Denis Drainas - Academia.edu (original) (raw)

Papers by Denis Drainas

European Journal of Biochemistry, 1993

Ribonuclease P from the fission yeast Schizosaccharomyces pombe has been purified to apparent hom... more Ribonuclease P from the fission yeast Schizosaccharomyces pombe has been purified to apparent homogeneity. A purification of 23 000-fold was achieved by four fractionation steps with DEAEcellulose chromatography, phosphocellulose chromatography, glycerol-gradient fractionation and finally tRNA-affinity chromatography. A 100-kDa protein was present in the most pure preparations in amounts approximately stoichiometric with the previously identified RNA components of the enzyme, K1-RNA and K2-RNA

European Journal of Biochemistry, 1985

Aspergillus nidulans asparaginase activity may be assayed conductimetrically. The method is based... more Aspergillus nidulans asparaginase activity may be assayed conductimetrically. The method is based on the increase of conductivity which is due to the production of ammonia and/or aspartate in a reaction mixture containing A. nidulans cell-free extract and asparagine or aspartate hydroxamate. This conductivity is linear with time and enzyme concentration and it follows Michaelis kinetics. Conductimetric activity was not detectable in mutants lacking asparaginase activity.

Molecular Biology Reports, 1996

Ribonuclease P (RNase P) is a key enzyme involved in tRNA biosynthesis. It catalyses the endonucl... more Ribonuclease P (RNase P) is a key enzyme involved in tRNA biosynthesis. It catalyses the endonucleolytic cleavage of nearly all tRNA precursors to produce 5'-end matured tRNA. RNase P activity has been found in all organisms examined, from bacteria to mammals. Eubacterial RNase P RIgA is the only known RNA enzyme which functions in trans in nature. Similar behaviour has not been demonstrated in RNase P enzymes examined from archaebacteria or eukaryotes. Characterisation of RNase P enzymes from more diverse eukaryotic species, including the slime mold Dietyostelium discoideum, is useful for comparative analysis of the structure and function of eukaryotic RNase E

The kinetic behaviour of Bee venom phospholipase A2 was studied using both long and short chain s... more The kinetic behaviour of Bee venom phospholipase A2 was studied using both long and short chain substrates in the presence and absence of organic solvents. The activity was controlled by a variety of activators and in particular the behaviour in dilute organic solvents was dominated by fatty acid activation. The enzyme was activated irreversibly by a variety of acylating agents derived from long-chain fatty acids. The most effective derivatives were azides and imidazolides and the reactivity was proportional to the hydrocarbon chain length, in contrast to the general chemical reactivity. Oleoyl and linoleoyl imidazolide were shown to be the most effective activators and the oleoyl derivative gave full activation corresponding to addition of one acyl residue per protein molecule. Studies of maximally activated enzyme showed that earlier models in which activation was proposed to rely on fatty acid modification of the substrate surface could be ruled out. Activation does not appear to...

Medicinal chemistry (Shariqah (United Arab Emirates)), Jan 11, 2018

Background RNase P-mediated cleavage of target RNAs has been proposed as a promising tool for gen... more Background RNase P-mediated cleavage of target RNAs has been proposed as a promising tool for gene silencing. Ets-2 proto-oncogene controls the expression of a wide variety of genes involved in cancer and immunity. Objective Construction of a functional RNase P-based ribozyme (M1GS303) that targets Ets-2 mRNA Method The accessible sites for targeting of Ets-2 mRNA were identified by footprinting analysis. M1GS303 ribozyme was constructed by cloning. The activity of the ribozyme in the presence or absence of spiramysin in E. coli cells and human cell lines was quantified by RT-PCR. The efficiency of the ribozyme in silencing the endogenous expression of Ets-2 in human cell lines was examined by RT-PCR, western blot and immunofluorescence analysis. Results In E. coli cells co-transformed with plasmids bearing M1GS303 and the ets-2 target gene, Ets-2 mRNA was decreased by 93% 12h after IPTG induction in the absence, and after 4h in the presence of spiramycin. Ets-2 was rapidly downregu...

European Journal of Biochemistry, 2000

The effects of cholesterol, 7-dehydrocholesterol, vitamin D 3 and several synthetic vitamin D 3 a... more The effects of cholesterol, 7-dehydrocholesterol, vitamin D 3 and several synthetic vitamin D 3 analogs on ribonuclease P (RNase P) were investigated using a cell-free system from the slime mold Dictyostelium discoideum. RNase P is an ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5 H end. Among the compounds tested, only calcipotriol was capable of affecting RNase P activity, and revealed a bimodal action at the kinetic phase of the reaction. Depending on the concentration of the drug, both activation and inhibition of tRNA maturation were observed, indicating that calcipotriol may have a direct effect on tRNA biogenesis, possibly associated with the presence of a highly reactive small ring on the side chain of its molecule.

Experimental dermatology, 2009

Ribonuclease P (RNase P) is ubiquitous and essential Mg(2+)-dependent endoribonuclease that catal... more Ribonuclease P (RNase P) is ubiquitous and essential Mg(2+)-dependent endoribonuclease that catalyzes the 5'-maturation of transfer RNAs. RNase P and the ribosome are so far the only ribozymes known to be conserved in all kingdoms of life. Eukaryotic RNase P activity has been detected in nuclei, mitochondria and chloroplasts and demonstrates great variability in sequence and subunit composition. In the last few years we have developed methodologies and pursued projects addressing the occurrence, distribution and the potential physiological role of RNase P in human epidermal keratinocytes. In view of the vital importance of lymphocytes for an effective immune system and their successful application after transfection with RNase P-associated external guide sequences in gene therapy, we concerned ourselves with the isolation and characterization of RNase P of peripheral human lymphocytes. We developed a method described herein, that will enable the study of the possible involvement...

European Journal of Biochemistry, 1981

Bee venom phospholipase A2 causes a small increase in the sublytic leakiness of rabbit erythrocyt... more Bee venom phospholipase A2 causes a small increase in the sublytic leakiness of rabbit erythrocytes which is strongly inhibited by lysolecithin and potentiated by albumin. 2. Albumin extracts long-chain fatty acids from the erythrocyte membrane by a process which is faster than diffusion-determined efflux of fatty acid and during this process it greatly increases the sublytic leakiness of the membrane. In contrast, albumin rapidly terminates sublytic responses to lysolecithin. 3. These actions of albumin are abolished only when albumin is preloaded with four molecules of oleic acid per protein molecule. 4. A minor proportion of the response of phospholipase-Az-treated cells to albumin is due to accumulated fatty acid products; the combination of active enzyme and accumulated fatty acids is much more effective than either alone. Membrane partially depleted of phospholipids by enzyme attack is not highly leaky. 5. Albumin may, in part, act by removing lysolecitihin, the inhibitory reaction product. A model is proposed in which susceptibility to phospholipase A2 attack is not uniform on the cell surface and is used to explain the inhibitory action of lysolecithin and the high sensitivity of the membrane in the presence of albumin to damage by newly released fatty acids.

Skin Pharmacology and Physiology, 2003

The ever-growing resistance of pathogens to antibiotics and the lack of potent antibacterial drug... more The ever-growing resistance of pathogens to antibiotics and the lack of potent antibacterial drugs constitute major problems in the treatment of infectious diseases. Thus, the better understanding of the mode of action of antibiotics at the molecular level is of essential importance. Accumulating evidence points towards RNA as being a crucial target of antibacterial and antiviral drugs. Interestingly, aminoglycosides, one of the most important families of antibiotics, apart from their inhibitory effect on ribosome function, reportedly interfere with various RNA molecules and in vitro suppress the proliferation of human keratinocytes. In this study we investigated the effect of the aminoglycosides neomycin B, paromomycin, tobramycin and gentamycin on ribonuclease P activity from normal human epidermal keratinocytes. All aminoglycosides tested revealed a dose-dependent inhibition of tRNA maturation, which was reduced by increasing Mg2+ ion concentrations, indicating competition of the...

Skin Pharmacology and Physiology, 2002

The purpose of this double-blind randomised parallel-group study was to compare the efficacy and ... more The purpose of this double-blind randomised parallel-group study was to compare the efficacy and safety of short-contact treatment with dithranol ointment (2%) with its combination with calcipotriol ointment (50 µg/g) in 2 groups of in-patients with chronic plaque psoriasis. The patients of the first group (n = 23) topically applied dithranol once daily for 30 min and the vehicle of calcipotriol twice daily. The patients of the second group (n = 23) used a single topical application of dithranol for 30 min daily and additionally applied calcipotriol twice daily. The extent and the severity of psoriasis were assessed by means of psoriasis area and severity index score (PASI score) before the onset of the 6-week therapy and weekly thereafter. The difference between the two groups with regard to the mean PASI score became statistically significant already after the first week of treatment and remained so until the end of the trial. No significant differences were observed between the t...

Skin Pharmacology and Physiology, 2000

The effect of five different anthralin concentrations on tRNA biogenesis was investi- gated emplo... more The effect of five different anthralin concentrations on tRNA biogenesis was investi- gated employing the ribonuclease P (RNase P) of the slime mold Dictyostelium discoideum as an in vitro cell-free experimental system. RNase P is an ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5′ end. Anthralin revealed a dose-dependent inhibition of RNase P activity indicating that this compound may have a direct effect on tRNA biogenesis. Taking into account that anthralin has no structural similarities to the substrate (pre-tRNA) of RNase P, it seems reasonable to suggest that this compound may bind to allosteric inhibition sites of the enzyme.

IUBMB Life, 2008

RNA molecules play critical roles in cell biology, and novel findings continuously broaden their ... more RNA molecules play critical roles in cell biology, and novel findings continuously broaden their functional repertoires. Apart from their well-documented participation in protein synthesis, it is now apparent that several noncoding RNAs (i.e., micro-RNAs and riboswitches) also participate in the regulation of gene expression. The discovery of catalytic RNAs had profound implications on our views concerning the evolution of life on our planet at a molecular level. A characteristic attribute of RNA, probably traced back to its ancestral origin, is the ability to interact with and be modulated by several ions and molecules of different sizes. The inhibition of ribosome activity by antibiotics has been extensively used as a therapeutical approach, while activation and substrate-specificity alteration have the potential to enhance the versatility of ribozyme-based tools in translational research. In this review, we will describe some representative examples of such modulators to illustrate the potential of catalytic RNAs as tools and targets in research and clinical approaches.

Current Medicinal Chemistry, 2004

RNase P is an ubiquitous and essential endonuclease in tRNA biogenesis, which generates the matur... more RNase P is an ubiquitous and essential endonuclease in tRNA biogenesis, which generates the mature 5-termini of tRNAs. RNase P activities have been identified in all three kingdoms of life (Bacteria, Archaea, Eukarya). Most forms of RNase P are ribonucleoproteins, i.e., they consist of an essential RNA and protein subunits. In bacteria and in some archaea, the catalytic function of this enzyme resides entirely in its RNA subunit, which is one of firstly identified ribozymes. Its high structural and functional diversity among representatives of a vast variety of phylogenetic domains indicates that RNase P could also serve as a molecular target of and a useful screening system for the biological activity of different compounds and give more insight into the molecular mechanisms of their action inside the cell. The emerged information from recent studies on the mechanism and structural idiosyncrasies of RNase P provides a convenient platform for designing specific inhibitors for this r...

Biological Chemistry, 2003

Ribonuclease P (RNase P) is a ubiquitous and essential enzyme that endonucleolytically cleaves al... more Ribonuclease P (RNase P) is a ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5'-end. We have investigated the effect of synthetic rertinoids (all-trans retinoic acid, acitretin) and arotinoids (Ro 13-7410, Ro 15-0778, Ro, 13-6298 and Ro 15-1570) on RNase P activity isolated for the first time from normal human epidermal keratinocytes (NHEK). All tested compounds but one (Ro 15-1570) revealed a dose-dependent inhibition of RNase P activity, indicating that they may have a direct effect on tRNA biogenesis. Detailed kinetic analysis showed that all retinoids behave as classic competitive inhibitors. On the basis of the Ki values Ro 13-7410 was found to be the strongest inhibitor among all compounds tested.

Archives of Biochemistry and Biophysics, 1993

Archives of Biochemistry and Biophysics, 1992

Analytical Biochemistry, 1997

unit have been suggested to be involved in the peptidyl-We have developed an in vitro system for ... more unit have been suggested to be involved in the peptidyl-We have developed an in vitro system for the determitransferase center (3). Although ribosome reconstitunation of peptidyltransferase activity in rabbit reticulotion experiments have demonstrated an absolute cyte ribosomes. Using this system, a detailed kinetic analrequirement for a restricted number of ribosomal proysis of a model reaction for peptidyltransferase is teins, direct evidence for their involvement in the catadescribed, with AcPhe-tRNA as the peptidyl donor and lytic process is still lacking (3). Moreover, it was obpuromycin as the acceptor. The [AcPhe-tRNA-poly(U)served that the peptidyltransferase activity of the large 80S ribosome] complex (complex C) is isolated and then subunit of Thermus aquaticus ribosomes shows an unreacted with excess puromycin to give AcPhe-puromycin. usual resistance to protein extraction procedures, in This reaction (puromycin reaction) follows first-order kicontrast to its sensitivity to ribonuclease (4). Several netics at all concentrations of puromycin tested. At satulines of evidence indicate the involvement of rRNA in rating concentrations of puromycin, the first-order rate the process of translation (5). rRNA participates in (k 3) constant is identical to the catalytic rate constant tRNA binding and may play a crucial role in the cataly-(k cat) of peptidyltransferase. This k 3 of peptidyltransfersis of the peptidyl transfer (4, 5). Recently, it has been ase is equal to 2.9 min 01 at 37ЊC. Moreover, the ratio k 3 / shown that a Watson-Crick G-C base pair between K s , which is an accurate measure of peptidyltransferase 23S rRNA and the acceptor end of tRNA is required activity, was increased 80-fold when salt-washed ribofor proper functional interaction of the CCA end of somes were replaced by unwashed ribosomes. Finally, the tRNA with the ribosomal P site. These findings estabpuromycin reaction was inhibited by several well-known lish a direct role for 23S rRNA in protein synthesis (6). antibiotics acting on the eukaryotic peptidyltransferase. The standard reaction for determining peptidyltran-᭧ 1997 Academic Press sferase activity in vitro is the puromycin reaction, in which a peptide bond is formed between the amino group of puromycin and the ester group of the donor peptidyl-tRNA. The donor can be an N-protected ami-Peptide bond formation, the major function of the noacyl-tRNA such as AcPhe-tRNA (7, 8). This method ribosome, is an inherent property of the large ribosomal has been applied in a prokaryotic system derived from subunit. This reaction is catalyzed by the main enzy-Escherichia coli, to recognize certain types of inhibition matic activity associated with the ribosome, the soof this reaction by several ligands which are reminiscalled peptidyltransferase activity. Although considercent of the types of inhibition encountered in typical able progress has been made in recent years on the enzyme reactions (9, 10). In eukaryotic systems, the structure and function of the prokaryotic ribosomes (1), puromycin reaction has been mainly applied for the relatively little is known about eukaryotic ribosomes evaluation of the distribution of peptidyl-tRNA beand eukaryotic peptidyltransferase. The components of tween A or P site, and not for the determination of the peptidyltransferase center have not yet been identipeptidyltransferase activity. In the present study we fied (2). A number of ribosomal proteins from the large used a method of analyzing a certain variant of the subunit, as well as some proteins from the small subpuromycin reaction such as the reaction between puromycin and a ribosomal complex bearing the donor pre-† Professor C. Coutsogeorgopoulos passed away about two years bound at the P site (puromycin reactive state) by using ago. a cell-free system derived from rabbit reticulocytes. By

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1985

Assay methods for bee venom phospholipase A z are presented which respond to different aspects of... more Assay methods for bee venom phospholipase A z are presented which respond to different aspects of enzymic behaviour and which allow basal activi~, fatty, acid activation and acyl-group activation to be distinguished. The stability, of the enzyme to thiols and proteinases is dramatically increased by activation with the selective acylating agent, oleoyl imidazolide. These results support the model of activation by conformation change. Limited-fixation studies indicate that enzyme conformation is determined by interaction with the substrate. The oleoyi-enzyme is partially inactivated by t~psin, but its electrophoretic mobility is unchanged. This protective effect is highly selective and only one other component of the venom is protected against t~psin by oleoyl imidazolide. Combination of trypsin and thiol treatment produces a large fragment of the activated enzyme which could be used for structural studies of the activation site.

International Journal of Biochemistry, 1992

1. The presence of polyamines in the growth medium of Escherichia coli can modulate the activity ... more 1. The presence of polyamines in the growth medium of Escherichia coli can modulate the activity of the RNA-processing enzyme, ribonucleoprotein ribonuclease P (RNase P), by altering the expression of the rnpA and rnpB genes, which encode its C5 protein and M1 RNA subunits, respectively. 2. Following growth in the presence of 1 mM spermidine the levels of C5 protein mRNA and catalytic M1 RNA were significantly elevated in the wild type E. coli K-12 strain MG1655. 3. The rnpA mRNA, together with the ribosomal protein L34 (rpmH) mRNA, was found to constitute a dicistronic rpmH-rnpA message whose half-life did not change upon Escherichia coli growth in the presence of spermidine. 4. This suggests that the spermidine effect is on the transcriptional level. 5. Increased expression of the rnpA and rnpB genes was reflected in the activity of RNase P, which almost doubled. 6. These results identify yet another component of the protein synthetic machinery which is specifically affected by polyamines.