Biochemical and biophysical studies on the folding of the core region of the origin of replication of bacteriophage M13.

DNA oligonucleotides with the sequence corresponding to the plus strand origin of replication of the filamentous bacteriophage M13 are studied. Biochemical structure probing and UV melting studies, supplemented with initial NMR experiments, are used to investigate structural features of a 51-nucleotides long synthetic oligonucleotide and two oligonucleotides that are integral parts of this latter molecule. The results demonstrate the feasibility and complementarity of the use of methidiumpropyl.EDTA-Fe(II) and nuclease S1 in the structural analysis of small oligonucleotides. The bacteriophage origin region appears to comprise two hairpins. The first hairpin, which contains a cleavage site for the bacteriophage gene II protein, has a large and probably flexible loop. NMR as well as UV melting studies demonstrate that the second hairpin contains a stable three-membered loop. Both hairpins are present in the 51-mer, which forms a stable tertiary structure.     

Effects of base sequence on the loop folding in DNA hairpins

High-resolution NMR and UV-melting experiments have been used to study the hairpin formation of partly self-complementary DNA fragments in an attempt to derive rules that describe the folding in these molecules. Earlier experiments on the hexadecanucleotide d(ATCCTA-TTTT-TAGGAT) had indicated that within the loop of four thymidines a wobble T-T pair is formed (Blommers et al., 1987). In the present paper it is shown that if the first and the last thymines of the intervening sequence are replaced by complementary bases, sometimes base pairs can be formed. Thus for the intervening sequences -CTTG- and -TTTA- with the pyrimidine in the 5'-position and the purine in the 3'-position, a base pair is formed leading to a loop consisting of two residues. For the intervening sequences -GTTC- and -ATTT- with the purine in the 5'-position and the pyrimidine in the 3'-position, this turns out not to be the case. It was found that it made no difference when the four-membered sequence was closed by a G-C base pair or an A-T base pair. Replacement of the two central thymidine residues by the more bulky adenine residues limits the hairpin to a four-membered loop scheme. Very surprisingly, it was found from 2D NOE experiments that the T-A base pair, formed in the loop consisting of the -TTTA- sequence, is a Hoogsteen pair. It is argued that the pairing of the bases in this scheme may facilitate the formation of a loop of two residues, since the distance of the C1' atoms in this base pair is 8.6 A instead of 10.4 A found in the canonical Watson-Crick base pair. Combination of the data obtained for the series of DNA fragments studied shows that the results can be explained by a simple, earlier proposed, loop folding principle which assumes that the folding of the four-membered loop is dictated by the stacking of the double-helical stem of the hairpin.     

Nuclear magnetic resonance studies on yeast tRNAPhe I. Assignment of the iminoproton resonances of the acceptor and D stem by means of Nuclear Overhauser Effect experiments at 500 MHz

Resonances of the water exchangeable iminoprotons of the acceptor and D stem of yeast tRNAPhe have been assigned by means of Nuclear Overhauser Effects (NOE's). Assignments were made for spectra recorded from tRNA dialysed against a buffer with 110 mM sodium and 5 mM magnesium ions and against a buffer with 430 mM sodium and no magnesium ions. Remarkable is the assignment of a resonance at 13.6 - 13.7 ppm to the iminoproton of C11G24. This assignment as well as those of G1C72, G3C70, U7A66, U12A23 and C13G22 are different from those made previously on the basis of less direct evidence. NOE experiments performed at 45 degrees C support the view that the D stem together with the tertiary interaction U8A14 is one of the most stable parts of the molecule in the presence of magnesium ions. A comparison of the spectra recorded under the two different buffer conditions shows that an excess of 320 mM sodium ions is not capable to force the tRNA in the same conformation as 5 mM magnesium ions can do.     

The iminoproton NMR spectrum of yeast tRNA-Phe predicted from crystal coordinates.

The ring current effects on the base paired iminoprotons in yeast tRNA-Phe have been calculated from crystal coordinates. The results in conjunction with independently determined intrinsic positions of the iminoprotons in various base pairs enable us to predict the low field NMR spectrum of yeast tRNA-Phe. It turns out that the calculated NMR spectra are very sensitive to slight changes in structure. Moreover the crystal and solution structure are identical as far as the present methods go.     

Studies on DNA unwinding. Proton and phosphorus nuclear-magnetic-resonance studies of gene V protein from bacteriophage M13, interacting with d(pC-G-C-G)

The interaction of gene V protein from bacteriophage M13 with the self-complementary tetranucleotide d(pC-G-C-G) was studied by 1H and 31P nuclear magnetic resonance. It is shown, using the hydrogen-bonded proton resonances of the Watson-Crick base pairs as a probe, that the protein is able to unwind the small double-helical fragment even at 0 degrees C. Binding of the tetranucleotide causes changes in the aromatic part of the 1H NMR spectrum of the complex, suggesting that aromatic residues, most likely tyrosines, take part in the protein.nucleic-acid interaction. From the 31P NMR spectra of the protein.nucleic-acid complex it follows that the pK value of the 5'-terminal phosphate is lower than for the free nucleic acid species. Moreover, it could be shown that the exchange of the protein between nucleic acid substrates is fast. Combination of these measurements has led us to derive a mechanism of unwinding on the tetranucleotide level. To a large extent the unwinding is determined by fluctuations in the double-helical DNA structure.     

Prevalence of methotrexate intolerance in rheumatoid arthritis and psoriatic arthritis

Introduction

The aim of this study was to determine the prevalence of gastrointestinal and behavioural symptoms occurring before (anticipatory/associative) and after methotrexate (MTX) administration, termed MTX intolerance, in rheumatoid (RA) and psoriatic arthritis (PsA).

Methods

Methotrexate Intolerance Severity Score (MISS), previously validated in juvenile idiopathic arthritis patients, was used to determine MTX intolerance prevalence in 291 RA/PsA patients. The MISS consisted of four domains: abdominal pain, nausea, vomiting and behavioural symptoms, occurring upon, prior to (anticipatory) and when thinking of MTX (associative). MTX intolerance was defined as ≥6 on the MISS with ≥1 point on anticipatory and/or associative and/or behavioural items.

Results

A total of 123 patients (42.3%) experienced at least one gastrointestinal adverse effect. The prevalence of MTX intolerance was 11%. MTX intolerance prevalence was higher in patients on parenteral (20.6%) than on oral MTX (6.2%) (p < 0.001).

Conclusion

Besides well-known gastrointestinal symptoms after MTX, RA and PsA patients experienced these symptoms also before MTX intake. RA and PsA patients on MTX should be closely monitored with the MISS for early detection of MTX intolerance, in order to intervene timely and avoid discontinuation of an effective treatment.     

Dermatological conditions during TNF-α-blocking therapy in patients with rheumatoid arthritis: a prospective study

Various dermatological conditions have been reported during tumor necrosis factor (TNF)-α-blocking therapy, but until now no prospective studies have been focused on this aspect. The present study was set up to investigate the number and nature of clinically important dermatological conditions during TNF-α-blocking therapy in patients with rheumatoid arthritis (RA). RA patients starting on TNF-α-blocking therapy were prospectively followed up. The numbers and natures of dermatological events giving rise to a dermatological consultation were recorded. The patients with a dermatological event were compared with a group of prospectively followed up RA control patients, naive to TNF-α-blocking therapy and matched for follow-up period. 289 RA patients started TNF-α-blocking therapy. 128 dermatological events were recorded in 72 patients (25%) during 911 patient-years of follow-up. TNF-α-blocking therapy was stopped in 19 (26%) of these 72 patients because of the dermatological event. More of the RA patients given TNF-α-blocking therapy (25%) than of the anti-TNF-α-naive patients (13%) visited a dermatologist during follow-up (P < 0.0005). Events were recorded more often during active treatment (0.16 events per patient-year) than during the period of withdrawal of TNF-α-blocking therapy (0.09 events per patient-year, P < 0.0005). The events recorded most frequently were skin infections (n = 33), eczema (n = 20), and drug-related eruptions (n = 15). Other events with a possible relation to TNF-α-blocking therapy included vasculitis, psoriasis, drug-induced systemic lupus erythematosus, dermatomyositis, and a lymphomatoid-papulosis-like eruption. This study is the first large prospective study focusing on dermatological conditions during TNF-α-blocking therapy. It shows that dermatological conditions are a significant and clinically important problem in RA patients receiving TNF-α-blocking therapy.     

Analysis of 1H chemical shifts in DNA: Assessment of the reliability of 1H chemical shift calculations for use in structure refinement

The reliability of ¹H chemical shift calculations for DNA is assessed by comparing the experimentally and calculated chemical shifts of a reasonably large number of independently determined DNA structures. The calculated chemical shifts are based on semiempirical relations derived by Giessner-Prettre and Pullman [(1987) Q. Rev. Biophys., 20, 113–172]. The standard deviation between calculated and observed chemical shifts is found to be quite small, i.e. 0.17 ppm. This high accuracy, which is achieved without parameter adjustment, makes it possible to analyze the structural dependencies of chemical shifts in a reliable fashion. The conformation-dependent ¹H chemical shift is mainly determined by the ring current effect and the local magnetic anisotropy, while the third possible effect, that of the electric field, is surprisingly small. It was further found that for a double helical environment, the chemical shift of the sugar protons, H2 to H5, is mainly affected by the ring current and magnetic anisotropy of their own base. Consequently, the chemical shift of these sugar protons is determined by two factors, namely the type of base to which the sugar ring is attached, C, T, A, or G, and secondly by the -angle. In particular, the H2 shift varies strongly with the -angle, and strong upfield H2 shifts directly indicate that the -angle is in the syn domain. The H1 shift is not only strongly affected by its own base, but also by its 3-neighboring base. On the other hand, base protons, in particular H5 of cytosine and methyl protons of thymine, are affected mainly by the 5-neighboring bases, although some effect (0.2 ppm) stems from the 3-neighboring base. The H2 protons are mainly affected by the 3-neighboring base. As a result of these findings a simple scheme is proposed for sequential assignment of resonances from B-helices based on chemical shifts.     

Structure of the 3''-hairpin of the TYMV pseudoknot: preformation in RNA folding.

The solution structure of an RNA-hairpin present in the pseudoknot, which is found at the 3'-terminus of turnip yellow mosaic virus genomic RNA, has been solved by nuclear magnetic resonance spectroscopy. The loop, which contains the sequence 5'-GGGUCA-3', was found to be highly structured and, contrary to expectations, does not attain its stability through GA or GC base pair formation but by triple interactions between the tilted adenosine and the minor groove sides of the first two guanosines. Interestingly, a very similar conformation was found for the cognate pseudoknot, implying that the 3'-hairpin is preformed for folding into a pseudoknotted structure. These findings suggest a mechanism of 'predetermined-fit' as a principle in RNA folding.     

Residue-specific assignments of resonances in the 1H nuclear magnetic resonance spectrum of ribosomal protein E-L30 by systematic application of two-dimensional Fourier transform nuclear magnetic resonance methods

A two-dimensional Fourier transform nuclear magnetic resonance study of the ribosomal protein E-L30 is reported. Five two-dimensional techniques, namely: nuclear magnetic resonance J-resolved spectroscopy, correlated spectroscopy, double quantum spectroscopy, relayed coherence transfer and nuclear Overhauser enhancement spectroscopy were used. Qualitative inspection of the spectra obtained by these techniques provided evidence that the E-L30 molecule has a well-defined structure in solution. This analysis indicated that, despite the fact that the protein is stable only at moderate temperatures and neutral pH, a structural analysis of the molecule would be feasible. A detailed analysis of the spectra permitted unambiguous discrimination between the spin systems of different amino acids, resulting in residue-specific resonance assignments. We were able to assign all resonances of all six threonine, four valine, five alanine, two histidine, two serine, one phenylalanine, one asparagine and one aspartic acid residue of E-L30. Complete resonance assignment was obtained for two glycine residues. Partial assignments became available for all six isoleucine, three glycine and one glutamine residue. These results form a sound basis for the structure determination of the protein described in the accompanying paper.