Genetic heterogeneity of familial hypercholesterolaemia in two populations from two different countries.

BACKGROUND: Familial hypercholesterolemia (FH) is a genetically determined monogenic disorder of predominantly autosomal dominant inheritance. A number of studies on differences in the genetic profile of patients with FH have demonstrated the importance of a more substantive evaluation of genetic features. The aim of this study was to evaluate the genetic profile of patients with clinical FH among Italian and Russian patients. METHODS: We included 144 Italian and 79 Russian FH patients; clinical diagnosis was based on the same criteria. Patients were divided in: positive to genetic test (one causative variant), inconclusive (only variants of uncertain clinical significance [VUS]), and negative (with likely benign/benign variants, heterozygous variants in LDLRAP1 gene, or without causative variants). RESULTS: The genetic test was positive in 76.4 % of the Italian patients and in 49.4 % of the Russian patients. The presence of VUS alone was detected in 7.6 % and in 19.0 % (p < 0.001), respectively. Among patients with positive genetic diagnosis, pre-treatment LDL-C levels were higher in the Russian cohort (353.5 ± 111.3 vs. 302.7 ± 52.1 mg/dL, p = 0.009), as well as the percentage of treated patients (53.8 % vs. 14.5 %, p < 0.001) and the prevalence of premature coronary heart disease (12.8 % vs. 3.6 %, p = 0.039). Among patients carrying only VUS, mean pre-treatment LDL-C levels were similar between the cohorts (299.5 ± 68.1 vs. 295.3 ± 46.8 mg/dL, p = 0.863). Among pathogenic/likely pathogenic variants and VUS, only 5 % and 4 % was shared between the two cohorts, respectively. CONCLUSION: The genetic background of patients clinically diagnosed with FH in two different countries is characterized by high variability.

Chlorophyll fluorometry in evaluating photosynthetic performance: key limitations, possibilities, perspectives and alternatives.

Non-destructive methods for the assessment of photosynthetic parameters of plants are widely applied to evaluate rapidly the photosynthetic performance, plant health, and shifts in plant productivity induced by environmental and cultivation conditions. Most of these methods are based on measurements of chlorophyll fluorescence kinetics, particularly on pulse modulation (PAM) fluorometry. In this paper, fluorescence methods are critically discussed in regard to some their possibilities and limitations inherent to vascular plants and microalgae. Attention is paid to the potential errors related to the underestimation of thylakoidal cyclic electron transport and anoxygenic photosynthesis. PAM-methods are also observed considering the color-addressed measurements. Photoacoustic methods are discussed as an alternative and supplement to fluorometry. Novel Fourier modifications of PAM-fluorometry and photoacoustics are noted as tools allowing simultaneous application of a dual or multi frequency measuring light for one sample.

Chlorophyll fluorescence kinetics and oxygen evolution in Chlorella vulgaris cells: Blue vs. red light.

Oxygen evolution and chlorophyll fluorescence kinetics in cells of the Chlorella vulgaris strain (Europolytest, Russia) were studied under low, moderate and high photosynthetic photon flux densities (PPFD 40, 130 and 350 µmol photons m-2 s-1) of the red and blue actinic light. A novel method of a pulse amplitude modulated (PAM) Fourier chlorophyll fluorometry was applied to obtain photoinduction curves simultaneously for the red and blue measuring light for one sample. It was found that the red light did not induce oxygen evolution at low and moderate PPFD, whereas at high PPFD it caused a declining oxygen release. There was only a trace fluorescence kinetics at the low PPFD, but noticeable fluorescence kinetics under the red light was observed at the low and moderate PPFD. Particularly, the moderate red illumination of Chlorella cells excited a high chlorophyll fluorescence kinetics along with the absence of oxygen evolution that suggests anoxygenic photosynthesis. In contrast, the blue light induced a significant oxygen evolution as well as fluorescence kinetics already at low PPFD which were both further increased with the PPFD increasing. In addition, a high value of the chromatic divergence of quantum yield of photosystem II was revealed between the red and blue measuring light under high PPFD of the red actinic light.

Combination therapy with nifedipine GITS 60 mg: subanalysis of a prospective, 12-week observational study (AdADOSE).

BACKGROUND: AdADOSE was a 12-week, international, observational study conducted in the Middle East and Russia where patients received nifedipine gastrointestinal therapeutic system (GITS) at a daily dose of 30, 60, or 90 mg as part of an antihypertensive combination therapy. This subgroup analysis of the AdADOSE study assesses the efficacy and tolerability of nifedipine GITS combination therapy when used specifically at the 60-mg strength. METHODS: Patients with hypertension who received a daily nifedipine GITS dose of 60 mg, either at constant dose (n = 686) or up-titrated from 30 mg (n = 392), were analyzed. Target blood pressure (BP) was <140/90 mmHg (or <130/80 mmHg for those at high/very high cardiovascular risk). RESULTS: Following nifedipine GITS combination therapy, target BP was achieved by 33.7% patients in the 60 mg group (previously untreated, 42.5%; previously treated, 32.0%) and 32.4% patients in the 30-60 mg group (previously untreated, 45.2%; previously treated, 30.7%). Mean systolic BP/diastolic BP changes were -40.3/-20.7 mmHg and -35.6/-18.5 mmHg, respectively, and were similar regardless of previous antihypertensive treatment or the number of concomitant diseases. Incidences of drug-related adverse events (AEs) were low (3.2%, 60 mg; 2.0%, 30-60 mg group), few patients discontinued because of AEs (0.6% and 1.0%, respectively), and there were no serious AEs. CONCLUSION: Combination therapy with nifedipine GITS 60 mg in a real-life observational setting was effective and well tolerated in hypertensive patients, with low rates of treatment-related AEs.

Effectiveness of combination therapy with nifedipine GITS: a prospective, 12-week observational study (AdADOSE).

BACKGROUND: Observational studies can provide important information on the efficacy and safety of antihypertensive agents in the real-life clinical setting. AdADOSE was a large observational study to assess the effectiveness of nifedipine GITS in combination with other antihypertensive agent(s). The study was also the first to examine the role of combination therapy with nifedipine GITS in the Middle East, Pakistan and Russia, regions that are associated with particularly high cardiovascular risk. METHODS: AdADOSE was a 12-week, international, multicenter, prospective, observational study. Patients with hypertension (ie, blood pressure [BP] >140/90 mm Hg, or >130/80 mm Hg in patients at high or very high cardiovascular risk) received once-daily nifedipine GITS (30, 60 or 90 mg) in combination with another antihypertensive or as add-on to existing therapy. The primary study endpoint was the proportion of patients who achieved the target BP of <140/90 mm Hg (or <130/80 mm Hg for those at high or very high cardiovascular risk). Study outcomes are reported by descriptive statistics. RESULTS: The study enrolled 4497 patients (n = 4477, safety population; n = 3430, efficacy population). Baseline mean systolic/diastolic BP (SBP/DBP) was 166.4/99.7 mm Hg; 85.2 % of patients had received prior antihypertensive treatment, and 90.6 % had ≥ 1 concomitant diseases. Following combination treatment with nifedipine GITS, target BP was achieved by 64.8% of patients without concomitant diseases, and by 56.5%, 32.3% and 22.6% with 1, 2-3 and >3 concomitant diseases, respectively. The proportion of patients achieving target BP was 51.5% in previously untreated and 33.7% in previously treated patients. Nifedipine GITS combination treatment provided mean SBP/DBP changes of -36.1/-18.8 mm Hg in all patients, -40.2/-21.5 mm Hg in previously untreated patients, and -35.6/-18.4 mm Hg in previously treated patients, with similar BP reductions irrespective of the number of concomitant diseases. Drug-related adverse events (AEs) were reported in 2.6% patients. There were no serious AEs and only 0.8% of patients discontinued due to drug-related AEs. CONCLUSIONS: Combination therapy with nifedipine GITS in a real-life observational setting was highly effective in reducing SBP/DBP in a range of hypertensive patients, with low rates of treatment-related AEs. TRIAL REGISTRATION: at ClinicalTrials.gov registration number NCT01118286 .

2'-deoxy-4'-azido nucleoside analogs are highly potent inhibitors of hepatitis C virus replication despite the lack of 2'-alpha-hydroxyl groups.

RNA polymerases effectively discriminate against deoxyribonucleotides and specifically recognize ribonucleotide substrates most likely through direct hydrogen bonding interaction with the 2'-alpha-hydroxy moieties of ribonucleosides. Therefore, ribonucleoside analogs as inhibitors of viral RNA polymerases have mostly been designed to retain hydrogen bonding potential at this site for optimal inhibitory potency. Here, two novel nucleoside triphosphate analogs are described, which are efficiently incorporated into nascent RNA by the RNA-dependent RNA polymerase NS5B of hepatitis C virus (HCV), causing chain termination, despite the lack of alpha-hydroxy moieties. 2'-deoxy-2'-beta-fluoro-4'-azidocytidine (RO-0622) and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187) were excellent substrates for deoxycytidine kinase and were phosphorylated with efficiencies up to 3-fold higher than deoxycytidine. As compared with previous reports on ribonucleosides, higher levels of triphosphate were formed from RO-9187 in primary human hepatocytes, and both compounds were potent inhibitors of HCV virus replication in the replicon system (IC(50) = 171 +/- 12 nM and 24 +/- 3 nM for RO-9187 and RO-0622, respectively; CC(50) >1 mM for both). Both compounds inhibited RNA synthesis by HCV polymerases from either HCV genotypes 1a and 1b or containing S96T or S282T point mutations with similar potencies, suggesting no cross-resistance with either R1479 (4'-azidocytidine) or 2'-C-methyl nucleosides. Pharmacokinetic studies with RO-9187 in rats and dogs showed that plasma concentrations exceeding HCV replicon IC(50) values 8-150-fold could be achieved by low dose (10 mg/kg) oral administration. Therefore, 2'-alpha-deoxy-4'-azido nucleosides are a new class of antiviral nucleosides with promising preclinical properties as potential medicines for the treatment of HCV infection.

Hydrophilically enhanced 3-carboranyl thymidine analogues (3CTAs) for boron neutron capture therapy (BNCT) of cancer.

Five novel 3-carboranyl thymidine analogues (3CTAs) were designed and synthesized for boron neutron capture therapy (BNCT) of cancer. Phosphorylation of all five 3CTAs was catalyzed by recombinant human thymidine kinase (hTK1) using adenosine triphosphate (ATP) as the phosphate donor. The obtained phosphorylation rates ranged from 4% to 64.5% relative to that of thymidine. The compound with the most favorable hTK1 binding properties had a k(cat)/K(M) value of 57.4% relative to that of thymidine and an IC(50) of inhibition of thymidine phosphorylation by hTK1 of 92 microM. Among the five synthesized 3CTAs, this agent had also the overall most favorable physicochemical properties. Therefore, it may have the potential to replace N5-2OH, the current lead 3CTA, in preclinical studies. An in silico model for the binding of this compound to hTK1 was developed.

Fluorescence energy transfer studies of human deoxycytidine kinase: role of cysteine 185 in the conformational changes that occur upon substrate binding.

Human deoxycytidine kinase (dCK) phosphorylates both pyrimidine and purine deoxynucleosides, including numerous nucleoside analogue prodrugs. Energy transfer studies of transfer between Trp residues of dCK and the fluorescent probe N-(1-pyrene)maleimide (PM), which specifically labels Cys residues in proteins, were performed. Two of the six Cys residues in dCK were labeled, yielding a protein that was functionally active. We determined the average distances between PM-labeled Cys residues and Trp residues in dCK in the absence and presence of various pyrimidine and purine nucleoside analogues with the Trp residues as energy donors and PM-labeled Cys residues as acceptors. The transfer efficiency was determined from donor intensity quenching and the Förster distance R(0) at which the efficiency of energy transfer is 50%, which was 19.90 A for dCK-PM. The average distance R between the Trp residues and the labeled Cys residues in dCK-PM was 18.50 A, and once substrates bound, this distance was reduced, demonstrating conformational changes. Several of the Cys residues of dCK were mutated to Ala, and the properties of the purified mutant proteins were studied. PM labeled a single Cys residue in Cys-185-Ala dCK, suggesting that one of the two Cys residues labeled in wild-type dCK was Cys 185. The distance between the single PM-labeled Cys residue and the Trp residues in Cys-185-Ala dCK was 20.75 A. Binding of nucleosides had no effect on the pyrene fluorescence of Cys-185-Ala dCK, indicating that the conformational changes observed upon substrate binding to wild-type dCK-PM involved the "lid region" of which Cys 185 is a part. The substrate specificity of Cys-185-Ala dCK was altered in that dAdo and UTP were better substrates for the mutant than for the wild-type enzyme.

Human deoxycytidine kinase as a deoxyribonucleoside phosphorylase.

Human deoxycytidine kinase (dCK) is a key enzyme in the 5'-phosphorylation of purine and pyrimidine deoxynucleosides with deoxycytidine as the most efficient substrate. The ability of dCK to degrade 2'-deoxyribonucleosides to free nucleobases and 2-deoxy-alpha-d-ribofuranose-1-phosphate was demonstrated by 1H-31P correlation spectroscopy and by isotope enzyme kinetic methods. The reaction depended on inorganic phosphate, and dCK showed maximum cleavage activity between pH 7 and pH 8. In this pH range, [HPO4(2-)] is the dominant phosphate species, most likely being the phosphate donor. All natural deoxyribonucleosides could be cleaved and the Vmax of the phosphorylytic reaction compared to the kinase reaction was about 2-10%. The formation of free nucleobases occurred only with reduced dCK, because the reaction was highly dependent on the presence of reducing agents such as dithiotreitol. Thus, recombinant dCK can act as a phosphorylase, similar to the nucleoside phosphorylase family of enzymes. This catalytic activity is important for the design of in vitro experiments with dCK, such as crystallization and NMR spectroscopy.

Hydrodynamic and spectroscopic studies of substrate binding to human recombinant deoxycytidine kinase.

Deoxycytidine kinase (dCK), a cytosolic enzyme with broad substrate specificity, plays a key role in the activation of therapeutic nucleoside analogues by their 5'-phosphorylation. The structure of human dCK is still not known and the current work was undertaken to determine its oligomeric and secondary structure. Biophysical studies were conducted with purified recombinant human dCK. The Mr determined by low-speed sedimentation equilibrium under nondenaturing conditions was 60,250 +/- 1,000, indicating that dCK, which has a predicted Mr of 30,500, exists in solution as a dimer. Analysis of circular dichroism spectra revealed the presence of two negative dichroic bands located at 222 and 209 nm with ellipticity values of -11,900 +/- 300 and -12,500 +/- 300 deg x cm2 x dmol(-1), respectively, indicating the presence of approximately 40% alpha-helix and 50% beta-structure. Circular Dichroism studies in the aromatic and far-ultraviolet range and UV difference spectroscopy indicated that binding of substrates to dCK reduced its alpha-helical content and perturbed tryptophan and tyrosine. Steady-state fluorescence demonstrated that deoxycytidine (the phosphate acceptor) and ATP (the phosphate donor) bound to different sites on dCK and fluorescence quenching revealed bimodal binding of deoxycytidine and unimodal binding of ATP. Spectroscopic studies indicated that substrate binding induced conformational changes, with the result that dCK exhibited different affinities for various substrates. These results are consistent with a random bi-bi kinetic mechanism of phosphorylation of dCyd with either ATP or UTP.

Identification of residues involved in the substrate specificity of human and murine dCK.

Deoxycytidine kinase (dCK) is a salvage pathway enzyme that can phosphorylate both pyrimidine and purine deoxynucleosides, including important antiviral and cytostatic agents. Earlier studies showed that there are differences in kinetic properties between human and murine dCK, which may explain differences in toxic effects of nucleoside analogs. To determine if certain substitutions in amino acid sequences between human and mouse dCK give these differences in substrate specificity the 14 mutants and hybrid forms of human dCK were studied. All variants were characterised with dCyd, dAdo and dGuo as phosphate acceptors and ATP and UTP as phosphate donor. The relative activities with dCyd, dAdo and dGuo were about 70, 20, 30%, respectively, with UTP as compared to ATP for human dCK and 40, 60, 70% for mouse dCK. Among all tested mutants only the triple combination of substitutions Q179R-T184K-H187N (RKN) had a kinetic behaviour very similar to mouse dCK. The kinetic patterns with several important nucleoside analogs, such as AraC, CdA, ddC and AraG have also been studied. Results demonstrated 50-70% low relative capacities of the recombinant mouse and triple mutant RKN to phosphorylate this nucleoside analogs compare with human dCK. A model for dCK was used to try to explain the functional role of these amino acid substitutions. According to this model the triple mutant RKN have altered amino acids in a region necessary for conformational changes during catalyses. This may affects the substrate selectivity both for the nucleosides and the phosphate donors.