Hypoplastic form of myelodysplastic neoplasm.

BACKGROUND: Hypoplastic myelodysplastic neoplasm (MDS-h) is a rare hematopoietic disorder characterized by peripheral cytopenia, hypoplasia (cellularity ≤ 25%) and dysplastic changes in the bone marrow. Compared to normo- /hypercellular MDS, in addition to hypocellularity, MDS-h patients have more profound neutropenia and thrombocytopenia, a lower percentage of blasts, and less frequent abnormal karyotype. It is difficult to distinguish MDS-h from aplastic anemia in differential diagnosis. Abnormal karyotype is found in 15-50% of MDS-h patients and the most common chromosomal aberrations include -5/del (5q), -7/del (7q), +8, 17pLOH, del (20q), UPD at 4q, 11q, 13q, and 14q. Approximately 35% of MDS-h patients harbour somatic mutations that are most often detected in PIGA, TET2, DNMT3A, RUNX1, NPM1, ASXL1, STAG2, and APC genes. An autoimmune destruction of hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) mediated by abnormally activated T cells plays a key role in the pathophysiology of MDS-h. Expanded T cells overproduce proinflammatory cytokines (IFN- g and TNF-a), which inhibit proliferation and induce apoptosis of HSC/HPCs. The antigens that trigger the immune response are not known, but potential candidates have been suggested such as WT1 protein and HLA class I molecules. MDS-h does not represent a phenotypically homogeneous subtype of MDS, but rather it is a mixed entity comprising both patients showing features similar to myelodysplastic neoplasm and patients with features of non-malignant bone marrow failure. Determining the prevailing phenotype in MDS-h is important for choosing the optimal treatment and prognosis prediction. PURPOSE: The aim of this article is to point out an interesting hypoplastic MDS, the diagnosis of which is difficult, and to provide an overview of its main clinical-pathological features, genetic background, and mechanisms of aberrant immune response.

Microarray profiling defines circulating microRNAs associated with myelodysplastic syndromes.

Circulating microRNAs (miRNAs) are non-coding RNAs secreted into body fluids, and aberrant levels of these miRNAs correlate with diseases of various origins, making them highly potential clinical biomarkers. We investigated the spectrum of circulating miRNAs in the plasma of myelodysplastic syndrome (MDS) patients to identify miRNAs showing discriminatory levels in the patients with different prognosis. Plasma samples were analyzed with microarrays to define miRNA profiles, and the deregulated miRNAs were further studied using droplet digital PCR. With regard to the prognosis, the levels of miR-27a-3p, miR-150-5p, miR-199a-5p, miR-223-3p and miR-451a were reduced in higher-risk MDS. Multivariate analysis indicated miR-451a level as an independent predictor of progression-free survival (HR = 0.072, P = 0.006) and revealed a significant association of miR-223-3p level with overall survival (HR = 0.039, P = .032). Our data demonstrate that plasma levels of specific miRNAs are associated with MDS patient outcome and may add information beyond the currently used scoring systems.

Circulating MicroRNAs: Methodological Aspects in Detection of These Biomarkers.

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that regulate expression of protein-coding genes involved in important biological processes and (patho)physiological states. Circulating miRNAs are protected against degradation, indicating their relevant biological functions. Many studies have demonstrated an association of the specific profile of circulating miRNAs with a wide range of cancers as well as non-malignant diseases. These findings demonstrate the implication of circulating miRNAs in the pathogenesis of diseases and their potential as non-invasive disease biomarkers. However, methods for measurement of circulating miRNAs have critical technical hotspots, resulting in a discrepancy of the reported results and difficult definition of consensus disease biomarkers that may be implicated in clinical use. Here, we review functions of circulating miRNAs and their aberrant expression in particular diseases. Further, we discuss methodological aspects of their detection and quantification as well as our experience with the methods.

The Hans algorithm failed to predict outcome in patients with diffuse large B-cell lymphoma treated with rituximab.

Diffuse large B-cell lymphoma (DLBCL) consists of at least two biologically and pathogenetically different subtypes, the germinal centre B-cell (GCB) and the activated B cell type (ABC). It has been suggested that immunohistochemistry can discriminate these subtypes as well. The aim of this study was to verify the validity of the most commonly used Hans algorithm in patients with DLBCL treated with anthracycline- based chemotherapy with rituximab. Immunohistochemical staining using standard protocols was performed on formalin fixed paraffin-embedded tissues. CD20, CD5, CD23, BCL2, CD10, BCL6, MUM1 and Ki67 antibodies were applied. Out of 120 examined cases 52 patients were evaluated as GCB type and 68 patients as having non-GCB, out of a set of 99 patients treated with immunochemotherapy 45 patients with GCB and 54 patients with non-GCB DLBCL were identified. In this set of patients, there was no statistically significant difference neither in overall survival (OS) (HR 1.47 95% CI 0.51-2.63; p=0.45) nor in progression free survival (PFS) (HR 1.57, 95 % CI 0.76-3.22; p=0.731) between both groups.

Transcriptome alterations in maternal and fetal cells induced by tobacco smoke.

OBJECTIVES: Maternal smoking has a negative effect on all stages of pregnancy. Tobacco smoke-related defects are well established at the clinical level; however, less is known about molecular mechanisms underlying these pathologic conditions. We thus performed a comprehensive analysis of transcriptome alterations induced by smoking in maternal and fetal cells. STUDY DESIGN: Samples of peripheral blood (PB), placenta (PL), and cord blood (UCB) were obtained from pregnant smokers (n = 20) and gravidas without significant exposure to tobacco smoke (n = 52). Gene expression profiles were assayed by Illumina Expression Beadchip v3 for analysis of 24,526 transcripts. The quantile method was used for normalization. Differentially expressed genes were analyzed in the Limma package and the P-values were corrected for multiple testing. Unsupervised hierarchical clustering was performed using average linkage and Euclidean distance. The enrichment of deregulated genes in biological processes was analyzed in DAVID database. RESULTS: Comparative analyses defined significant deregulation of 193 genes in PB, 329 genes in PL, and 49 genes in UCB of smokers. The deregulated genes were mainly related to xenobiotic metabolism, oxidative stress, inflammation, immunity, hematopoiesis, and vascularization. Notably, functional annotation of the affected genes identified several deregulated pathways associated with autoimmune diseases in the newborns of smokers. CONCLUSIONS: The study demonstrated maternal smoking causes significant changes in transcriptome of placental and fetal cells that deregulate numerous biological processes important for growth and development of the fetus. An activation of fetal CYP genes showed a limited ability of the placenta to modulate toxic effects of maternal tobacco use.

Advanced rai stage in patients with chronic lymphocytic leukaemia correlates with simultaneous hypermethylation of plural tumour suppressor genes.

Hypermethylation of CpG islands within gene promoters is one of various mechanisms of gene silencing involved in the pathogenesis of human cancer. By using methylation-specific polymerase chain reaction we explored aberrant promoter methylation of five tumour suppressor genes in 29 patients with chronic lymphocytic leukaemia. Aberrant methylation of DLC1, SHP1, p15 and p16 occurred, respectively, in 89.7 %, 70 %, 62.1 % and 31 % of patients at diagnosis. Lamin A/C was unmethylated in all the samples. Hypermethylation of at least one gene was detected in 96.6 % of patients. Concurrent methylation of two or more genes correlated with Rai stage at diagnosis.

Distinguishing of primary mediastinal B-cell lymphoma and diffuse large B-cell lymphoma using real-time quantitative polymerase chain reaction.

Primary mediastinal B-cell lymphoma (PMBL) seems to be reliably distinguished from diffuse large B-cell lymphoma (DLBCL) with microarray technology. We measured expression of Fcer2, Pdl2 and Blk genes using real-time quantitative polymerase chain reaction (RTqPCR) on formalin fixed, paraffin embedded material (FFPE) and suggested a formula to discriminate PMBL from DLBCL. For 39/82 included patients the diagnosis of PMBL was expected clinico-pathologically. Diagnosis of 10/39 and 2/43 of clinically considered PMBLs and DLBCLs, respectively, was not genetically confirmed. Compared to confirmed PMBLs, unconfirmed ones showed clinical features similar to DLBCLs, e.g. spleen infiltration (p=0,028) and decreased invasiveness in pericardium (p=0,045). They tended to have more common infradiaphragmatic involvement, less often tumor sclerosis or fluidothorax. There were no immunohistochemical differences between genetically confirmed and unconfirmed PMBLs. New approach of distinguishing PMBL and DLBCL is presented. It is based on expression of three genes in routinely available FFPE material using RTqPCR.

Synthesis and structure of 6-amino-2,3,6-trideoxy-D-erythro-hexono-1,6-lactam and 6-amino-3,6-dideoxy-D-xylo-hexono-1,6-lactam.

Solid-state conformations of 6-amino-2,3,6-trideoxy-D-erythro-hexono-1,6-lactam (3a) and 6-amino-3,6-dideoxy-D-xylo-hexono-1,6-lactam (7a) were determined using X-ray diffraction. Conformations of the compounds 3a, 7a, and their per-O-acetyl derivatives 4,5-di-O-acetyl-6-amino-2,3,6-trideoxy-D-erythro-hexono-1,6-lactam (3b) and 2,4,5-tri-O-acetyl-6-amino-3,6-dideoxy-D-xylo-hexono-1,6-lactam (7b) in solutions were deduced from the analysis of NMR spectra using a modified Karplus equation and compared with the results of circular dichroism measurement of lactams 3a and 7a. Conformation 4C(1,N) was revealed for solid lactams 3a and 7a and for lactams 7a and 7b in solution, while lactams 3a and 3b in solution exist in the approximately 1:1 equilibrium of the conformers 4C(1,N) and (1,N)C4.

Interaction of a bZip oligopeptide model with oligodeoxyribonucleotides modelling DNA binding sites. The effect of flanking sequences.

A leucine zipper (bZip) binding peptide BP1 was constructed based on the DNA binding sequence of the GCN4 protein, slightly modified to make it more similar to the sequence of other bZip proteins (Jun) with related DNA binding specificity. Self-complementary DNA hexadecanucleotides containing ATF/CRE, AP-1 and C/EPB target sites were used to study peptide-DNA complex formation. Conformation changes in both components that occur on complex formation were studied by circular dichroism (CD) spectroscopy. The results show that the amount of alpha-helix formed in the peptide strongly depends not only on the target site present, but also on the type of the sequence flanking the ATF/CRE target site. Highest amount of the alpha-helix induced in the peptide was observed when homopurine homopyrimidine flanking sequences were present, whereas the presence of alternating sequences, especially of the CA/TG type, showed considerably lower effects. The change in DNA conformation on complex formation was generally small, but also depended on the type of the flanking sequence. It appears that the sequences flanking the target site can considerably modify the ability of the target sequence to bind specifically the bZip peptide, probably by slightly varying the overall DNA conformation.

Selective binding of synthetic polypeptides to DNA of varying composition and sequence: effect of minor groove binding drugs.

Repetitive basic polypeptides containing lysine or arginine as every third amino acid were shown to cause DNA condensation at physiological salt concentration connected with selective DNA binding with respect to DNA composition and sequence. This selectivity is very similar to that existing in the case of histone H1 and other basic proteins and does not depend on polypeptide chain conformation. The effect of the minor groove binding drugs netropsin and distamycin was tested to elucidate the origin of the binding selectivity. The results suggest that the binding preferences are due to the variations in the conformation in various types of B-DNA that depend on DNA composition and sequence. The most important factor affecting the selectivity is probably the value of the negative electrostatic potential in the minor groove.

B-Z transition of synthetic oligonucleotides containing non-Z forming elements.

Ten oligonucleotides of the length 8-12 base pairs have been synthesized, which contain, in addition to the obligatory sequences CG/CG, sequences not favorable for the transition to the Z conformation (A.T pairs, GG/CC or AA/TT sequences). Conformational transitions of these oligonucleotides in high concentrations of NaClO4 in the absence and in the presence of Ni2+ were investigated using CD spectroscopy. The B-Z transition is affected by the length and sequence of the oligonucleotide. Increasing the NaClO4 concentration alone the transition of only one of the oligonucleotides studied. (CGCGCGTGCACGCGCG)2, can be induced. Other oligonucleotides remain in the B conformation or only partial transition to the Z conformation can be observed. Most other oligonucleotides can be converted into the Z conformation at intermediate concentrations of NaClO4 (2.0-3.2 M) by an addition of Ni2+ ions. In some cases, however, Ni2+ can destabilize the double stranded structure of the sample. We have studied the effect of the presence of A.T pairs in the G.C containing oligonucleotides and the effect of the presence of pu-pu/pyr-pyr sequences. The presence of the latter sequences in the Z form implicates the formation of a Z-Z'junction which makes the transition quite difficult. Despite the fact that some oligonucleotides contained several structural elements not favorable for the transition, we did not find any sequence which would completely block the ability of the oligonucleotide to adopt the Z conformation.

B-Z transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of basic oligopeptides.

The effect of basic oligopeptides (Lys-Ala-Ala)n and (Lys-Leu-Ala)n (n = 1-4) on the B-Z transition of poly(dG-m5dC).poly(dG-m5dC) in aqueous solution and in methanol-water mixtures was investigated by c.d. spectroscopy. In aqueous solution dimers and higher oligomers induce a transition to the Z conformation. These temperature dependent transitions are consistent with positive delta HB-Z values which depend on peptide composition. In the absence of peptides no transition can be observed. In the presence of peptides B-Z transition can be induced by small amount of methanol. The temperature dependence of this transition is consistent with a small, but definitely negative delta HB-Z. At high methanol concentration a transition to Z' type conformation was observed. In subcritical methanol concentrations B-Z transition can be induced by the addition of peptides. In this case the delta HB-Z values are again very small, but definitely positive. The effects of bulky hydrophobic peptide side chains, of the presence of methanol and the temperature dependences are consistent with an important contribution of hydrophobic interactions in maintaining the stability of the Z DNA-peptide complex.

Mechanisms of distamycin A/DAPI chromosome staining. I. Competition binding effects of nonintercalative DNA groove-binding agents in situ and in vitro.

The molecular mechanism underlying distamycin A-induced differential DAPI fluorescent staining of metaphase chromosomes was studied in Sus scrofa domestica both cytologically, using, besides DAPI, two isomeric derivatives of DAPI (D288.45 and D288.48), and molecularly, by in vitro competitive-binding studies using S. scrofa satellite DNA and synthetic DNA polymers. Significant differences in heterochromatin staining were observed between D288.45 and D288.48. Distinct distamycin A/DAPI bands were obtained with DAPI and D288.45 but not with D288.48. Circular dichroism measurements were performed to characterize the displacement of DAPI from its DNA binding sites by distamycin A and also netropsin. Distamycin A was most effective in displacing DAPI when DAPI was bound to contiguous clusters of AT base pairs and much less effective in displacing DAPI bound to GC or mixed AT/GC base-pair sequences. The results of these competitive-binding studies provide the basis of a molecular explanation of the quenching phenomenon of distamycin A counterstaining on chromosomal DAPI fluorescence.

Effect of basic oligopeptides on the B-Z transition of poly(dG-dC). poly(dG-dC) in water-methanol solutions.

The effect of basic oligopeptides (Lys-Ala-Ala)n (n = 1-5, 10) and (Lys-Leu-Ala)n (n = 1-4) on the B-Z transition of poly(dG-dC).poly(dG-dC) in water-methanol solutions was investigated using CD and uv spectroscopy. In the absence of peptides, the concentration of methanol at the midpoint of the B-Z transition is 64% at 25 degrees C. The transition is temperature dependent and the B conformation is preferred at higher temperatures. All peptides tested shift the midpoint of the B-Z transition to lower concentrations of methanol. For shorter peptides this effect increases with an increasing number of monomeric units, showing the importance of the number of positive charges in the peptide molecule. Al conditions of low methanol content, the trimer and tetramer of the (Lys-Leu-Ala)n series have a greater effect on the B-Z transition than the corresponding oligomers of the (Lys-Ala-Ala)n series. This indicates an important influence of the presence of hydrophobic groups in the peptide side chains on the binding. In the presence of peptides, the B-Z transition is also temperature dependent and the B conformation is preferred at higher temperatures. The addition of peptides results in an increase of the transition midpoint and of the transition width. These parameters were used for the calculation of the transition enthalpy delta HB-Z in 65% methanol, which is -1.15 +/- 0.25 kcal/base pair. Since the van't Hoff enthalpy delta HVH calculated from the temperature dependence of the B-Z transition in the absence of peptides is -130 kcal/mol, the length of the cooperative unit is about 110 base pairs. The results suggest that the mechanism of Z-DNA induction is similar but not identical with that involved in the action of metal cations in aqueous solution.

[Design and synthesis of peptides capable of specific binding to DNA]. / Konstruirovanie i sintez peptidov, sposobnykh spetsificheski sviazyvat'sia s DNK.

In the present communication, design, synthesis and DNA binding activities of the following two peptides are reported: Dns-Gly-Ala-Gln-Lys-Leu-Ala-Cly-Lys-Val-Gly-Thr-Lys-Val-Lys-Val-Gl y-Thr-Lys-Thr - Val-OH (I) and [(H-Ala-Lys-Leu-Ala-Thr-Lys-Ala-Gly-Val-Lys-Gln-Gln-Ser-Ile-Gln-Leu-Ile- Thr- Ala-Aca-Lys-Aca)2Lys-Aca]2Lys-Val-OH (II), where Aca = NH(CH2)5CO--; Dns is a residue of 5-dimethylaminonaphtalene-1-sulfonic acid. Peptide I contains a large fraction (ca.30%) of valyl and threonyl residues, which possess a high potential for beta structure formation. Peptide II contains four repeats of the amino acid sequence present in the presumed DNA binding helix-turn-helix unit of 434 Cro repressor. These four domains are linked in such a way that two domains can interact with two halves a 14 base pair long operator site on DNA. From CD studies we have found that peptide I is in a random coil conformation in the aqueous solution in the presence of 20% trifluoroethanol. By contrast, amino acid residues of peptide II assume alpha helical, beta and random coiled conformations under the same conditions. A change in the secondary structure of the two peptides upon binding to DNA is observed. The difference CD spectra obtained by subtracting the spectra of free DNA from the spectra of peptide I--DNA complexes gives rise to a beta-like pattern. The difference CD spectra obtained for complexes of peptide II with various natural and synthetic DNAs suggest that alpha-beta-transition takes place in the presumed helix-turn-helix repeat units of peptide II upon binding to DNA. Peptide I binds more strongly to poly(dG).poly(dC) than to poly(dA).poly(dT) and poly[d(GC)].poly[d(GC)]. The binding takes place in the minor DNA groove because minor groove binding antibiotic sibiromycin can displace peptide I from a complex with poly(dG).poly(dC). Analysis of footprinting diagramms shows that peptide I specifically protects phosphodiester bonds within operator sites OR1 and OR2 of phage lambda from nuclease cleavage. By contrast, peptide II does not react specifically with operators OR1, OR2 and OR3 of phage 434 although it forms very tight complexes with DNA which are stable in the presence of 1M NH4F.

B-X transition in synthetic and natural (dA-dT)n.(dA-dT)n sequences.

AB-X transition of polyh(dA-dT).poly(dA-dT) was observed to occur in methanol-water mixtures with methanol concentrations higher than 50% in the presence of a specific combination of monovalent and divalent cations. In the presence of Na+, divalent cations induce denaturation of poly(dA-dT).poly(dA-dT) accompanied by condensation and/or aggregation, and effect similar to that observed previously with random sequence DNA (Votavová, Kucerová, Felsberg and Sponar, J. Biomol. Struct. Dyn. 4,477-489, 1986). In the presence of Cs+ cations a B-X transition was induced by addition of Ca2+ or Mn2+ but not Mg2+ or Ni2+ ions. Circular dichroism and ultraviolet spectroscopy demonstrate that the X conformation is a double stranded form of poly(dA-dT).poly(dA-dT) belonging presumably to the B family which, however has an altered base stacking. The X conformation of poly(dA-dT).poly(dA-dT) found in methanol-water mixtures is a condensed and/or aggregated form. In contrast, the X conformation characterized by similar CD spectra observed in high salt concentrations is not aggregated up to a concentration of 6 M CsF. In methanol-water mixtures (A+T)-rich bacterial DNA behaves essentially as a random sequence DNA revealing no detectable amount of the X form. On the other hand crab (Cancer pagurus) satellite and crab non-satellite DNAs containing varying amounts of (dA-dT)n.(dA-dT)n sequences were shown to undergo a B-X transition, at least partly, in both methanol-water mixtures and 6 M CsF solutions.

Changes in conformation, stability and condensation of DNA by univalent and divalent cations in methanol-water mixtures.

Circular dichroism spectroscopy, absorption spectroscopy, measurements of Tm values, sedimentation analysis and electron microscopy were used to study properties of calf thymus DNA in methanol-water mixtures as a function of monovalent cation (Na+ or Cs+) concentration and also in the presence of divalent cations Ca2+, Mg2+, and Mn2+. In the absence of divalent cations only slight conformational changes occurred and no condensation and/or aggregation could be detected. The Tm values depend on the amount of methanol and on the nature and concentration of cations. In methanol-water mixtures higher thermal stability was observed in solutions containing Cs+ ions. Up to 40% (v/v) methanol the addition of divalent ions leads to DNA stabilization. At methanol concentration higher than 50% the presence of divalent cations causes DNA condensation and denaturation even at room temperature. The denaturation is reversible with respect to EDTA addition indicating that no separation of complementary strands occurred and the resulting form of DNA is probably similar to the P form. DNA destacking appears to be a direct consequence of stronger cation binding by the condensed DNA in methanol-water mixtures.

Two binding modes of netropsin are involved in the complex formation with poly(dA-dT).poly(dA-dT) and other alternating DNA duplex polymers.

Using CD measurements we show that the interaction of netropsin to poly(dA-dT).poly(dA-dT) involves two binding modes at low ionic strength. The first and second binding modes are distinguished by a defined shift of the CD maximum and the presence of characteristic isodichroic points in the long wavelength range from 313 nm to 325 nm. The first binding mode is independent of ionic strength and is primarily determined by specific interaction to dA.dT base pairs. Employing a netropsin derivative and different salt conditions it is demonstrated that ionic contacts are essential for the second binding mode. Other alternating duplexes and natural DNA also exhibit more or less a second step in the interaction with netropsin observable at high ratio of ligand per nucleotide. The second binding mode is absent for poly(dA).poly(dT). The presence of a two-step binding mechanism is also demonstrated in the complex formation of poly(dA-dT).poly(dA-dT) with the distamycin analog consisting of pentamethylpyrrolecarboxamide. While the binding mode I of netropsin is identical with its localization in the minor groove, for binding mode II we consider two alternative interpretations.

Binding of netropsin to DNA in complexes with polypeptides containing repetitive lysine sequences.

The interaction of the antibiotic netropsin with calf thymus DNA, T4 DNA and poly(dA-dT) . poly(dA-dT) in complexes with sequential polypeptides containing repetitive lysine sequences and histone H1 was investigated using circular dichroism spectroscopy and equilibrium dialysis. Both soluble DNA-polypeptide complexes and insoluble complexes showed binding of netropsin. The possibility of displacement of polypeptides from DNA binding sites by competition with netropsin molecules was eliminated by experiments using 14C-labelled polypeptides. From the analysis of CD titration behavior as well as from the results of equilibrium dialysis studies it follows that netropsin does not compete with polypeptides for DNA binding sites, which suggests that these two ligands occupy different sites. Various explanations for minor differences in the CD behavior of the bound netropsin in the saturation region are also discussed.

A comparative study of G+C-rich satellite components of sheep and goat DNA.

Restriction fragment patterns of G+C-rich satellites of sheep and goat DNA were compared. The 1,712 g/cm3 satellites of both species appear homologous, consisting of repeats 760 base pairs long and showing coincidence of position of primary+ EcoRI, BamHI and most BspRI restriction target sites. The EcoRI and BamHI endonucleases produce mostly monomers of the repeating unit, while oligomers prevail in the A1uI and Bg1II digests. Species-specific differences in the frequency, position and mode of distribution of secondary+ restriction target sites for EcoRI, Bg1II and A1uI were observed. Unlike the 1,712 g/cm3 satellites, the 1,723 g/cm3 component of sheep DNA and the 1,719 g/cm3 material from goat DNA appear species--specific, since no homologous material could ever be detected in the DNA of the other species.