Sequence-dependence of the CD of synthetic double-stranded RNAs containing inosinate instead of guanylate subunits

The CD spectra and melting profiles have been measured for nine synthetic double-stranded RNAs containing I · C instead of G · C base pairs: poly[r(I) · r(C)], poly[r(I-C) · r(I-C)], poly[r(A-I-C) · r(I-C-U)], poly[r(A-C) · r(I-U)], poly[r(A-I) · r(C-U)], poly[r(A-C-C) · r(I-I-U)], poly[r(A-A-C) · r(I-U-U)], poly[r(A-C-U) · r(A-I-U)], and poly[r(A-U-C) · r(I-A-U)]. CD spectra have not previously been reported for the latter six of these polymers. The substitution of inosinate for guanylate led to recognizable CD differences, with all but two of the polymers having two resolved positive bands above 230 nm. Also, the I-containing RNAs differed from their G-containing counterparts in the almost complete absence of negative CD bands at long wavelengths and in the reduction of negative CD bands near 210 nm. First-neighbor comparisons showed that the CD spectra of the I-containing RNAs were consistent with the nearest-neighbor sequences of the polymers, as previously shown for G-containing RNAs (D. M. Gray, J.-J. Liu, R. L. Ratliff, and F. S. Allen, Biopolymers (1981) 20 , 1337–1382). Moreover, two of the first-neighbor comparisons involved spectra of poly[r(A) · r(U)] and poly[r(I) · r(C)], polymers known to be in the A family of conformations in fibers (S. Arnott, D. W. L. Hukins, S. D. Dover, W. Fuller, and A. Hodgson, (1973) J. Mol. Biol. 81 , 107–122). Thus, differences in the CD spectra of I- and G-containing RNAs could be simply explained as resulting from differences in the hypoxanthine and guanine chromophores, without invoking differences in conformation. Finally, melting temperatures of the I-containing RNAs were found to vary much less with base composition than do the melting temperatures of G-containing RNAs, since A · U base pairs are closer to I · C than to G · C base pairs in stability.     

CD of six different conformational rearrangements of poly[d(A-G).d(C-T)] induced by low pH.

On the basis of circular dichroism (CD) data, we have now identified six different conformational states (other than the duplex) of poly[d(A-G).d(C-T)] at pH values between 8 and 2.5 (at 0.01M Na+; 20 degrees C). Three of these structural rearrangements were observed as the pH was lowered from 8 to 2.5, and three additional rearrangements were observed as the pH was raised from 2.5 back to neutral pH. The major components of the six conformational states were defined using appropriate combinations of the CD spectra of the duplex, triplex, and denatured forms of this polymer, as well as the CD spectra of the individual single strands and their respective acid-induced self-complexes. Our results show that the acid-induced rearrangements of poly[d(A-G).d(C-T)] include not only the poly[d(C+-T).d(A-G).d(C-T)] triplex, but also include the poly[d(C-T)] loop-out structure and a self-complexed form of the poly[d(A-G)] strand that is pH-dependent.     

Circular dichroism spectra of DNA oligomers show that short interior stretches of C.C+ base pairs do not form in duplexes with A.T base pairs

Circular dichroism (CD) experiments were carried out on a series of DNA oligomers to determine if short internal stretches of protonated cytosine-cytosine (C.C+) base pairs could coexist with adenine-thymine (A.T) base pairs. (1) C.C+ base pairs did form in the absence of A.T base pairs in the individual oligomers d(AACC)5 and d(CCTT)5, as indicated by the appearance of a long-wavelength CD band centered at 282-284 nm, when the pH was lowered to 6 or 5 at 0.5 M Na+. A comparison of measured with calculated spectra showed that d(CCTT)5 at pH 5, 0.5 M Na+, 20 degrees C, likely adopted a structure with a central core of stacked C.C+ base pairs and looped-out thymines. Under the same conditions, it appeared that C.C+ base pairs also formed in d(AACC)5, but with the adenines remaining intrahelical. Each of these oligomers showed a cooperative transition for formation of C.C+ base pairs as the temperature was lowered, with C.C+ base pairs forming at a higher temperature in d(CCTT)5 than in d(AACC)5. A.T base formed in equimolar mixtures of d(AACC)5 plus d(CCTT)5 as monitored by an increase in the negative magnitude of the 250-nm CD band. However, a large increase did not appear at about 285 nm in CD spectra of the mixtures, showing that there were no stacked C.C+ base pairs in the d(AACC)5.d(CCTT)5 duplex even though they formed under the same conditions in the individual strands. Thus, in this duplex, A.T base pairs prevented the formation of neighboring internal C.C+ base pairs. (2) CD measurements were also made of d(A10C4T10).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of soluble fibronectin binding to Bacille Calmette-Guérin

Fibronectin (FN), a 420 kDa glycoprotein, consists of two similar subunits linked by a disulphide bond near the C-terminal end. FN is present in soluble and matrix forms in various body fluids and tissues and has been shown to bind to variety of organisms. We characterized the conditions required for 125I-FN binding to Bacille Calmette-Guérin (BCG). The binding was dose-dependent, reached saturation within 3 min, and was essentially irreversible for at least 24 h under optimal binding conditions at pH 6.0. In contrast, the binding was reversible (greater than 90% in 24 h) when the pH was increased to 10.0. Scatchard analysis of the dose-response experiments produced a straight line, suggesting the presence of a single class of FN receptor on BCG. 125I-FN binding was trypsin-sensitive, suggesting that the BCG-binding molecule is a protein. The number of FN receptors was determined to be 8000-15,000 per bacterium. 125I-FN binding was pH dependent, with maximal binding at acidic pH. 125I-FN binding was sensitive to the presence of NaCl, with 0.08 M-NaCl inhibiting binding by 85%. These data demonstrate that soluble FN binds to a trypsin-sensitive cell-surface component of BCG in an essentially irreversible manner.     

Imprinting the fate of antigen-reactive B cells through the affinity of the B cell receptor

Long-lived plasma cells (PCs) and memory B cells (B(mem)) constitute the cellular components of enduring humoral immunity, whereas short-lived PCs that rapidly produce Ig correspond to the host's need for immediate protection against pathogens. In this study we show that the innate affinity of the BCR for Ag imprints upon naive B cells their differentiation fate to become short- or long-lived PCs and B(mem). Using BCR transgenic mice with varying affinities for Ag, naive B cells with high affinity lose their capacity to form germinal centers (GCs), develop neither B(mem) nor long-lived PCs, and are destined to a short-lived PC fate. Moderate affinity interactions result in hastened GC responses, and differentiation to long-lived PCs, but B(mem) remain extinct. In contrast, lower affinity interactions show tempered GCs, producing B(mem) and affinity-matured, long-lived PCs. Thus, a continuum of elementary to comprehensive humoral immune responses exists that is controlled by inherent BCR affinity.     

A KV2.1 gating modifier binding assay suitable for high throughput screening

Gating modifier peptides alter gating of voltage-gated potassium (KV) channels by binding to the voltage sensor paddle and changing the energetics of channel opening. Since the voltage sensor paddle is a modular motif with low sequence similarity across families, targeting of this region should yield highly specific channel modifiers. To test this idea, we developed a binding assay with the KV2.1 gating modifier, GxTX-1E. Monoiodotyrosine-GxTX-1E (125I-GxTX-1E) binds with high affinity (IC50 = 4 nM) to CHO cells stably expressing hKV2.1 channels, but not to CHO cells expressing Maxi-K channels. Binding of 125I-GxTX-1E to KV2.1 channels is inhibited by another KV2.1 gating modifier, stromatoxin (IC50 = 30 nM), but is not affected by iberiotoxin or charybdotoxin, pore blocking peptides of other types of potassium channels, or by ProTx-II, a selective gating modifier peptide of the voltage-gated sodium channel NaV1.7. Specific 125I-GxTX-1E binding is not detectable when CHO-KV2.1 cells are placed in high external potassium, suggesting that depolarization favors dissociation of the peptide. The binding assay was adapted to a 384-well format, allowing high throughput screening of large compound libraries. Interestingly, we discovered that compounds related to PAC, a di-substituted cyclohexyl KV channel blocker, displayed inhibitory binding activity. These data establish the feasibility of screening large libraries of compounds in an assay that monitors the displacement of a gating modifier from the channel's voltage sensor. Future screens using this approach will ultimately test whether the voltage sensor of KV channels can be selectively targeted by small molecules to modify channel function.     

Structure-based virtual screening for novel inhibitors of the sarco/endoplasmic reticulum calcium ATPase and their experimental evaluation

A public compound library with 260,000 compounds was screened virtually by computational docking for novel inhibitors of the transmembrane enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA). Docking was performed with the program GOLD in conjunction with a high resolution X-ray crystal structure of SERCA. Compounds that were predicted to be active were tested in bioassays. Nineteen novel compounds were discovered that were capable of inhibiting the ATP hydrolysis activity of SERCA at concentrations below 50 μM. Crucial enzyme/inhibitor interactions were identified by analyzing the docking-predicted binding poses of active compounds. Like other SERCA inhibitors, the newly discovered compounds are of considerable medicinal interest because of their potential for cancer chemotherapy.