Proliferative and Differentiation Potential of Multipotent Mesenchymal Stem Cells Cultured on Biocompatible Polymer Scaffolds with Various Physicochemical Characteristics.

Biocompatibility of film and fibrous scaffolds from polylactide-based polymers and the relationship between their architecture and the functional characteristics of mesenchymal stem cells were studied. Cell culturing on polylactide-based film and fibrous matrixes did not deteriorate cell morphology and their proliferation and differentiation capacities. The rate of cell proliferation and penetration in microporous 3D matrices with the same porosity parameters and pore size depended on their spatial organization. The above materials can be used as scaffolds for mesenchymal stem cells for creation of tissue engineering implants. The scaffold size and structure should be determined by the defects in the organs in which the regeneration processes have to be stimulated.

[Biodistribution of Rifabutin Polymeric Transport Form].

BACKGROUND: One way to increase drug efficacy is to provide a drug delivery transport system to the target organ. A widely used method is to incorporate the drug in a biodegradable polymer composition with forming nanosized drug's transport forms. Objective: Our aim was to investigate the tissue biodistribution of antibiotic rifabutin transport system based on lactic and glycolic acids copolymer, and to compare it with the pure substance of rifabutin. METHODS: These substances were administered to two groups of rats intragastrically in the doses of 10 mg/kg. After a certain period of time, the animals were sacrificed by cervical dislocation. Samples preparation for analysis was carried out of the liquid-liquid extraction. Active substance's concentrations were measured by high performance liquid chromatography method. RESULTS: The study included 8-week-aged Wistar rats of both sexes weighing 0.22 ± 0.02 kg. Animals were divided into 2 groups. The study group received polymer form of antibiotic, and the comparison group received substance of rifabutin. In intervals of 10 min, 30 min, 1 h, 2 h, 4 h, 7h, 15 h, 24 h after drug administration liver, lung, spleen, kidney, intestines, stomach, heart and brain were resected respectively. Organs were measured by their weight. The drug was not detected in the brain. Rifabutin was determined in other examined tissues within 10 minutes and the maximum drug concentration in organs was fixed in 1.5-3.5 hours after administration. The rifabutin concentrations defined in the lungs were significantly higher in polymerform (p < 0.05). The polymer form's distribution coefficient was higher in the liver and lungs (15.83 and 10.14 µg/g respectively) in comparison with the substance one. The minimum amount of the active ingredient was observed in the heart (0.02 µg/g). CONCLUSION: It is shown that the inclusion of the drug in a polymeric form substantially alters its localization in organs and tissues. Extensive biodistribution nanorifabutin in lung tissue, liver and spleen is established.

Telomerase as a tumor marker in diagnosis of prostatic intraepithelial neoplasia and prostate cancer.

BACKGROUND: Early diagnosis of prostate cancer (CaP) can be addressed by studying prostatic intraepithelial neoplasia (PIN) as precancer (high-grade PIN or HGPIN). This article attempts to analyze the diagnostic role of telomerase as an early marker of carcinogenesis. METHODS: Complex urological patient evaluation and assessment of telomerase activity. RESULTS: Out of 92 patients 44% were diagnosed with CaP, 49% with low-grade PIN (LGPIN) in association with benign prostatic hyperplasia (BPH), and 7% with HGPIN in association with BPH. Active telomerase (AT) in prostate biopsy specimens was detected in 98% of patients with CaP, in 33% of patients with HGPIN, and in 20% of patients with LGPIN. In the event of simultaneous detection of AT and PIN in initial prostate biopsy specimens, further monitoring for 0.5-4.0 years revealed CaP development in 50-56% of cases. Further follow-up of patients with PIN and absent telomerase activity in initial biopsy specimens did not demonstrate the development of CaP. The PSA level was significantly higher in patients with active telomerase in the prostate tissue than in telomerase negative patients. CONCLUSIONS: Telomerase activity in the prostate tissue increases the risk of CaP development in patients with PIN. Detection of telomerase activity in prostate biopsy specimens from patients with PIN enables selection of a group of patients with high risk of CaP development and reduction of the number of prostate biopsies performed in other patients.

[The role of adiponectin in the pathogenesis of the metabolic syndrome and approach to therapy].

In this adiponectin-focused review, the pathophysiological role and the potential therapeutic benefits of adiponectin in metabolic syndrome (MetS) are analysed. MetS is recognized as clusters several metabolic abnormalities and the leading cause of cardiovascular diseases. Insulin resistance (IR) is a key factor in the pathogenesis MetS. Adiponectin is the most abundant and adipose-specific adipokine. Adiponectin acts through the activation of AMP-activated protein kinase and peroxisome proliferator-activated receptor-alpha (PPARalpha) pathways. The wide distribution of adiponectin receptors in various organs and tissues suggests that adiponectin has pleiotropic effects on numerous physiological processes. Its well-known insulin-sensitizing, anti-inflammatory and antiatherosclerotic properties, accumulating evidence suggests that adiponectin may have cardioprotective properties. There is an evidence that adiponectin decreases systematic IR and generally predicts cardiovascular diseases. Recent therapeutic strategies have focused on the indirect upregulation of adiponectin through the administration of various therapeutic agents and/or lifestyle modifications. Weight loss, diet, lifestyle changes and/or medications including orlistat, sibutramine, rimonabant, increase level of adiponectin. Also insulin sensitizers, including thiazolidinediones, and lipid-lowering agents, including statins and fibrates, upregulate adiponectin and may improve IR. The wider use of new treatment approaches appears to signal of a new era in the management of cardiovascular diseases, diabetes mellitus and MetS.

[Duodenal bleeding due to metastasis from lung adenocarcinoma controlled by radiotherapy: A case report and literature review]. / Saignement duodénal dû à une métastase d'un cancer pulmonaire contrôlé par radiothérapie : cas clinique et revue de la littérature.

INTRODUCTION: Gastrointestinal (GI) metastases in lung cancer rarely occur. CASE REPORT: We report here the case of a 43-year-old male active smoker who was admitted to our hospital for cough, abdominal pain and melena. Initial investigations revealed poorly differentiated adenocarcinoma of the superior-right lobe of the lung: positive for thyroid transcription factor-1 and negative for protein p40 and for antigen CD56, with peritoneal, adrenal and cerebral metastasis, as well as anemia requiring major transfusion support. Over 50% of cells were positive for PDL-1, and ALK gene rearrangement was detected. GI endoscopy showed a large ulcerated nodular lesion of the genu superius with active intermittent bleeding, as well as an undifferentiated carcinoma with positivity for CK AE1/AE3 and TTF-1, and negativity for CD117, corresponding to metastatic invasion originating from lung carcinoma. Palliative immunotherapy with pembrolizumab was proposed, followed by targeted therapy with brigatinib. Gastrointestinal bleeding was controlled with a single 8Gy dose of haemostatic radiotherapy. CONCLUSION: GI metastases are rare in lung cancer and present nonspecific symptoms and signs but no characteristic endoscopic features. GI bleeding is a common revelatory complication. Pathological and immunohistological findings are critical to diagnosis. Local treatment is usually guided by the occurrence of complications. In addition to surgery and systemic therapies, palliative radiotherapy may contribute to bleeding control. However, it must be used cautiously, given a present-day lack of evidence and the pronounced radiosensitivity of certain gastrointestinal tract segments.

Effect of anthralin on cell viability in human prostate adenocarcinoma.

The study revealed the key role of serine protease hepsin activity in transition of in situ prostate adenocarcinoma into the metastasizing form. Inhibition of hepsin activity suppresses the invasive growth of the tumor. Hepsin is an convenient target for pharmacological agents, so the study of its inhibitory mechanisms is a promising avenue in drug development. Assay of proteolytic activity in various tumor cell lines in vitro showed that this activity in prostate adenocarcinoma cells significantly surpasses proteolytic activity in other examined tumor cell lines. Selective cytotoxic action of anthralin, an inhibitor of hepsin activity, on human adenocarcinoma cells was demonstrated in comparison with other tumor cell lines.

[The distribution of iodine-125 labeled alpha-fetoprotein in the animal organism and its accumulation in the tumor].

The distribution of iodine-125 labeled human alpha-fetoprotein in mice was studied after its intravenous injection. The maximal accumulation of alpha-fetoprotein in different tissues and organs of animals was observed mainly 5 hours after injection. Then the protein was gradually eliminated from the body. In the liver, intestine and blood of intact animals 125I-alpha-fetoprotein persists for at least three days. Accumulation of alpha-fetoprotein in various tissues and organs may determine the different biological effects of this protein. In the mice with transplanted lymphatic leukemia cells P388 the high level of alpha-fetoprotein accumulation was detected in the tumor tissue, reaching 6% of the injected amount per 1 g of tissue. This allows considering the radionuclide-labeled alpha-fetoprotein as a promising medical radionuclide marker for the radiological detection of malignant tumors.

[Characterization of recombinant human HSP70's domains functions and interdomain interactions].

We investigated ATP-ase and peptide-binding activity of recombinant human heat shock protein HSP70(A1B), HSC70, and two hybrid proteins derived from those. The UV-spectral recorded data was used to characterize conformational rearrangements, which were induced by domain replacement or HSP70-peptide interaction. We have shown that N-terminal domain dramatically affect substrate specificity of C-terminal peptide-binding domain. This proposes new hypothesis for HSP70 chaperone machinery. The linear dependence between ATP-ase activity and peptide complex ratio was found. This relationship could be used for unlabeled peptide-HSP70 complex quantification.

[High-efficient renaturation of immobilized recombinant C-terminal fragment of human alpha-fetoprotein].

C-terminal fragment of a human oncofetal alpha-fetoprotein (AFP) may be used in targeted cytostatics delivery to malignant cells of many tumors. AFP fragment (from 404 to 595 amino acids residues of a full-sized protein) was cloned and produced in Escherichia coli cells, BL21 strain (DE3) in the form of inclusion bodies. To obtain a functionally active protein, is it necessary to renature the protein. The renaturation procedure of the AFP third domain (rAFP3D) is considerably complicated by the fact that the protein is hydrophobic and contains a large number of S-S bonds. A renaturation technique of rAFP3D immobilized on silicic metal chelate resin has been developed. The yield of renatured C-terminal fragment was no less than 60% with purity on the order of 98%. The developed technique has been applied for the first time for hydrophobic protein with a large number of S-S bonds. The approach can be applied for efficient renaturation of other hydrophobic proteins with a large number of disulfide bonds for scientific and practical purposes.

Isolation, purification, and study of properties of recombinant hepsin from Escherichia coli.

A recombinant hepsin-producing strain of Escherichia coli was obtained and the conditions for hepsin expression in a bacterial system were optimized. To study the physicochemical properties of the enzyme, a procedure for purification of active recombinant hepsin using metal-chelate affinity chromatography and ion-exchange chromatography was developed. The interaction of recombinant hepsin with various peptide substrates is characterized. The dose-dependent inhibition of the recombinant hepsin enzyme activity by anthralin in vitro and an increase in the hepsin enzymatic activity in the presence of resveratrol were revealed.

[Purification and characterization of recombinant human alpha-fetoprotein fragment, corresponding to the C-terminal structural domain].

Human alpha-fetoprotein (hAFP) is the main human oncofetal protein. Receptor of hAFP is expressed on the surface of different types of cancer cells, but not produced by normal cells of the adult organism. The hAFP interacts with the receptor via its third domain. The conjugates of native hAFP with a variety of natural cytostatic agents inhibit growth of cancer cells in vivo and in vitro. The C-terminal hAFP fragment comprising amino acids from 404 to 595 of the native hAFP was expressed in E. coli BL21 (DE3) strain. The level of the protein expression was no less than 150 mg/l. Highly efficient purification and refolding procedures were developed. The final protein yield was no less than 50% with purity of about 95%. Refolded rAFP3D bound specifically with cancer cells carrying hAFP receptor and was accumulated by them. This rAFP3D can be used as a carrier for the targeted drug delivery to cancer cells.

[Urokinase-type plasminogen activator and its inhibitor type 1 in the diagnosis of evolving complications in coronary heart disease].

Fibrinolytic system components are important in the regulation of thrombogenesis therefore the aim of the investigation was to compare the level of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1) in the monocytes of patients with stable coronary heart disease (CHD) and acute coronary syndrome (ACS) to reveal an association of the content of these proteins with the severity of disease, by applying two different techniques: immunocytochemistry and flow cytofluorimetry. The counts of uPA- and PAI-1-expressing monocytes were equal in each case and accounted for 81.9-99.9% in all groups. At the same time, the level of PAI-1 was higher than that of uPA and significantly higher in patients with ACS than in those without ACS and in the controls. No significant differences were found in uPA levels between the ACS and stable CHD groups; however, it was significantly higher in the patient groups than in the control one. The detection of the higher expression of PAI-1 in the peripheral blood monocytes of patients with ACS suggests that it can be used as a marker of disease severity.

[Live xenogenic vaccine in the prevention of metastases of uveal melanoma].

By the results of experimental studies and by being guided by Protocol No. 22 dated January 22, 2004, by the Ministry of Health of the Russian Federation, the authors have conducted clinical trials of biotherapy used in oncological care. They describe the first experience in using xenovaccination in 35 patients with uveal melanoma to prevent hematogenic metastases. The follow-ups of the patients lasted within 14-49 months after initiation of the vaccination. No recurrences were observed in 30 patients during follow-ups whose durations averaged 41.3 months. The authors consider that this is reason to regard xenovaccination as an available method for the prevention of metastases of uveal melanoma.

Cdc42-dependent F-actin dynamics drive structuration of the demarcation membrane system in megakaryocytes.

UNLABELLED: Essentials Information about the formation of the demarcation membrane system (DMS) is still lacking. We investigated the role of the cytoskeleton in DMS structuration in megakaryocytes. Cdc42/Pak-dependent F-actin remodeling regulates DMS organization for proper megakaryopoiesis. These data highlight the mandatory role of F-actin in platelet biogenesis. SUMMARY: Background Blood platelet biogenesis results from the maturation of megakaryocytes (MKs), which involves the development of a complex demarcation membrane system (DMS). Therefore, MK differentiation is an attractive model for studying membrane remodeling. Objectives We sought to investigate the mechanism of DMS structuration in relationship to the cytoskeleton. Results Using three-dimensional (3D) confocal imaging, we have identified consecutive stages of DMS organization that rely on F-actin dynamics to polarize membranes and nuclei territories. Interestingly, microtubules are not involved in this process. We found that the mechanism underlying F-actin-dependent DMS formation required the activation of the guanosine triphosphate hydrolase Cdc42 and its p21-activated kinase effectors (Pak1/2/3). Förster resonance energy transfer demonstrated that active Cdc42 was associated with endomembrane dynamics throughout terminal maturation. Inhibition of Cdc42 or Pak1/2/3 severely destructured the DMS and blocked proplatelet formation. Even though this process does not require containment within the hematopoietic niche, because DMS structuration was observed upon thrombopoietin-treatment in suspension, integrin outside-in signaling was required for Pak activation and probably resulted from secretion of extracellular matrix by MKs. Conclusions These data indicate a functional link, mandatory for MK differentiation, between actin dynamics, regulated by Cdc42/Pak1/2/3, and DMS maturation.

ATP-binding cassette transporter 1 (ABCA1) deficiency decreases platelet reactivity and reduces thromboxane A2 production independently of hematopoietic ABCA1.

UNLABELLED: ESSENTIALS: The role of ATP-binding cassette transporter 1 (ABCA1) in platelet functions is poorly characterized. We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and two Tangier patients. ABCA1-deficient platelets exhibit reduced positive feedback loop mechanisms. This reduced reactivity is dependent on external environment and independent of hematopoietic ABCA1. BACKGROUND: The ATP-binding cassette transporter ABCA1 is required for the conversion of apolipoprotein A-1 to high-density lipoprotein (HDL), and its defect causes Tangier disease, a rare disorder characterized by an absence of HDL and accumulation of cholesterol in peripheral tissues. The role of ABCA1 in platelet functions remains poorly characterized. OBJECTIVE: To determine the role of ABCA1 in platelet functions and to clarify controversies concerning its implication in processes as fundamental as platelet phosphatidylserine exposure and control of platelet membrane lipid composition. METHODS AND RESULTS: We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and in two Tangier patients. We show that platelets in ABCA1-deficient mice are slightly larger in size and exhibit aggregation and secretion defects in response to low concentrations of thrombin and collagen. These platelets have normal cholesterol and major phospholipid composition, granule morphology, or calcium-induced phosphatidylserine exposure. Interestingly, ABCA1-deficient platelets display a reduction in positive feedback loop mechanisms, particularly in thromboxane A2 (TXA2) production. Hematopoietic chimera mice demonstrated that defective eicosanoids production, particularly TXA2, was primarily dependent on external environment and not on the hematopoietic ABCA1. Decreased aggregation and production of TXA2 and eicosanoids were also observed in platelets from Tangier patients. CONCLUSIONS: Absence of ABCA1 and low HDL level induce reduction of platelet reactivity by decreasing positive feedback loops, particularly TXA2 production through a hematopoietic ABCA1-independent mechanism.

[Inactivation and sensitization of the tumor cells by the gene Bax transfection].

After the transfection of the gene Bax into the cultured tumor cells of human ovary adenocarcinoma SKOV3 and uterus carcinoma HeLa in vitro the high sensitivity of the cells SKOV3 to the protein Bax produced after the gene Bax transfection was found. The sensitivity of the cells HeLa to the gene Bax transfection was much smaller. The hyperexpression of gene Bax and hypersensitivity to doxorubicin were seen in HeLa cells received as a result of the gene Bax transfection and subsequent selection. All cells of the line SKOV3 with the increased expression of the transfected gene Bax died. In the cell line SKOV3 the mutation in a gene Bax was found which has a genotype G7/G9 against a native type of a gene Bax--G8/G8. It was concluded that the found in the exone 3 of the gene Bax mutation G7/G9 in cells SKOV3 results in an inactivation of proapoptotic activity of the protein Bax.

[Molecular mechanisms of niclosamide antitumor activity].

In this review the recent data regarding the antitumor activity of niclosamide and the molecular mechanisms of its antitumor activity are presented. Niclosamide has been used in the clinic for the treatment of intestinal parasite infections. In recent years in several screening investigations of various drugs and chemical compounds niclosamide was identified as a potential anticancer agent. Niclosamide not only inhibits the Wnt/ß-catenin, mTORC1, STAT3, NF-κB and Notch signaling pathways, but also targets mitochondria in cancer cells to induce growth inhibition and apoptosis. A number of studies have established the anticancer activity of niclosamide in both in vitro and in vivo in xenotransplantation models using human tumors and immunodeficient mice. It is important that niclosamide is active not only against tumor cells but also cancer stem cells. Normal cells are resistant to niclosamide. The accumulated experimental data suggest niclosamide is a promising drug for the treatment of various types of cancer.

Polyphosphoinositides as activators of PKC-dependent synapsin I phosphorylation.

The effect of PIP2 and diacylglycerol (products of polyphosphoinositide turnover) on the activation level of phosphorylation of the human brain neurospecific protein SI by PKC from the same source was studied. The apparent activation constant of the phosphorylation process was shown to decrease in the presence of PIP2 from 1.1 micrograms/ml for PI and from 0.8 micrograms/ml to 0.6 microgram/ml for PS; the value of 0.4 microgram/ml in the latter case was detected merely after the addition of DOG into the reaction mixture. Polyphosphoinositides are suggested to play a role in activating PKC-mediated phosphorylation of SI in nerve terminals.

Synapsin I phosphorylated by Ca2+, calmodulin-dependent protein kinase II is able to self-degrade.

Phosphorylation of the neurospecific protein synapsin I (SI) by various cellular protein kinases was studied. The analysis of functional properties of phosphosynapsins showed that in the case of PK II-mediated modification of the protein, it becomes capable of self-degradation. The latter process was found to be specific and did not appear to be characteristic of the phosphoforms emerging after the protein modification by PK C or PK A. A possible involvement of the process in the regulation of neurotransmitter secretion is discussed.

The effect of phosphorylation on pyruvate dehydrogenase.

Phosphorylation of the pyruvate dehydrogenase component (E1) of the muscle pyruvate dehydrogenase complex (PDC) by E1-kinase inhibits substrate conversion both in oxidative and non-oxidative reactions. Circular dichroism spectra were used to monitor the effect of phosphorylation on the following stages of the process: holoform formation from apo-E1 and thiamine pyrophosphate (TPP), substrate binding and active site deacetylation. It has been shown that phosphorylation of E1 reduces its affinity for TPP and prevents holo-E1 interaction with pyruvate. Phosphorylated and dephosphorylated PDC convert 2-hydroxyethyl-TPP in similar ways involving half of their active sites; all active sites of E1 function in the presence of deacetylating agents. The data obtained suggest that the phosphorylation and substrate binding sites interact with each other.