RET/PTC1 fusion oncogene is the most common genetic alteration identified to date in thyroid papillary carcinomas (PTC) and represents a good target for small interfering RNA (siRNA). Our aim was: i) to target the RET/PTC1 oncogene by siRNAs, ii) to assess the knockdown effects on cell growth and cell cycle regulation and iii) to vectorize it in order to protect it from degradation. Methods. Human cell lines expressing RET/PTC1 were transfected by siRNA RET/PTC1, inhibition of the oncogene expression was assessed by qRT-PCR and by Western blot. Conjugation of siRNA RET/PTC1 to squalene was performed by coupling it to squalene. In vivo studies are performed in nude mice. Conclusion. In this short communication, we report the main published results obtained during last years.
Aim. To examine neutrophil F-actin, phagocytosis and macropinocytosis dymanics in patients with Familial Mediterranean Fever (FMF), in an effort to understand the mechanisms that regulate switch of neutrophil activation program. Methods. Whole blood neutrophils obtained from 37 attack-free FMF patients and 20 normal donors (ND) were activated with N-formyl-Met-Leu-Phe (fMLP), phorbolmyristate acetate (PMA) or lipopolysaccharide (LPS) and cellular F-actin content, phagocytosis and macropinocytosis determined by flow cytometry. F-actin oscillation amplitude and period were calculated from the curves generated by mathematical simulation giving the assumption that in neutrophil F-actin oscillates about a fixed point in a harmonic motion. Results. Unstimulated neutrophil F-actin content was markedly increased in FMF patients. fMLP- but not PMA- or LPS-stimulated and Col-pretreated neutrophils where characterized by different pattern of F-actin dynamics and delayed time period of F-actin oscillation during FMF. Neutrophils from FMF patients failed to induce chemoattractant receptor desensitization during repeated action of fMLP, while in ND it occurred with significant reduction of F-actin oscillation amplitude and period. In FMF patients we observed significant enhancement of phagocytosis but not macropinocytosis amplitude and frequency. Conclusions. Impaired neutrophil F-actin, phagocytosis and macropinocytosis oscillations amplitude and frequency that tightly regulate switch of neutrophil activation program during its encounter with increasing concentration of chemoattractants may be a potential pathogenic mechanism causing aberrant resolution of inflammation during FMF
Aim. For the purpose of local chemotherapy in patients with malignant brain tumors after partial or total removal of tumors we used biodegradable film with methotrexate. The film, due to its properties, is firmly attached to the wound surface. With its composition of chemotherapeutic agents over specified time, the dosed film immediately affects diseased tissue, playing the role of a local chemotherapy depo. Methods. Results of treatment of 74 patients with low differentiated gliomas of the brain, which have undergone a comprehensive treatment that includes surgical removal of tumor and implantation of methotrexate depo in the wall of postoperative cerebral injury followed by radiotherapy in the pig on a bed of the tumor removed. Results. A retrospective analysis of the results of complex treatment of patients with glial brain tumors with local chemotherapy showed a significant increase in disease-free period in patients who have undergone, consistently, surgery, chemotherapy and adjuvant local radiation therapy. Conclusions. Our research has shown that the use of local chemotherapy greatly improves the results of treatment of patients not only for total removal of intracerebral tumors, but also for subtotal removal, as well as in patients with prolonged tumor growth, when there is no possibility of radiation treatment
Aim. We do analyze of the dynamics of morphometabolic changes in transformed cells (of susceptoible lines) demonstrating resistance to picrnaviral infection. Methods. The study was performed by application of cell culture technology and a complex of cytochemical and cytophotometric assays. Were used picornaviruses from various genu. Results. According to the results obtained, resistant to picornavirus infection cells of different susceptible lines have similar changes in the phenotype. They have decreased number of nucleoli and increased percentage of euploidy (and near euploid). In resistant cells of all cultures the reduction in amount of DNA and RNA both in nucleus and in cytoplasm was found. All these data correlated with the increased euploidy (and near euploid) of the resistant population. All picornavirus resistant cells had a less transformed phenotype, and decreased proliferative activity. Decreased nucleolar status becomes apparent by reduction of all nucleolar indices. Conclusions. Picornaviruses on the susceptible cells produce 2 types of changes – selection and modification. Whatever the mechanism, it is specific for an individual virus, since no restrictions occur in case of infection caused by another picornavirus
Aim. Analyze the interaction between Lysyl-tRNA synthetase (LysRS) and HIV-1 GagPol to know whether a particular N-terminal sequence of mitochondrial LysRS triggers a specific recognition with GagPol. Methods. Yeast two-hybrid analysis, immunoprecipitation. Results. We have shown that LysRS associates with the Pol domain of GagPol. Conclusions. A model of the assembly of the LysRS:tRNA3Lys:GagPol packaging complex is proposed.
Aim. The main objective of this study was the comparative investigation of diverse lipid second messenger (LSM) generation by human peripheral blood lymphocytes (HPBL) at different (5, 10, 30 and 60 s) time points of cell co-stimulation by anti-CD3 and anti-CD28 monoclonal antibodies in norm and breast cancer (BC). Methods. Ficoll-Hypaque gradient centrifugation. Results. The data obtained indicate that some mechanisms of LSM generation/utilization in stimulated crude HPBL were significantly altered in BC compared to norm. Particularly, the reliable generation of arachidonyl-1,2-diacylglycerol (1,2-DAG) at the initial step (5 s) of cell stimulation observed in norm was depressed in BC and reached the value below the basal level in unstimulated cells. It is important that the disturbances in 1,2-DAG formation in HPBL obtained from patients with BC were identical with those observed earlier in other forms of cancer. Conclusions. We conclude that the regularities revealed are common characteristics for all the types of malignancy studied and can be used as additional testing parameters for cancer definition and individual correction of the chemotherapy programs for disease treatment
The review is devoted to the inhibitors of cysteine proteinases which are believed to be very important in many biochemical processes of living organisms. They participate in the development and progression of numerous diseases that involve abnormal protein turnover. One of the main regulators of these proteinases is their specific inhibitors: cystatins. The aim of this review was to present current knowledge about endogenous inhibitors of lysosomal cysteine proteases and their synthetic analogs.
Some of the pathogenetic mechanisms which cause the state of cellular insulin resistance on receptor and postreceptor levels were analyzed. Special attention is given to the analysis of factors which influence the activity of the components of phosphatidylinositol chain of insulin signaling cascade.
Aim. Cloning of Er. rhusiopathiae surface antigen – SPAA gene into plasmid vector and expression of SpaA protein in E. coli. Methods. Oligonucleotide primers design and synthesis, polymerase chain reaction, cloning, recombinant proteins expression in E. coli, electrophoresis in agarose, PAGE. Results. Full-size and truncated Er. rhusiopathiae SPAA genes have been cloned into expression plasmid pET24a(+). Expression of full-size gene hasn’t accompanied with the significant recombinant protein amount synthesis, presumably due to its toxicity for E. coli cells. Elimination of the fragment coding for proline-rich region and tandem repeats domain from full-size gene led to the obtaining of the truncated variant which produced stable synthesis of the recombinant protein with expected molecular mass. Conclusions. recombinant plasmid containing fragment of Er. rhusiopathiae SPAA gene has been cloned. It allows to obtain in E. coli recombinant protein with m. m. 47 kDa in preparative amount.
The article provides a brief overview of the literature on target design, exploration properties and effectiveness of the application of recombinant baculoviruses in model systems in vivo. The results of experiments with wild and recombinant baculoviruses are analysed in regard to the priority areas of biomedicine such as tissue regeneration, gene therapy of cancer, development of vaccines against infectious diseases and malignancies
Aim. To study the association of the polymorphic variants of ACE (I/D), AT2R1 (A1166C), TNF-alpha (G308A), MTHFR (C677T) genes and their combinations with the risk of perinatal pathology and gestation reduction. Methods. The polymorphic variants of genes were analyzed by PCR and RFLP in 235 newborns with severe perinatal pathology and 110 clinically healthy term newborns. Results. An increased risk of severe perinatal pathology was associated with such genotypes: DD and ID (ACE), 1166AC, 1166CC (AT2R1), 677CT (MTHFR), 308AA and 308AG (TNF-alpha), this risk for homozygotes is almost 2-fold higher than for heterozygotes. Reduction of terms of gestation is associated with the genotype 677TT (MTHFR), and resistance to diseases in the perinatal period – with the genotype II (ACE) and 1166AA (AT2R1), 677CC (MTHFR) and the 308GG (TNF-alpha), particularly when combined. Conclusions. The identified associations evidence the role of polymorphic variants of ACE, AT2R1, TNF-alpha, MTHFR genes in the development of severe perinatal pathology and can be used for its early prediction with subsequent correction of treatment.
In spite of the strong prognostic value of all traditional cardiovascular risk factors, still striking differences exist in the prevalence of clinical events between patients at apparently similar risk. One of the main reasons is different genetic background. One of recently discussed candidate genes for cardiovascular disease is the gene for the protein Connexin 37 (Cx37). This protein is a part of gap junctions responsible for communications between cells including cells in the vessel wall. Studies focused on the association between Cx37 gene polymorphism (1019C > T; Pro319Ser) and cardiovascular disease demonstrate inconsistent results. Our findings in 1.316 men and women indicated that the Cx37 gene polymorphism (genotype CC) is significantly associated with acute coronary syndrome in non-smoking women. In addition, in urban and rural women from general population (n = 1.056) with impaired fasting glycaemia the same genotype is associated with increased intima media thickness of carotid arteries measured by ultrasound. Finally, in 289 women with diabetes type 1 or 2, and in 208 women from general population with central obesity, the CC genotype was associated with lower ankle brachial blood pressure index. These data indicate that Cx37 gene polymorphism could have gender- and smoking-dependent effects on acute coronary events and glucose dependent effect on atherosclerosis in women.
In the last hundred years modern society went through numerous changes in life style, dietary habits, work load, physical activity and other environmental factors. As a species we are not well adapted to new de- mands. Higher levels of stress hormones provoke various effects, especially gradual change in the sensitivity of adrenergic, glucocorticoid and insulin receptors. All these changes are mutually associated and they gradually lead to metabolic syndrome, obesity, diabetes, heart failure and other types of pathology depending on genetic makeup and environmental factors. The aim of this paper is to summarize current knowledge concerning the impact of stress on cardiac function. Whereas stress response is sex specific we would emphasize a potential difference in pathophysiology of ischemic heart failure in men and women. Modern medicine has misinterpreted autonomous nervous system functions for years and this was reflected in heart failure (HF) and arterial hypertension therapy. Stress before the onset of menopause has a lesser effect on cardiac function compared to stress after menopause. Postmenopausal women have a significantly higher risk of heart disease, which is related to the diminished protection of the female hormonal cycle, but low doses of estrogen have not proven protective in postmenopausal women. Potential new targets of sex- specific cardiac therapy would come from better understanding of the molecular mechanisms exerted by nuclear receptors for steroid hormones, transcription factors involved in heart remodeling, cross-talk in adrenergic signaling pathways and their down-stream molecules.
Aim. To synthesise NaYF4 nanocrytstals doped or co-doped with different lanthanide ions (Eu, Tb, Gd) and to investigate them optically to achieve efficient optical markers. Methods. Samples have been synthesized by using co-thermolizys method and optical properties have been investigated by using photoluminescence (PL), PL excitation and PL decay spectroscopies. Results. Efficient emission in visible spectra range has been observed for all investigated samples. The excitation mechanism of the main emission centre has been explained. Conclusions. It has been shown that the main excitation mechanism of Eu ions is through energy transfer from Tb or Gd ions. Moreover, it has been shown that obtained by us nanocrystals characterize by strong emission which make them potential as efficient optical markers in biology or medicine
Aim. To study the molecular mechanisms of action of novel anticancer antibiotic landomycin E (LE). Methods. Annexin V/propidium iodide, DAPI (4',6-diamidino-2-phenylindole) staining, Western-blot analysis. Results. LE applied in 2 µg/ml dose (IC50), induced reactive oxygen species (ROS)-dependent splitting of poly [ADP-ribose] polymerase 1 (PARP-1) and DNA Fragmentation Factor 45 (DFF45) proteins involved in DNA reparation. This effect was observed 6 h after the start of treatment and it positively correlated with phosphatidyl serine externalization (early morphological marker of apoptosis). We suggest that cleavage of PARP-1 and DFF45 was mediated by active caspase-7 which is a key effector caspase in the LE-induced apoptosis in leukemia cells. We found that activation of initiator procaspase-10 (involved in receptor- mediated apoptosis) was the earliest detected event in LE-induced apoptotic signaling pathways; however, this activation was shown to be ROS-independent. We also demonstrated that the induction of apoptosis by LE is accompanied by activation of apoptosis-inducing factor (AIF) in mitochondria. Conclusions. Our data suggest that LE-induced cascade of apoptotic events is started by the initiator caspase-10 which leads to activation of the effector caspase-7 and AIF that is known to induce caspase-independent apoptosis involving ROS generation.
The aim of our study was to follow the persistence of viral RNA in selected organs of experimentally infected with coxsackievirus (CV) B5 strains from different sources such as a patient’s sample, an environmental sample and a prototype virus strain. Methods . CD-1 mice were infected with CVB5 strain Faulkner the prototype, CVB5 – isolate from treated sewage waste and isolate from patient’s stool sample both identified as CVB5. The viral RNA was detected by RT-PCR using enterovirus primers specific for the non-coding 5' region. Results . We observed presence of RNA in the brain and heart of mice infected with isolate from patient’s stool at day 45 post infection (p. i.). Conclusion. We conclude that CVB5 persists in the brain and heart after oral infection of CD1 mice. The relevance of viral persistence maybe related viral origin and the genetics
Aim. In the elderly subjects metabolic syndrome (MetS) seems to be associated with low levels of circulating protective soluble receptor for advanced glycation end products (sRAGE). This secondary study aimed to answer whether this phenomenon is manifested from early childhood. Methods. 73 mothers and their 77 infants (4-to-12-months of age) were included in the study. Mothers were classified according to the presence of MetS components as negative (n = 32), those with pre-MetS (insulin resistance + 1 sign of MetS, n = 27) and overt MetS (n = 14). sRAGE and carboxymethyllysine (CML) were determined in the mothers and the infants. Results. Mothers with pre- and overt MetS displayed lower sRAGE levels, while in their children only a trend towards decline was observed. sRAGE levels significantly and inversely correlated with insulin sensitivity and BMI/body weight. No difference in CML levels across the groups was observed. Conclusions. Metabolic syndrome is associated with decreased levels of sRAGE in the mothers and a tendency towards decline of sRAGE in their offspring. Infants of mothers with MetS maintain normoglycemia on the account of higher insulin levels.
All cellular RNA molecules are damaged at the scale of DNA molecules, or even more. In the present review the RNA damaging agents, some mechanisms of RNA repair and editing, their difference from DNA repair mechanisms have been discussed.