond Lys-15--Ala-16 is cleaved, to alpha-chymotrypsin and beta-trypsin.

Equilibrium measurements of the binding of reactive-site-cleaved (modified) bovine trypsin-kallikrein inhibitor (Kunitz) to alpha-chymotrypsin and beta-trypsin show a stoichiometric 1:1 association with high binding constants. At least in the case of chymotrypsin much evidence is presented that the reaction with modified inhibitor leads to the same complex as the reaction with virgin inhibitor does. The association rate constant of modified inhibitor with chymotrypsin at pH 7, 22.5 degrees C is 15.8 M-1 S-1. This is about 2 x 10(4) times slower than the binding of virgin inhibitor to that enzyme. In the analogous reaction of modified inhibitor with beta-trypsin, however, the association rate constant (1.2 x 10(4) M-1 s-1 at pH 6.9, 22.5 degrees C) is of about the same order of magnitude as it is in the reaction of virgin inhibitor and trypsin. These and analogous phenomena observed in the reactions of virgin and modified soybean trypsin inhibitor (Kunitz) with alpha-chymotrypsin and beta-trypsin suggest that the specificity of both inhibitors to trypsin is strongly reflected in the association rate constants of the modified forms. The dissociation rate constants of the complexes of trypsin-kallikrein inhibitor with chymotrypsin or with trypsin towards the modified inhibitor are estimated to be unmeasurably slow (half-life times of 45 or 1.5 x 10(4) years, respectively).     

Distinct binding sites of Ala48-hirudin1-47 and Ala48-hirudin48-65 on alpha-thrombin

The interaction of alpha-thrombin with Ala48-hirudin, Ala48-hirudin1-47, and Ala48-hirudin48-65 was analyzed. Mutations at Pro48 were found to cause only slight changes in the kon (human: 3.1 +/- 0.3 x 10(8) M-1 s-1; bovine: 1.03 +/- 0.3 x 10(8) M-1 s-1) and koff (human: 0.4 +/- 0.2 x 10(-3) s-1; bovine: 2.9 +/- 0.4 x 10(-3) s-1) rate constants for the formation of the thrombin-hirudin complex. The amino-terminal fragment Ala48-hirudin1-47 containing the three disulfide bridges and the carboxyl-terminal fragment Ala48-hirudin48-65 were derived from the Ala48 mutant by proteolysis with endoproteinase Lys-C. These fragments inhibit bovine alpha-thrombin clotting activity with IC50 values of 0.6 and 4.9 microM, respectively (2.4 nM for r-hirudin). By mapping the interaction of Ala48-hirudin-derived fragments with bovine alpha-thrombin by limited proteolysis with trypsin and pancreatic elastase distinct binding sites for each fragment were determined. The carboxyl-terminal fragment was found to bind to the proposed anion-binding exosite in the region B62-74, whereas the amino-terminal fragment binds to a region around the elastase cleavage site at residues 150-151 of the alpha-thrombin B-chain.     

A modified dinucleotide motif specifies tRNA recognition by TLR7

RNA can function as a pathogen-associated molecular pattern (PAMP) whose recognition by the innate immune system alerts the body to an impending microbial infection. The recognition of tRNA as either self or nonself RNA by TLR7 depends on its modification patterns. In particular, it is known that the presence of a ribose methylated guanosine at position 18, which is overrepresented in self-RNA, antagonizes an immune response. Here, we report that recognition extends to the next downstream nucleotide and the effectively recognized molecular detail is actually a methylated dinucleotide. The most efficient nucleobases combination of this motif includes two purines, while pyrimidines diminish the effect of ribose methylation. The constraints of this motif stay intact when transposed to other parts of the tRNA. The results argue against a fixed orientation of the tRNA during interaction with TLR7 and, rather, suggest a processive type of inspection.