Denis Drainas

Calcipotriol Plus Short-Contact Dithranol: A Novel Topical Combination Therapy for Chronic Plaque Psoriasis

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 two groups with respect to the cutaneous adverse events. These findings indicate that the addition of calcipotriol ointment to short-contact dithranol markedly augments the therapeutic efficacy of the latter in chronic plaque psoriasis and impressively accelerates the response of psoriatic plaques to this well-tolerated regimen.

Inhibition of Ribonuclease P Activity by Retinoids

The effect of two naturally occurring (retinol and all-trans retinoic acid) and two synthetic (isotretinoin and acitretin) analogs of vitamin A (retinoids) on tRNA biogenesis was investigated employing the RNase P ofDictyostelium discoideum as an in vitroexperimental system. RNase P is an ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5′ end. All retinoids tested revealed a dose-dependent inhibition of RNase P activity, indicating that these compounds may have a direct effect on tRNA biogenesis. Detailed kinetic analysis showed that all retinoids behave as classical competitive inhibitors. The K i values determined were 1475 μmfor retinol, 15 μm for all-trans retinoic acid, 20 μm for isotretinoin, and 8.0 μmfor acitretin. On the basis of these values acitretin is a 184, 2.5, and 1.9 times more potent inhibitor, as compared with retinol, isotretinoin, and all-trans retinoic acid, respectively. Taking into account that retinoids share no structural similarities to precursor tRNA, it is suggested that their kinetic behavior reflects allosteric interactions of these compounds with hydrophobic site(s) ofD. discoideum RNase P.

Inhibition of eukaryotic ribonuclease P activity by aminoglycosides: kinetic studies

The effect of several aminoglycoside antibiotics on ribonuclease P (RNase P) was investigated using an in vitro experimental system from Dictyostelium discoideum. Detailed kinetic analysis showed that all aminoglycosides tested (tobramycin, gentamicin, kanamycin, paromomycin, neomycin) behave as classical non‐competitive inhibitors, with neomycin being the strongest inhibitor. The inhibition effect is attributed to the electrostatic competition of the cationic aminoglycosides with magnesium ions required for catalysis. Increasing Mg2+ ion concentrations reduced the effect of aminoglycosides on RNase P activity. Detailed kinetic analysis showed that aminoglycosides compete with Mg2+ for common binding sites on RNase P holoenzyme.

Two-codon T-box riboswitch binding two tRNAs

T-box riboswitches control transcription of downstream genes through the tRNA-binding formation of terminator or antiterminator structures. Previously reported T-boxes were described as single-specificity riboswitches that can bind specific tRNA anticodons through codon–anticodon interactions with the nucleotide triplet of their specifier loop (SL). However, the possibility that T-boxes might exhibit specificity beyond a single tRNA had been overlooked. In Clostridium acetobutylicum, the T-box that regulates the operon for the essential tRNA-dependent transamidation pathway harbors a SL with two potential overlapping codon positions for tRNAAsn and tRNAGlu. To test its specificity, we performed extensive mutagenic, biochemical, and chemical probing analyses. Surprisingly, both tRNAs can efficiently bind the SL in vitro and in vivo. The dual specificity of the T-box is allowed by a single base shift on the SL from one overlapping codon to the next. This feature allows the riboswitch to sense two tRNAs and balance the biosynthesis of two amino acids. Detailed genomic comparisons support our observations and suggest that “flexible” T-box riboswitches are widespread among bacteria, and, moreover, their specificity is dictated by the metabolic interconnection of the pathways under control. Taken together, our results support the notion of a genome-dependent codon ambiguity of the SLs. Furthermore, the existence of two overlapping codons imposes a unique example of tRNA-dependent regulation at the transcriptional level.

Aminoglycosides suppress tRNA processing in human epidermal keratinocytes in vitro

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 cationic aminoglycosides with magnesium ions required for catalysis. Our in vitro findings suggest that the inhibitory effects of aminoglycosides on tRNA processing may be implicated in the mechanisms of their antiproliferative action on human epidermal keratinocytes.

Bimodal action of spermine on ribosomal peptidyltransferase at low concentration of magnesium ions

At 6 mM Mg2+, submillimolar concentrations of spermine affect the end-point as well as the kinetic phase of puromycin reaction in a cell-free system from Escherichia coli. When the ternary complex AcPhe-tRNA-poly(U)-ribosome (complex C) is formed in the absence of ribosomal wash (FWR fraction), the final degree of AcPhe-puromycin synthesis is raised from 12% to 60%, as the concentration of spermine increases from zero to 200 microM. However, spermine displays partial noncompetitive inhibition at the kinetic phase of the reaction. The inhibitory effect of spermine is related with its binding to AcPhe-tRNA. When complex C is formed in the presence of FWR fraction, spermine slightly affects the final degree of puromycin synthesis is markedly stimulated by the addition of relatively low concentrations of spermine. Kinetic analysis of the activation phase revealed that spermine attached on a specific site of complex C, acts as a nonessential, partial noncompetitive activator. The stimulatory effect of spermine seems to be due to its interaction with ribosomes. Further additions of spermine cause partial noncompetitive inhibition on the puromycin reaction. This result suggests that complex C possesses a second binding site, responsible for the inhibitory effect of spermine. Both activator and inhibitor sites can be occupied by spermine at the same time.

Kinetics of inhibition of peptide bond formation on bacterial ribosomes.

A cell-free system derived from Escherichia coli has been used in order to study the kinetics of inhibition of peptide bond formation with the aid of the puromycin reaction in solution. A similar study has been carried out earlier on a solid support matrix with the same inhibitors. We find that the overall pattern of the kinetics of inhibition is the same in the two systems. At low concentrations of inhibitor there is a competitive phase of inhibition, whereas at higher concentrations of inhibitor the type of inhibition becomes mixed noncompetitive. The values of Ki of the competitive phase in the system in solution are: 5.8 microM (amicetin), 0.2 microM (blasticidin S), 0.5 microM (chloramphenicol), and 0.5 microM (tevenel). The inhibitors amicetin, blasticidin S, and tevenel interact with the ribosome in a reaction which is slower than that of the substrate puromycin, showing clear-cut characteristics of slow-onset inhibition in both systems. Chloramphenicol, on the other hand does not easily show such a delay in solution. It interacts with the ribosome relatively faster than the other three antibiotics. Despite this, chloramphenicol too shows characteristics of time-dependent inhibition.

Effect of spermine on peptide-bond formation, catalyzed by ribosomal peptidyltransferase

The effect of spermine on the binding of AcPhe-tRNA to poly(U)-programmed ribosomes (step 1) and on the puromycin reaction (step 2) has been studied in a cell-free system, derived from E. coli.In the absence of ribosomal wash (FWR fraction) and at suboptimal concentration of Mg++ (6 mM), spermine stimulated the binding of AcPhe-tRNA at least five fold, while at 10 mM Mg++ there was a three fold stimulation. The above stimulatory effect was decreased at 6 mM Mg++, or was abolished at 10 mM Mg++ by the presence of FWR during the binding. Beside the stimulatory effect, spermine enhanced the stability of initiation complex AcPhe-tRNA-poly(U)-ribosome.In step 2, spermine affected the final degree of puromycin reaction and the activity status of peptidyltransferase. Both stimulatory and inhibitory effects have been observed, depending on the experimental conditions followed during the binding of the donor and during the peptide bond formation.

Preparation of spermine conjugates with acidic retinoids with potent ribonuclease P inhibitory activity.

Novel mono- and diacylated spermines, readily obtained using isolable succinimidyl active esters of acidic retinoids for the selective acylation of free spermine or in situ activated acidic retinoids for acylating selectively protected spermine followed by deprotection, were shown to inhibit the ribozyme ribonuclease P more strongly than the parent retinoids.

Effect of conjugates of all-trans-retinoic acid and shorter polyene chain analogues with amino acids on prostate cancer cell growth

In the present work, a series of conjugates of amino acids with all-trans-retinoic acid (ATRA) and shorter polyene chain analogues were rationally designed, synthesized by coupling the succinimidyl active esters of the acidic retinoids with appropriately protected amino acids or peptides followed by deprotection, and examined for their possible effect on viability of human prostate cancer LNCaP cells. In contrast to ATRA, all conjugates bearing amino acids with polar side chains showed no inhibitory effect on LNCaP cell proliferation, while conjugates with alpha-amino acids with lipophilic side chain, such as 7, or linear amino acids, such as 9, significantly decreased prostate cancer LNCaP cell number. Interestingly, while the effect of ATRA was RARalpha-dependent, the effect of its active analogues was not inhibited by a selective RARalpha antagonist. Cell cycle analysis showed no effect on cell cycle, while quantitative analysis by annexin V-propidium iodide staining revealed that neither ATRA nor its analogues affected LNCaP cell apoptosis or necrosis. These results demonstrate that compounds 7 and 9 are potentially useful agents that warrant further preclinical development for treatment of prostate cancer.