[Determination of calcium metabolism markers concentration in patients with calcium oxalate nephrolithiasis].

THE AIM OF THE RESEARCH: to reveal the relationship of various markers of calcium metabolism (osteopontin (OPN), parathyroid hormone-related protein (PTHrP), vitamin D, parathyroid hormone (PTH)) on the course of urolithiasis (Urolithiasis) in patients with calcium oxalate nephrolithiasis. MATERIALS AND METHODS: 100 people were examined, the following groups were included: group 1 - patients with calcium oxalate primary nephrolithiasis (n=41), group 2 - with calcium oxalate recurrent nephrolithiasis (n=39). Group 3 included conditionally healthy volunteers (n=20). The studies were carried out by the immunoenzymometric ELIZA and biochemical methods using appropriate test systems. RESULTS: in patients with recurrent nephrolithiasis, the serum PTHrP level is 54.6 (25.4-78.2) pg / ml, which is 3.7 times higher than in conventionally healthy individuals; the level of osteopontin is more than 1.5 times higher and amounts to 1.820 (0.991-2.212) pg / ml. In the group of primary nephrolithiasis, the level of PTHrP is 2-2.5 times higher than in conventionally healthy people. In patients with primary nephrolithiasis, the blood calcium level does not correlate with the level of PTHrP in the blood (r=- 0.0173, p> 0.05), as in the group with recurrent nephrolithiasis (r=0.0223, p>0.05). DISCUSSION: in patients with recurrent nephrolithiasis in the preoperative period, the serum levels of osteopontin and PTHrP in the blood serum were higher than in patients who were first diagnosed with urolithiasis, the data obtained can be used as a criterion for predicting the risk of recurrence of urolithiasis in the postoperative period. The blood calcium level does not have a statistically significant relationship with PTHrP, which allows us to assume that PTHrP has other mechanisms of influence on the development of urolithiasis, given the data obtained that the PTHrP level in patients with primary and recurrent nephrolithiasis is higher than in conditionally healthy people. CONCLUSION: Determination of the level of PTHrP and osteopontin in patients with urolithiasis allows predicting the risk of recurrence of urolithiasis at the stage of primary calcium oxalate nephrolithiasis. Determination of the level of PTHrP makes it possible to predict the risks of developing urolithiasis in conventionally healthy individuals, which can be used for targeted prevention of an unfavorable course of urolithiasis by prescribing timely adequate rational therapy and correcting the patients diet. At the same time, no correlation was found between the level of PTHrP and the level of blood calcium in patients with calcium oxalate nephrolithiasis; therefore, further studies of the role of this protein in the pathogenesis of urolithiasis are needed.

[The use of herbal supplement Renotinex based on terpenes for the complex treatment of patients with urinary stone disease].

AIM: to evaluate the efficiency of dietary supplements Renotinex for the complex treatment of patients with urinary stone disease who undergone to extracorporeal shock wave lithotripsy (ESWL). MATERIALS AND METHODS: a total of 60 patients with uncomplicated form of the urinary stone disease with the first stone episode (of size up to 1 cm) were evaluated. Patients were divided into two groups of 30 people and treated by ESWL. In the first group, patients additionally received Renotinex. In the second group, standard complex therapy (antispasmodics, analgesics, non-steroidal anti-inflammatory drugs) was prescribed. The urinary level of 2-microglobulin and serum level of tocopherol were considered as markers of damage and inflammation. Pain intensity was assessed using visual analogue scale. Follow-up studies were carried out on the 1st, 7th and 14th day of therapy. Duration of treatment and follow-up was 1 month. The average number of ESWL sessions was 2.6 in both groups. RESULTS: according to the study, it was established that components of Renotinex had antiseptic, antispasmodic, anti-inflammatory effects on the genitourinary system, enhancing renal blood flow and decreasing the permeability of the kidney capillaries. In addition, Renotinex has diuretic effect and nephroprotective effect, and improves renal function, alleviating aggressive therapeutic influence of ESWL. Antioxidant and nephroprotective effect are among the main mechanisms of action of Renotinex. CONCLUSION: In patients who received Renotinex as dietary supplements fragments after ESWL pass twice as fast, while in patients who did not receive Renotinex, there was more pronounced damage to the kidney parenchyma diagnosed by urine level of 2-microglobulin. The serum concentration of vitamin E increases, while taking Renotinex, which may prevent the peroxidation of polyunsaturated lipids in cell membranes and enhances the nephroprotective effect of Renotinex.

[Sestrins are Gatekeepers in the Way from Stress to Aging and Disease].

Sestrins belong to a family of evolutionary conserved proteins which are found in the majority of animal species. While invertebrate genomes contain only one sestrin gene, mammalian and other vertebrate genomes comprise three highly homologous genes that encode Sestrin 1, 2 and 3 proteins (Sesn1, Sesn2 and Sesn3). Sestrins are activated in response to a variety of stimuli and trigger metabolic shifts promoting cell survival under stress conditions. Although cellular stress within an organism is often caused by external stimuli it can be induced by excess of cytokines, chemokines, reactive oxygen species which are produced during aberrant metabolic or immune processes and are involved in regulation of cell physiological states including cell death. Activation of sestrins facilitates cell adaptation to stress through stimulation of antioxidant response and autophagy through regulation of the signaling pathways mediated by AMPK and mTOR kinases. These activities are involved in protection of the organism during physical exercise and certain level of sestrins activity contributes to the development of age-related diseases. However, prolonged activation of sestrins under chronic stress may cause negative effects for the organism.

Sensing the Environment Through Sestrins: Implications for Cellular Metabolism.

Sestrins are a family of stress-responsive genes that have evolved to attenuate damage induced by stress caused to the cell. By virtue of their antioxidant activity, protein products of Sestrin genes prevent the accumulation of reactive oxygen species within the cell, thereby attenuating the detrimental effects of oxidative stress. In parallel, Sestrins participate in several signaling pathways that control the activity of the target of rapamycin protein kinase (TOR). TOR is a crucial sensor of intracellular and extracellular conditions that promotes cell growth and anabolism when nutrients and growth factors are abundant. In addition to reacting to stress-inducing insults, Sestrins also monitor the changes in the availability of nutrients, which allows them to serve as a key checkpoint for the TOR-regulated signaling pathways. In this review, we will discuss how Sestrins integrate signals from numerous stress- and nutrient-responsive signaling pathways to orchestrate cellular metabolism and support cell viability.

Sestrin2 integrates Akt and mTOR signaling to protect cells against energetic stress-induced death.

The phosphoinositide-3 kinase/Akt (PI3K/Akt) pathway has a central role in cancer cell metabolism and proliferation. More importantly, it is one of the cardinal pro-survival pathways mediating resistance to apoptosis. The role of Akt in response to an energetic stress is presently unclear. Here, we show that Sestrin2 (Sesn2), also known as Hi95, a p53 target gene that protects cells against oxidative and genotoxic stresses, participates in the protective role of Akt in response to an energetic stress induced by 2-deoxyglucose (2-DG). Sesn2 is upregulated in response to an energetic stress such as 2-DG and metformin, and mediates the inhibition of mammalian target of rapamycin (mTOR), the major cellular regulator of energy metabolism. The increase of Sesn2 is independent of p53 but requires the anti-apoptotic pathway, PI3K/Akt. Inhibition of Akt, as well as loss of Sesn2, sensitizes cells to 2-DG-induced apoptosis. In addition, the rescue of Sesn2 partially reverses the pro-apoptotic effects of 2-DG. In conclusion, we identify Sesn2 as a new energetic stress sensor, which appears to be protective against energetic stress-induced apoptosis that integrates the pro-survival function of Akt and the negative regulation of mTOR.

[Transcriptional inhibition of human papilloma virus in cervical carcinoma cells reactivates functions of the tumor suppressor p53].

Inactivation of tumor suppressor p53 accompanies the majority of malignant diseases in humans. Restoration of p53 functions in tumor results in death of cancer cells, which can be used in cancer therapy. In cervical cancer a product of E6 gene of the human papilloma virus promotes accelerated degradation of p53 in proteasome system. Therefore, one of the approaches to reactivation of p53 in cervical carcinoma cells could be the use of small molecules that inhibit functions of viral proteins. By using as a test system human cervical carcinoma cells (HeLa cell line bearing human papilloma virus type 18, HPV-18) with introduced reporter construct that expresses beta-galactosidase under control of a p53-dependent promoter we carried out screening of a library of small molecules to select small molecules capable of reactivating transcriptional activity of p53. We then characterized the effects of two most active compounds in cell lines that differ in the status of p53-dependent signaling pathway. Both of the compounds caused specific activation of p53 in the cell lines expressing HPV-18, to a lesser extent--HPV-16, and do not cause any effect in control p53 negative cells, or in the cells with undisrupted p53 pathway. Activation of p53 in cervical carcinoma cells was accompanied by the induction of the p53-dependent gene CDKN1 (p21), by inhibition of proliferation, and by the induction of apoptosis. Both of the compounds were capable of deep inhibition of transcription from the HPV genome, which apparently was the cause for p53 reactivation in response to decreased expression of the E6 protein. The observed low toxicity for normal cells allows considering these chemical compounds as prototypes for future anticancer drugs.

[Retroviral reporter systems for the assessment of activity of stress-induced signal transduction pathways controlled by p53, HIF-1 and HSF-1 transcription factors].

Tumor suppressor p53, hypoxia-inducible factor 1 (HIF-1) and heat-shock factor 1 (HSF-1) are involved as the key transcription factors in cellular response to stress, induced by genetic material damage, hypoxia and heat shock respectively. The protein factors listed above also play an integral part in tumor development and progression. Thus, modulation of their activity may be important for treatment of cancer. In our work we obtained the reporter constructs for quantitative assessment of p53, HIF-1 and HSF-1 transcriptional activity on the basis of retro- and lentiviruses, allowing to obtain reporter cell lines almost out of any cell type. Induction of beta-galactosidase reporter gene expression, reflecting the activity of p53 and HIF-1 factors, depends on dose of treatment and also correlates with the induction of the endogenous target genes expression. The observed effect of activating treatments completely disappeared when the expression of p53 and HIF-1 genes was inhibited with specific siRNAs. The obtained reporter constructs may find the application in the screening of chemical and genetic (such as siRNA- and cDNA-libraries) modulators of transcriptional activity along with the investigation of components of signal transduction pathways modulating the transcriptional activity of those factors.

Virus-based reporter systems for monitoring transcriptional activity of hypoxia-inducible factor 1.

Being key regulator of oxygen homeostasis hypoxia-inducible factor 1 (HIF-1) plays significant roles in cancer progression as well as in cardiovascular diseases. The modulation of HIF-1alpha activity in vivo may represent a valuable therapeutic approach to these disorders. In order to monitor HIF-1 transcriptional activity, we have developed HIF-1alpha-responsive reporter constructs, in which lacZ gene expression is driven by minimal Hsp70 gene promoter or minimal immediate early promoter of cytomegalovirus (CMV) and a combination of hypoxia response elements from regulatory regions of PGK1, ENO1 and LDHA genes. For the efficient delivery to a wide variety of cell types we chose retroviral and lentiviral vectors as carriers of the reporter cassette. We demonstrate that the obtained reporter system i) has a high inducibility in response to treatments leading to HIF-1alpha activation, ii) shows upregulation in response to HIF-1 activation and downregulation following inhibition of HIF-1alpha expression by small interfering RNA, iii) follows the dynamics of endogenous HIF-1 target gene expression. The retrovirus- and lentivirus-based reporters can be used for high-throughput screening of HIF-1alpha modulators and for the study of crosstalk between HIF-1 and different related signal transduction pathways. Potential applications for the reporters are discussed.

High constitutive level of NF-kappaB is crucial for viability of adenocarcinoma cells.

Most of cells exhibit low nuclear level of NF-kappaB. However, in some cell lines and tissues aberrantly activated NF-kappaB is playing an important role in cell motility, growth control and survival. Here we describe the result of decrease of constitutive NF-kappaB level in different adenocarcinoma cell lines. Treatment of mouse adenocarcinoma cell line CSML-100 with both synthetic (TPCK or PDTC) or natural (I(kappaB)-alpha) NF-kappaB inhibitors caused apoptotic death. Low doses of TPCK were harmless for CSML100 cells but sensitized them to TNF-induced apoptosis. Death of CSML100 cells in the presence of high concentration TPCK was not accompanied with significant changes in c-myc activity but strongly correlated with rapid decrease in p53 level. Thus, mutual behavior p53 and NF-kappaB represented a unique feature of TPCK-induced apoptosis in CSML-100 adenocarcinoma cells.

[Construction by using retrovirus vector of recombinant with synthetic bradykinin "gene" for investigations of human gene expression in mammalian cells]. / Konstruirovanie na osnove retrovirusnogo vektora rekombinanta s sinteticheskim "genom" bradikinina s tsel'iu izucheniia ékspressii genov cheloveka v kletkakh mlekopitaiushchikh.

The synthetic gene of bradykinin was built into the retrovirus vector pPS-3-neo under the guidance of LTR promotor, followed by pPS-3-neo (brd) vector transfection of strain 293 cells. The physiological activity of the expressed bradykinin was tested on cultured neonatal rat cardiomyocytes. The culture medium of strain 293 cells transferred by pPS-3-neo (brd) produces a positive chronotropic effect that is directly related to the time parameters of preparation of recombinant bradykinin, which are comparable with the curve of chronotropic effect of synthetic bradykinin at concentrations of 10(-17) to 10(-16) M. The control of bradykinin "gene" expression was due to the lack of chronotropic responses of cardiomyocytes to the kinin receptor blocker parmidine and the transfection of strain 293 cells with the retrovirus vector without bradykinin "gene".