The effective of methotrexate on immune response cells in rheumatoid arthritis.

Methotrexate (MTX) is one of the most used medicines in rheumatoid arthritis [r. a.] treatment. There are many scientific reports which present influence of MTX on inflammatory process. In many centers influence of MTX on cytokines level have been investigated. Thanks to clinical studies it has been performed that MTX holds up activity and productions of Il-8, releasing of TNF-alpha and reduction of concentration Il-6. MTX holds up proliferation of monocytes, macrophages and synoviocytes. It has been indicated that MTX decreases synthesis of B-4 leucotrien in neutrophils, decreases level of neutral proteases, holds up cellular immunity and has antiproliferative influence on endothelial cells. There are reports, that MTX reduces expression of endothelial cells adhesive proteins and synthesis of chemotactic factors, stopping migration of leucocytes to tissues.

Favourable interactions of cytostatic and steroid drugs in treatment of systemic lupus erythematosus.

Systemic lupus erythematosus (referred to below as SLE) is a disorder associated with formation of immune complexes in the circulatory system and their deposition in the internal organs, as well as in the skin. The disease can appear at any age, but the vast majority of cases are women between 15 and 40 years of age. The paper presents the case of a thirty-year-old woman with systemic lupus erythematosus. She was admitted to hospital because of erythematous and haemorrhagic lesions in the skin of her face, hands and feet and febrile states. SLE was diagnosed in the patient on the basis of criteria established by the American Rheumatological Association. The following abnormalities were observed in the patient: skin lesions of erythematous type on the face, hypersensitivity to UV light, erosions of the mucous membranes of the oral cavity, changes in the kidneys confirmed by biopsy, haematological symptoms, immune disorders. Additional criteria to establish the diagnosis included: febrile states. Raynaud's sign, hair loss, presence of immunoglobulin complexes in the skin, decreased complement level. The paper discusses modern therapy of visceral lupus erythematosus, based on corticosteroids and immunosuppressants. The interactions of these drugs were observed in the patient. The woman was treated with combined corticosteroid and cytostatic therapy, which led to the remission of the pathologic process confirmed by the clinical condition of the patient and laboratory tests.

Comparative calorimetric studies on the dynamic conformation of plant 5S rRNA: II. Structural interpretation of the thermal unfolding patterns for lupin seeds and wheat germ.

Thermal unfolding of 5S rRNA from wheat germ (WG) and lupin seeds (LS) was studied in solution. Experimental curves of differential scanning calorimetry (DSC) were resolved into particular components according to the thermodynamic model of two-state transitions. The DSC temperature profiles for WG and LS differ significantly in spite of very high similarities in the sequence of both molecules. Those results are interpreted according to a model of the secondary and tertiary molecular structure of 5S rRNA. A comparison of the 'nearest neighbour' model of interaction with the experimental thermodynamic results enables a complete interpretation of the process of the melting of its structures. In light of our observations, the crucial differences between both DSC melting profiles are mainly an outcome of different thermodynamic properties of the first helical fragment 'A' made up of 9 complementary base pairs. It contains 6 differences in the nucleotide sequence of both types of molecules, which still retain 9-meric double helixes. The temperature stability of his helix in WG is much lower than of the LS one. Moreover, the results supply evidence for a strong specific tertiary interaction between the two hairpin loops 'c' and 'e' in both 5S rRNA molecules, modulated by small differences in the thermodynamic properties of both 5S rRNA.

Use of lead(II) to probe the structure of large RNA's. Conformation of the 3' terminal domain of E. coli 16S rRNA and its involvement in building the tRNA binding sites.

The present work shows that lead(II) can be used as a convenient structure probe to map the conformation of large RNA's and to follow discrete conformational changes at different functional states. We have investigated the conformation of the 3' domain of the E. coli 16S rRNA (nucleotides 1295-1542) in its naked form, in the 30S subunit and in the 70S ribosome. Our study clearly shows a preferential affinity of Pb(II) for interhelical and loop regions and suggests a high sensitivity for dynamic and flexible regions. Within 30S subunits, some cleavages are strongly decreased as the result of protein-induced protection, while others are enhanced suggesting local conformational adjustments. These rearrangements occur at functionally strategic regions of the RNA centered around nucleotides 1337, 1400, 1500 and near the 3' end of the RNA. The association of 30S and 50S subunits causes further protections at several nucleotides and some enhanced reactivities that can be interpreted in terms of subunits interface and allosteric transitions. The binding of E. coli tRNA-Phe to the 70S ribosome results in message-independent (positions 1337 and 1397) and message-dependent (1399-1400, 1491-1492 and 1505) protections. A third class of protection (1344-1345, 1393-1395, 1403-1409, 1412-1414, 1504, 1506-1507 and 1517-1519) is observed in message-directed 30S subunits, which are induced by both tRNA binding and 50S subunit association. This extensive reduction of reactivity most probably reflects an allosteric transition rather than a direct shielding.

Specificity and mechanism of the cleavages induced in yeast tRNAPhe by magnesium ions.

The specificity of magnesium ion-induced hydrolysis of yeast tRNAPhe in solution was studied as a function of the excess of Mg(II) ions and pH. The major cuts at phosphates 16 and 20 as well as minor cleavages at phosphates 17, 18, 21, 34 and 36 occur at all pH values in the range of 8.0-9.5, and at a molar excess of magnesium ions over the tRNA ranging from 125 to 5000. In yeast tRNA(Phe)-Y the efficiency of the anticodon and D-loop cleavages is considerably decreased while the differently modified Y-base of yellow lupin tRNA(Phe) lowers the specificity of the weak anticodon loop cleavages. The mechanism of the Mg(II)-induced cleavages is discussed on the basis of yeast tRNA(Phe) crystal structure data, and the two major D-loop cleavages are thought to be effected from two distinct magnesium binding sites. The possibility of probing the environments of magnesium binding sites in tRNAs by the induced cleavages is demonstrated, and the relevance of magnesium-induced tRNA cleavages to RNA catalysis is discussed.

Characterization of the lead(II)-induced cleavages in tRNAs in solution and effect of the Y-base removal in yeast tRNAPhe.

The specificity of lead(II)-induced hydrolysis of yeast tRNA(Phe) was studied as a function of concentration of Pb2+ ions. The major cut was localized in the D-loop and minor cleavages were detected in the anticodon and T-loops at high metal ion concentration. The effects of pH, temperature, and urea were also analyzed, revealing a basically unchanged specificity of hydrolysis. In the isolated 5'-half-molecule of yeast tRNAPhe not cut was found in the D-loop, indicating its stringent dependence on T-D-loop interaction. Comparison of hydrolysis patterns and efficiencies observed in yeast tRNA(Phe) with those found in other tRNAs suggests that the presence of a U59-C60 sequence in the T-loop is responsible for the highly efficient and specific hydrolysis in the spatially close region of the D-loop. The efficiencies of D-loop cleavage in intact yeast tRNA(Phe) and in tRNA(Phe) deprived of the Y base next to the anticodon were also compared at various Pb2+ ion concentrations. Kinetics of the D-loop hydrolysis analyzed at 0, 25, and 37 degrees C showed a 6 times higher susceptibility of tRNA(Phe) minus Y base (tRNA(Phe)-Y) to lead(II)-induced hydrolysis than in tRNA(Phe). The observed effect is discussed in terms of a long-distance conformational transition in the region of the interacting D- and T-loops triggered by the Y-base excision.

Comparative calorimetric studies on the dynamic conformation of plant 5S rRNA. I. Thermal unfolding pattern of lupin seeds and wheat germ 5S rRNAs, also in the presence of magnesium and sperminium cations.

An attempt has been made to correlate differential scanning calorimetry melting profiles of 5S rRNAs from lupin seeds (L.s.) and wheat germ (W.g.) with their structure. It is suggested that the observed differences in thermal unfolding are due to differences in RNA nucleotide sequence and as a consequence in higher order structures. Interesting effects induced by magnesium cation, perprotonated and permethylated sperminium tetracations are discussed. It is suggested that the difference in the stabilizing effect of the three cations results from different mode of their interactions with RNA. "Pure" electrostatic interactions expected for permethylated tetracations are rather weak due to the steric hindrance around each positively charged nitrogen atom. Electrostatic interactions of the other two cations are significantly enhanced by coordination bonding for magnesium and by hydrogen bonding for protonated sperminium cation.

Proton-coupled 15N NMR spectra of neutral and protonated ethenoadenosine and ethenocytidine.

The 15N chemical shifts and 15N, 1H spin coupling constants were determined in the title compounds using the INEPT pulse sequence and assigned with the aid of selective proton decoupling. The delta/15N/ and J/N, H/ values are discussed in terms of involvement of the imidazole ring created by ethenobridging in the electronic structure of the whole molecule. Both spectral parameters indicate that the diligant nitrogen in this ring is the primary site of protonation in these modified nucleosides. It is concluded that 15N NMR of nucleoside bases can be largely a complementary method to 1H and 13C NMR studies and, in addition, can serve as a direct probe for studies of nitrogen environment in oligomeric fragments of nucleic acids even at moderately strong magnetic fields due to the higher spectral dispersion compared with 1H and 13C NMR spectra.

Conservation of plant cytoplasmic tRNA structure. Nucleotide sequence of rape tRNAPhe.

The primary structure of rape seeds tRNAPhe has been determined. It is identical to that of wheat germ, pea and barley tRNAsPhe, and of the minor tRNAPhe species of yellow lupin seeds.

Comparative structural analysis of cytidine, ethenocytidine and their protonated salts III. 1H, 13C and 15N NMR studies at natural isotope abundance.

The 1H, 13C, 15N NMR spectra of cytidine /Cyd/, ethenocytidine /epsilon Cyd/ and their hydrochlorides /Cyd X HC1/ and /epsilon Cyd X HC1/ have been analysed to compare structural differences observed in solution with those existing in the crystalline state. The effects of ethenobridging and protonation of the hertero-aromatic base on the intramolecular stereochemistry, intermolecular interactions and electronic structure of the whole molecule are discussed on the basis of the NMR studies in DMSO solutions. Particular interest is devoted to the discussion of the conformation of the ribose ring, the presence of the intramolecular C-5'-0...H-6-C hydrogen bond, unambiguous assignment of the site of protonation, the mechanism of the 5C-H deuterium exchange in Cyd X HC1, and the intermolecular interactions in solution.

Organization and nucleotide sequence of nuclear 5S rRNA genes in yellow lupin (Lupinus luteus).

Genomic blots of yellow lupin (Lupinus luteus) DNA digested with restriction nucleases and probed with 32P-labelled Lupinus 5S RNA reveal that 5S DNA is organized as tandemly repeated sequences of one size class, 342 bp. The DNA is extensively methylated. Two cloned BamHI ribosomal repeats were sequenced, revealing sequence divergence within both the coding and spacer regions.

Comparative structural analysis of cytidine, ethenocytidine and their protonated salts. II. IR spectral studies.

The IR spectra of crystalline cytidine (Cyd), ethenocytidine (epsilon Cyd), and their hydrochlorides (Cyd-Hcl and epsilon CyD-HCl) have been analyzed to determine the spectroscopic manifestations of the structural differences that were previously established for these nucleosides from X-ray studies. O,N-Deuteration of the samples turned out to be a successful approach to obtaining interpretable spectra. The analysis was carried out in three frequency ranges: (i) The 2600-1900 cm-1 range originating from the vO-D and VN-D vibrations. All intermolecular hydrogen bonds could be recognized here. The positions of the individual vO-D (vN-D) bands were correlated with the geometrical delta HB parameters presenting the strengths of hydrogen bonds in which these groups act as donors (ii) The 1750-1500 cm-1 region originating from the stretching vibrations of double bonds. All absorption bands in this region were interpreted in terms of electronic structures of the base fragments. (iii) The region of the C-H stretching vibrations of the base fragments (3200-3000 cm-1) and sugar moieties (3000-2800 cm-1). The Csp2-H vibrations also reflect the electronic structures of the base fragments, whereas the vCsp-H frequencies seem to be sensitive to etheno-bridging and to the presence of an intramolecular C6-H...05' hydrogen bond.

Comparative structural analysis of cytidine, ethenocytidine, and their protonated salts. I. Crystal and molecular structure of ethenocytidine.

The X-ray crystal and molecular structure of 3,N4-ethenocytidine (comes from Cyd) has been solved and refined on counter data to R = 0.038. A detailed discussion of the base electronic structure, molecular conformation and intermolecular interactions is the starting point for a comparative analysis of the series: Cyd, epsilon Cyd, Cyd . HCll and epsilon Cyd . HCl. Protonation changes the base electronic structure and results in a completely different molecular conformation and intermolecular interactions. Etheno-bridging does not alter the molecular conformation but it also changes the intermolecular interactions.

The reaction of adenine and cytosine residues in tRNA with chloroacetaldehyde.

The reaction of Lupinus luteus tRNAPhe with 1 M chloroacetaldehyde in the pH range of 4 - 6 at 25 degrees C was studied. It was found that earlier difficulties lowering the utility of the reagent for structural studies of nucleic acids were caused by the formation of the stable reaction intermediates. In order to eliminate these difficulties the simple procedure of so-called maturation of the chloroacetaldehyde-modifies tRNA is proposed. It consists in further incubation of the short-term modified tRNA in water at 50 degrees C in the absence of the reagent. During the maturation step the stable intermediates are quantitatively converted into final ethenoderivatives. New HPLC conditions on Aminex A-6 were worked out which enable rapid separation of both ethenoadenosine and ethenocytidine from natural tRNA nucleosides.

The case of sulfonation in the chemical synthesis of oligonucleotides.

The sulfonation of nucleosidic component, a side reaction during phosphotriester bond formation, as a function of the reactivity of the condensing agents and the kind of substituents in the starting phosphodiester is discussed. It was found that in the coupling reaction of nucleoside alkyl phosphodiesters, the degree of sulfonation of the nucleosidic component was very high; while under the same conditions when the aryl group was present in the corresponding phosphodiester, this side reaction practically did not occur.

Chromatography on Sephadex LH20 as an efficient purification step after removal of internucleotide 2,2,2-trichloroethyl protective groups from oligoribonucleotide phosphotriesters.

Chromatography on Sephadex LH20, in a linear gradient of methanol in 0.02M TEAB buffer pH 7.5, is proposed as a fast and efficient method for the isolation and purification of protected oligoribonucleotide phosphodiesters obtained by deprotection of internucleotide phosphotriesters, and for the monitoring of the deprotection step itself. Its utility is shown on the example of removal of 2,2,2-trichloroethyl groups from oligoribonucleotide phosphotriester I of sequence CCCAUAA by two methods: /1/ reductive elimination with zinc in the presence of acetylacetone modified as presented here, and /2/ hydrogenolytic dehalogenation over palladium in pyridine. This method of chromatography on Sephadex LH20 is used as a key purification step during the removal of 2,2,2-trichloroethyl groups from I by method /1/ and allows to raise the yield of III during fianl deprotection step from 5 to 65%.

The reactions of thiouridines and thiouracils with chloroacetaldehyde; mechanistic considerations.

2-Thiouridine, 4-thiouridine and the corresponding thiouracils were quantitatively modified with aqueous chloroacetaldehyde /37 degrees C, pH 3.0--6.5/. The rate-pH dependence found for the disappearance of the substrates suggested initial S-alkylation. The unstable S-acetaldehydyl intermediates were not detected due to their further rapid transformations. The following possibilities of such transformations are discussed: 1. intramolecular addition of the endocyclic nitrogen atom to the aldehyde carbonyl group to form the "hydroxyethano" bridged compounds, 2. hydrolysis to the corresponding "oxo" analogues of the substrates, 3. hydrolysis of the N-glycoside bond. The structures of new compounds formed in these reactions were assigned on the basis of their FD-MS, UV, IR and PMR spectra. The reaction rates were similar to those found for modification of adenosine and cytidine with chloroacetaldehyde.

Further studies on oligoribonucleotide synthesis.

Recent results concerning the synthesis of oligoribonucleotides via the phosphotriester method, such as functionalization of ribonucleosides, new phosphorylating agents, 5'-O-sulfonylation and chromatography on Sephadex LH-20 for monitoring the removal of internucleotide phosphotriester groups, are presented. To show that efficiency of a new approach to the synthesis of oligoribonucleotides the pentamer /Up/4U was obtained.