[CASC5 Gene Expression Changes Correlate with Targeted Mutations in Leukemia].

Dysfunction of genes that control mitosis and are responsible for the correct segregation of sister chromatids in anaphase is often accompanied by aneuploidy, which is frequently detected in leukemia. One of the components of the kinetochore complex, namely, the AF15q14/KNL1/CASC5 protein, is an important factor ensuring the correct binding of the pericentromeric region of chromosomes with the spindle microtubules. As shown recently, in some leukemias, the gene of this protein can be involved in the generation of the chromosomal translocation t(11;15)(q23;q14) or a variant of the chimeric MLL-AF15Q14 oncogene, which serves as a biomarker of poor prognosis. Despite the implication of mRNA of the CASC5 gene in oncogenesis of solid tumors, expression of this gene in hematopoietic neoplasms has not been studied. We analyzed expression levels of the CASC5 gene and the nearest regulatory genes, including WT1, APOBEC3A (A3A), and N-MYC. A pronounced decrease in CASC5 expression in bone marrow cells of primary leukemia patients compared with healthy donors was found. It was also shown that reduced expression of the CASC5 gene correlates with the detection of targeted mutations in patients composed two prognostic subgroups (favorable, unfavorable) with a significance level (p <0.05). It was noted that the change in the expression level of the CASC5 gene in acute myeloid leukemia is associated with overexpression of the genes WT1, A3A, and in some cases N-MYC and SPT16, which is consistent with the resistance to chemotherapy and leukemia progression. However, the question of which regulatory gene initiates leukemogenesis remains open.

[The experience of using the drug Reamberin and NPWT-therapy in preparing wounds for autodermoplasty]. / Opyt ispol'zovaniya preparata Reamberin i NPWT-terapii v podgotovke ran k autodermoplastike.

OBJECTIVE: To evaluate the effectiveness of preparing a wound for plastic closure of autoskin using a negative pressure apparatus and Reamberin. MATERIAL AND METHODS: . The results of preoperative preparation of 24 patients with chronic wounds of various etiologies were analyzed. Depending on the treatment regimen, the patients were divided into groups: the main one (n=18), the patients of which were divided into subgroups: in IA (n=9) they used NPWT therapy for 5 days, in IB (n=9) it was combined with Reamberin infusions (1.5% intravenously at a rate of 40-60 drops/min, 500 ml, once a day for 5 days). Patients of the comparison group (II, n=6) received standard preparation. In the dynamics of therapy, a morpho-histological study of wound biopsies was carried out with the determination of the area of fibroblasts, the area of the nucleus of fibroblasts, the number of vessels and the determination of their diameter. After autodermoplasty, the survival of the skin flap was determined. RESULTS: It was found that the combined use of NPWT therapy and Reamberin significantly increases the effectiveness of therapy: the greatest effect on fibroblastogenesis was exerted by the inclusion of NPWT therapy in the regimen - an increase of 1.5 times compared with the results of the traditional regimen. The vascularization of the wound surface (increase in the area and diameter of blood vessels) was best influenced by the combined preparation (NPWT-therapy and Reamberin) preparations - an increase of 3.4 times in comparison with the comparison group, which affected the dynamics of the survival of the skin flap: it was 1.3 times higher than in the comparison group (p<0.01). CONCLUSIONS: The results obtained, along with good tolerability of the drug, allows us to recommend the inclusion of Reamberin in the scheme of preoperative preparation of patients with this pathology.

Saturable absorber: transparent glass-ceramics based on a mixture of Co:ß-Zn2SiO4 and Co:ZnO nanocrystals.

We report on the development of novel saturable absorbers for erbium lasers based on transparent glass-ceramics (GCs) containing a mixture of cobalt-doped ß-willemite, Co2+:ß-Zn2SiO4, and zinc oxide, Co2+:ZnO, nanosized (10-14 nm) crystals. The structure of the parent glass and GCs is studied by x-ray diffraction, differential scanning calorimetry, transmission electron microscopy, and Raman spectroscopy. Variations of absorption spectra with heat-treatment reveal that Co2+ ions from the parent glass enter the crystals of ZnO and ß-willemite. GCs are characterized by a broad absorption band due to the A24(F4)→T14(F4) transition of Co2+ ions in tetrahedral sites spanning up to ∼1.74 µm, relatively low saturation fluence, FS=0.75 J/cm2 at 1.54 µm, short recovery time, τ=830 ns, and high laser damage threshold, ∼14 J/cm2. By using the developed GCs in a diode-side-pumped Er, Yb:glass laser, 0.77 mJ/45 ns pulses are generated.

Direct detection of chicken genomic DNA for gender determination by thymine-DNA glycosylase.

1. Birds, especially nestlings, are generally difficult to sex by morphology and early detection of chick gender in ovo in the hatchery would facilitate removal of unwanted chicks and diminish welfare objections regarding culling after hatch. 2. We describe a method to determine chicken gender without the need for PCR via use of Thymine-DNA Glycosylase (TDG). TDG restores thymine (T)/guanine (G) mismatches to cytosine (C)/G. We show here, that like DNA Polymerase, TDG can recognise, bind and function on a primer hybridised to chicken genomic DNA. 3. The primer contained a T to mismatch a G in a chicken genomic template and the T/G was cleaved with high fidelity by TDG. Thus, the chicken genomic DNA can be identified without PCR amplification via direct and linear detection. Sensitivity was increased using gender specific sequences from the chicken genome. 4. Currently, these are laboratory results, but we anticipate that further development will allow this method to be used in non-laboratory settings, where PCR cannot be employed.

[Significance of mitotic spindle checkpoint genes in leukemia].

Leukemia is a clonal proliferative disorder of the multipotent hematopoietic stem cells that leads to abnormal cell growth and (or) differentiation. The hallmark of the disease is the presence of oncogene expression in bone marrow or peripheral blood as a result of some chromosome translocations. The development of leukemia with transfer to disease progression presents a multistage process implicating series of molecular changes leading to chromosomal instability and aneuploidy. The most possible of these changes include the followings: appearance of additional chromosome translocations, activation of other not previously expressed oncogenes, loss of tumor suppressor genes, abnormal centrosome duplication, and dysfunction of the genes which coordinate the accurate chromosome alignment and chromosome segregation during mitosis. The two latter molecular changes which are controlled by mitotic spindle checkpoints play the role in leukemogenesis and are probably involved in apoptosis.

[Basic methods for extraction and molecular analysis of micromycetic DNA].

The methods for extraction of nucleic acids from the fungi and clinical materials containing various microorganisms have currently modified and make it possible to extract DNA with its highest yield and to minimize its destruction. In particular, this concerns mycelial fungi that have a rigid cell wall. Addition of glass microbeads to the lysed sample, followed by mixture stirring, gives rise to undamaged DNA from fungi and their spores. Extracted DNA is suitable for the identification of micromycetes by qualitative or quantitative polymerase chain reaction (PCR), real-time PCR-based hybridization, and LightCycler PCR and for the polymorphism analysis of the spacer regions of ribosomal genes by the sequencing technique. A combination of quantitative PCR and sequencing permits co-determination of the number of gene target copies in 9 different micromycetes in the clinical material being examined, which is of great importance for the diagnosis and treatment of patients with higher immunosuppression and, of those after transplantation of organs and tissues in particular.

[The involvement of c-Abl and D40 (AF15q14/CASC5) proteins in the regulation of cell proliferation and cancer].

Although c-Abl and D40 proteins are localized predominantly in nucleus, they are involved in different cellular processes. c-Abl is a tyrosine-kinase that takes part in protein phosphorylation on tyrosine. Recently D40 has been identified as a component of outer kinetochore complex. Despite of functional differences between c-Abl and D40 proteins, they have some similarities. First, high expression levels of c-Abl and D40 were observed not only in proliferating somatic cells, such as tumors, but also in healthy human testis. The increased expression levels of c-Abl and D40 protein in spermatocytes and acrosome of spermatids indicate their role in meiosis and spermatogenesis. Second, both proteins interact with specific regions of chromatin and are involved in the regulation of cell growth and division. Third, ABL and D40 (AF15q14) genes are involved in chromosomal translocations that subsequently form chimeric oncoproteins BCR-ABL, TEL-ABL and MLL-AF15q14 in human leukaemia. Finally, both proteins interact with the tumor suppressor pRb protein and subsequently can lead to regulation of the cell proliferation. The possible regulatory pathways that are controlled by c-Abl and D40 proteins are described here in details.

[Donor chimerism and minimal residual disease monitoring in leukemia patients after allo-HSCT]. / Monitoring donorskogo khimerizma i minimal'noi ostatochnoi bolezni u onkogematologicheskikh bol'nykh posle allogennoi transplantatsii gemopoéticheskikh stvolovykh kletok.

In present research the comparative analysis of donor chimerism (DC) using different tests was performed to improve the diagnostic tool in patients with malignant hematological disorders after allo-HSCT. The RBC antigen typing, identification of ABO blood type and quantitative analysis of InDel-, STR-, Y-polymorphisms were carried out for detection of DC. In addition, the expression of well-known oncogenes and CD-markers for monitoring MRD was evaluated to predict relapse and clinical outcome. According to our research, the analysis of InDel polymorphism using AlleleSEQR-PCR is more sensitive test for estimation of DC as compared with other assays. Moreover, the sensitivity of AlleleSEQR-PCR may be increased after isolation of the CD34 cell population in bone marrow. Nevertheless, observation of high levels in DC (³95%) in some leukemia patients (ALL, Ph+, bcr-abl/p190+) during first 6 months after HSCT cannot exclude the possibility of relapse. Thus, the combined monitoring of both DC (InDel) and MRD (oncogenes, WT1 and CD-markers) is a more advisable and useful test in managing hematologic malignancies and predicting relapse risk after allo-HSCT.

Sinoatrial nodal cell ryanodine receptor and Na(+)-Ca(2+) exchanger: molecular partners in pacemaker regulation.

The rate of spontaneous diastolic depolarization (DD) of sinoatrial nodal cells (SANCs) that triggers recurrent action potentials (APs) is a fundamental aspect of the heart's pacemaker. Here, in experiments on isolated SANCs, using confocal microscopy combined with a patch clamp technique, we show that ryanodine receptor Ca(2+) release during the DD produces a localized subsarcolemmal Ca(2+) increase that spreads in a wavelike manner by Ca(2+)-induced Ca(2+) release and produces an inward current via the Na(+)-Ca(2+) exchanger (NCX). Ryanodine, a blocker of the sarcoplasmic reticulum Ca(2+) release channel, in a dose-dependent manner reduces the SANC beating rate with an IC(50) of 2.6 micromol/L and abolishes the local Ca(2+) transients that precede the AP upstroke. In voltage-clamped cells in which the DD was simulated by voltage ramp, 3 micromol/L ryanodine decreased an inward current during the voltage ramp by 1.6+/-0.3 pA/pF (SEM, n=4) leaving the peak of L-type Ca(2+) current unchanged. Likewise, acute blockade of the NCX (via rapid substitution of bath Na(+) by Li(+)) abolished SANC beating and reduced the inward current to a similar extent (1.7+/-0.4 pA/pF, n=4), as did ryanodine. Thus, in addition to activation/inactivation of multiple ion channels, Ca(2+) activation of the NCX, because of localized sarcoplasmic reticulum Ca(2+) release, is a critical element in a chain of molecular interactions that permits the heartbeat to occur and determines its beating rate.

Sinoatrial node pacemaker activity requires Ca(2+)/calmodulin-dependent protein kinase II activation.

Cardiac beating arises from the spontaneous rhythmic excitation of sinoatrial (SA) node cells. Here we report that SA node pacemaker activity is critically dependent on Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In freshly dissociated rabbit single SA node cells, inhibition of CaMKII by a specific peptide inhibitor, autocamtide-2 inhibitory peptide (AIP, 10 micromol/L), or by KN-93 (0.1 to 3.0 micromol/L), but not its inactive analog, KN-92, depressed the rate and amplitude of spontaneous action potentials (APs) in a dose-dependent manner. Strikingly, 10 micromol/L AIP and 3 micromol/L KN-93 completely arrested SA node cells, which indicates that basal CaMKII activation is obligatory to the genesis of pacemaker AP. To understand the ionic mechanisms of the CaMKII effects, we measured L-type Ca(2+) current (I(Ca, L)), which contributes both to AP upstroke and to pacemaker depolarization. KN-93 (1 micromol/L), but not its inactive analog, KN-92, decreased I:(Ca, L) amplitude from 12+/-2 to 6+/-1 pA/pF without altering the shape of the current-voltage relationship. Both AIP and KN-93 shifted the midpoint of the steady-state inactivation curve leftward and markedly slowed the recovery of I(Ca, L) from inactivation. Similar results were observed using the fast Ca(2+) chelator BAPTA, whereas the slow Ca(2+) chelator EGTA had no significant effect, which suggests that CaMKII activity is preferentially regulated by local Ca(2+) transients. Indeed, confocal immunocytochemical imaging showed that active CaMKII is highly localized beneath the surface membrane in the vicinity of L-type channels and that AIP and KN-93 significantly reduced CaMKII activity. Thus, we conclude that CaMKII plays a vital role in regulating cardiac pacemaker activity mainly via modulating I(Ca, L) inactivation and reactivation, and local Ca(2+) is critically involved in these processes.

Necroptosis is preceded by nuclear translocation of the signaling proteins that induce it.

A signaling pathway that induces programmed necrotic cell death (necroptosis) was reported to be activated in cells by several cytokines and various pathogen components. The major proteins participating in that pathway are the protein kinases RIPK1 and RIPK3 and the pseudokinase mixed lineage kinase domain-like protein (MLKL). Recent studies have suggested that MLKL, once activated, mediates necroptosis by binding to cellular membranes, thereby triggering ion fluxes. However, our knowledge of both the sequence of molecular events leading to MLKL activation and the subcellular sites of these events is fragmentary. Here we report that the association of MLKL with the cell membrane in necroptotic death is preceded by the translocation of phosphorylated MLKL, along with RIPK1 and RIPK3, to the nucleus.

[Allele polymorphism analysis in coagulation factors F2, F5 and folate metabolism gene MTHFR by using microchip-based multiplex real time PCR].

Single nucleotide polymorphism (SNP) genotyping methods are widely used for the detection of hereditary thrombophilias caused by genetic defects in the coagulation system. The hereditary thrombophilias are frequently associated with higher incidences of point mutations in hemostasis (F2 20210G>A, F5 1691G>A) and folate metabolism (MTHFR 677C>Т, MTHFR 1298A>C) genes. Moreover, the combination of gene abnormalities in F2 or/and MTHFR with F5 Leiden mutation leads to increased risk of developing thrombosis. Thus, simultaneous detection of the multiple gene mutations in a sample has important clinical relevance. The microchip-based multiplex real time PCR for estimation of allele specific polymorphism in hemostatic and folate metabolism genes presented here has a high efficiency and may be used for laboratory diagnosis. The optimized protocol for estimation of 4 different types of genetic polymorphisms allowed PCR to be performed with minimal quantity of DNA template and PCR reagents including Taq polymerase and a short-term thermocycling.

[Study of lymphoid tissue cells by the method of optico-structural machine analysis]. / Izuchenie kletok limfoidnoi tkani metodom optiko-strukturnogo mashinnogo analiza.

The possibility to use the method of optically-structural machine analysis for automatical classification of lymphoid cells of tumour-bearing rats and lymphoid cells of malignant tumour was studied. The preparations were fixed by methanol, stained by azureosin and scanned on the automatical microscope-analysor "Protva-3". The isolated cells of 6 different groups were scanned. Histogramms of optical density distribution amplitudes were received for every cell and the phase analysis was carried out. The statistical characterization for every cell was based on the results of this analysis. The average values were calculated for 1) the area of the nucleus and cytoplasm: 2) the average density of nucleus and cytoplasm; 3) dispersion of densities of nucleus and cytoplasm; 4) nucleus-cytoplasm relation for every six groups. It was shown that every group of cells may be separated from each other, at least, on the basis of one of the measured parameters. The results are consistent with the morphological data.

[Model of the dynamics of the body's water metabolism under normal and pathologic conditions]. / Model' dinamiki vodnogo obmena organizma v norme i patologii

A theoretical and experimental study of metabolic processes is presented. Dynamics of water exchange in the living organism under normal and pathological conditions is described. The investigation performed states that the coefficients of analytical equations and constant removal of tritium water from the organism depends on the form and degree of pathology, i.e. pn the change of the constants of water passage from one water reservoir into another.

[The effect of acoustic cavitation on the contraction force and membrane potential of rat papillary muscles]. / Deistvie akusticheskoi kavitatsii na silu sokrashcheniia i membrannyi potentsial papilliarnoi myshtsy krysy.

Acoustic cavitation induced by continuous focused ultrasound (1.4 W/cm2, 543 Hz) was found to result in reversible membrane depolarization (by 54 mV), loss of excitability and contracture in the rat papillary muscles. The same intensities of impulse ultrasound had positive inotropic effects.

[Distribution of the density of defibrillating current in the chest and underneath the defibrillator's electrodes]. / Raspredelenie plotnosti defibrilliruiushchego toka v grudnoi kletke i pod elektrodami defibrilliatora

Results of experiments carried out in animals and of measurements made on electrophysical and analogue models are presented. It has been found that with routinely employed manipulations in the electric impulse therapy of arrhythmias the denstiy of the current in the chest and directly underneath the defibrillator's electrodes is distributed in a non-uniform fashion. This results in high defibrillation thresholds, possible damages of the heart and body teguments. The causes responsible for this non-uniformity are analyzed, the non-uniformity itself is assessed and ways of its elimination are indicated.

[Changes in cell membrane excitability during guinea pig papillary muscle after-contractions]. / Izmenenie vozbudimosti kletochnoi membrany vo vremia ostsilliatsii tonusa papilliarnoi myshtsy morskoi svinki.

In isolated guinea-pig papillary muscles, the excitability of cellular membrane was investigated during the after contractions induced by cooling of the perfusate to 13--15 degrees C, addition of noradrenaline (2 mg/l), and calcium to 16 MM. At stimulation rate 0.2 Hz the oscillation amplitude was 19 +/- 2% of the initial contraction. The activity of the fast sodium channels was estimated by the threshold for AP initiation and appeared to increase gradually after the end of AP. Slow channel response measured in the high potassium solution (29 MM), significantly decreased during the after contraction. Even at supramaximal amplitude of stimulation the slow responses were about 10--20 mV smaller than those after or before oscillation. Slow channel activity seems to depend on intracellular calcium concentration. Oscillations after contractions were abolished by addition of caffeine (2 mM). The oscillations seem to result from recirculation of calcium between myoplasma and sarcoplasmatic reticulum.

[Effect of brief papillary muscle stretch on 2 contraction components in the presence of noradrenaline in guinea pigs]. / Vliianie kratkovremennogo rastiazheniia papilliarnoi myshtsy morskoi svinki na dve komponenty sokrashcheniia v prisutstvii noradrenalina.

In isolated guinea-pig papillary muscles perfused with Tyrode's solution, quick stretching did not affect the isotonic contraction when applied at its fast shortening phase, and accelerated the relaxation if the stretching coincided with the later phases of the contractile response. In presence of noradrenaline the contraction revealed two components. The amplitude of the 1st component was augmented whereas its time to peak did not change when the stimulation frequency had been increased. The amplitude of the 2nd component and its time to peak both diminished at the increase of the stimulation frequency. The changes of time to peak were always closely related to the changes of the AP duration. The 1st contraction component seems to be calcium release from sarcoplasmatic reticulum whereas the 2nd component is determined by the AP duration.

[Relationship between isometric and isotonic contractile responses of the mammalian myocardium]. / Sootnoshenie mezhdu izometricheskim i izotonicheskim sokratitel'nymi otvetami miokarda mlekopitaiushchego.

Effects of 6 and 12 mM of caffeine, 10(-5) M of verapamil, variation in stimulation frequency between 0.1 and 0.7 Hz, cooling to 15 degrees C after elevation of perfusate Ca2+ to 15 mM on isometric (IM) and isotonic (IT) contractions of guinea pig papillary muscles were studied. The first and the last interventions mentioned above were found to slow down relaxation phase and increase the IT/IM response amplitude ratio by 50-70%. Under the action of both interventions the maximum of IT response appears after the maximum of IM response with delay of 50-100 msec. A close correlation (r = 0.89) between changes of IT/IM and delay of IT peak was revealed. Different sensitivity of IM and IT responses to inotropic interventions could not be explained by the change of membrane electrical activity because the AP did not change after a transition from IM to IT mode. An analysis of experimental data suggests that the delay between IT and IM peaks and the ratio of their amplitudes depend on the rate of calcium uptake within the myocardial cell.