[Clinical efficacy and safety of Ethoxidol in cerebrovascular diseases]. / Klinicheskaya effektivnost' i bezopasnost' Etoksidola pri tserebrovaskulyarnykh zabolevaniyakh.

OBJECTIVE: Evaluation of efficacy and clinical safety of ethylmethylhydroxypyridine malate (Ethoxidol) in cerebrovascular disease in out-patient, and in-patient setting. MATERIAL AND METHODS: In the observational study was included of the 70 patients (aged 62.84±6.54 y.o) diagnosed with unspecified cerebrovascular disease, hypertensive encephalopathy, atherosclerosis of cerebral vessels. To access neurological deficit Montreal Scale (MoCA test) was used, MFI-20 test for asthenia. Berg balancing (BBS-test), tinnitus severity score (THI), and general clinical condition score also were recorded. Quality assurance was evaluated by VAS, and EQ-VAS Scores (European life quality gr). Interventions were identical, except treated received Ethoxidol 200 mg IV once/day, or 200 mg IM once/day and 200 mg per os (400 mg/day) during 7 days, follow up by oral administration of Ethoxidol 600 mg/day (200 mg TID) for the period of 53 days. RESULTS: The results of the observational study has shown of high clinical efficacy and tolerability of the Ethoxidol therapy. Statistical significance between baseline and treatment period was recorded on the 7th and 60th day of observation (p<0.001). Stabilization of the archived condition persisted on the 60th day. Ethoxidol administration reduced asthenia cognitive dysfunction, dizziness, balance disorders, and tinnitus. CGI Score revealed reduction of the severity of patient's condition and total of clinical improvement; EQ-VAS Score showed growth of quality of life. The majority of patients were satisfied with performed therapy, attending physicians highlight Ethoxidol safety. Serious adverse events weren't recorded. CONCLUSION: Ethoxidol was considered as efficient and safe medication for in-patient and out-patient treatment of patient with cerebrovascular diseases (chronic cerebral ischemia).

[Efficacy of Mexidol in patients with type 2 diabetes mellitus with neurological complications]. / Effektivnost' preparata Meksidol u patsientov s nevrologicheskimi oslozhneniyami sakharnogo diabeta 2-go tipa.

OBJECTIVE: Complications of Diabetes mellitus (DM), depends on the severity of hypoxic tissue damage. Amelioration of hypoxic injury may improve current therapy approaches and enhance the quality of life in patient's cohort suffered from DM. MATERIAL AND METHODS: 30 pts which DM were enrolled into the study. Mexidol forte, was given IV 500 mg/day for 14 days, follow up with oral administration 250 TID. Clinical impression, neuropsychological scales and biochemical blood tests were recorded. RESULTS: Mexidol administration improved cognitive functions, decreased the level of asthenia, depressia, improved sleep disturbances, and normalized biochemical landscape. CONCLUSIONS: Mexidol administration for the period 75 days may serve as important therapeutic supplementation and considered as adjunctive therapy in the treatment of patients with DM.

[Diagnosis and treatment of neurogenic dysphagia after acute ischemic stroke]. / Diagnostika i lechenie neirogennoi disfagii u bol'nykh, perenesshikh ostryi karotidnyi ishemicheskii insul't.

AIM: To determine the efficacy of post-stroke dysphagia treatment by choline alfoscerate (ChA), succinate combination (SC), and their combination with sip, larynx, and swallowing exercises. MATERIAL AND METHODS: Four groups of primary ischemic stroke (IS) patients (n=80; 62±0.2 y.o., verified by MRI), including controls, admitted to Stroke Unit 24 h after stroke in the area of RAM (29.5%), and LAM (70.5%), were studied. Basic therapy was provided according to National Stroke Treatment Recommendations, treated groups received ChA 14 mg/kg (2st gr.), SC 0.5 mg/kg (3nd gr.), combination of two compounds (3d gr.). Controls (4th gr.) received placebo. Pharmacological treatment was provided for 10 days with continuation by oral administration. Dysphagia was measured semi-quantitively by MASA scale, three scale determinants were measured on admission, on 5 and 13 days of stay in the hospital. RESULTS AND CONCLUSION: The differences were significant and observed on the 5th day of treatment. ChA mostly improved sip control, and larynx movements (38% above controls; p<0.01), while SC improved the closure of vocal cords (55% above controls; p<0.01). This may reflect the differences in synaptic control of these functions. Combined treatment was more effective than monotherapy: 15% above ChA, and 21% above SC for swallowing function (p=0.01); 33 and 22% for vocal closure, 37% (p=0.05) and 76% (p=0.01) for larynx movement, which may be due to synergism between two medications. Therefore, sip, larynx, and swallowing exercises with pharmacological support of ChA and SC ameliorated dysphagia after IS.

Kruppel-like factor 4 signals through microRNA-206 to promote tumor initiation and cell survival.

Tumor cell heterogeneity poses a major hurdle in the treatment of cancer. Mammary cancer stem-like cells (MaCSCs), or tumor-initiating cells, are highly tumorigenic sub-populations that have the potential to self-renew and to differentiate. These cells are clinically important, as they display therapeutic resistance and may contribute to treatment failure and recurrence, but the signaling axes relevant to the tumorigenic phenotype are poorly defined. The zinc-finger transcription factor Kruppel-like factor 4 (KLF4) is a pluripotency mediator that is enriched in MaCSCs. KLF4 promotes RAS-extracellular signal-regulated kinase pathway activity and tumor cell survival in triple-negative breast cancer (TNBC) cells. In this study, we found that both KLF4 and a downstream effector, microRNA-206 (miR-206), are selectively enriched in the MaCSC fractions of cultured human TNBC cell lines, as well as in the aldehyde dehydrogenase-high MaCSC sub-population of cells derived from xenografted human mammary carcinomas. The suppression of endogenous KLF4 or miR-206 activities abrogated cell survival and in vivo tumor initiation, despite having only subtle effects on MaCSC abundance. Using a combinatorial approach that included in silico as well as loss- and gain-of-function in vitro assays, we identified miR-206-mediated repression of the pro-apoptotic molecules programmed cell death 4 (PDCD4) and connexin 43 (CX43/GJA1). Depletion of either of these two miR-206-regulated transcripts promoted resistance to anoikis, a prominent feature of CSCs, but did not consistently alter MaCSC abundance. Consistent with increased levels of miR-206 in MaCSCs, the expression of both PDCD4 and CX43 was suppressed in these cells relative to control cells. These results identify miR-206 as an effector of KLF4-mediated prosurvival signaling in MaCSCs through repression of PDCD4 and CX43. Consequently, our study suggests that a pluripotency factor exerts prosurvival signaling in MaCSCs, and that antagonism of KLF4-miR-206 signaling may selectively target the MaCSC niche in TNBC.

NEDD9/Arf6-dependent endocytic trafficking of matrix metalloproteinase 14: a novel mechanism for blocking mesenchymal cell invasion and metastasis of breast cancer.

NEDD9 is an established marker of invasive and metastatic cancers. NEDD9 downregulation has been shown to dramatically reduce cell invasion and metastasis in multiple tumors. The mechanisms by which NEDD9 regulates invasion are largely unknown. In the current study, we have found that NEDD9 is required for matrix metalloproteinase 14 (MMP14) enzymatic recovery/recycling through the late endosomes to enable disengagement of tissue inhibitor of matrix metalloproteinase 2 (TIMP2) and tumor invasion. Depletion of NEDD9 decreases targeting of the MMP14/TIMP2 complex to late endosomes and increases trafficking of MMP14 from early/sorting endosomes back to the surface in a small GTPase ADP ribosylation factor-6 (Arf6)-dependent manner. NEDD9 directly binds to Arf6-GTPase-activating protein, ARAP3 and Arf6-effector GGA3, thereby facilitating the Arf6 inactivation required for MMP14/TIMP2 targeting to late endosomes. Re-expression of NEDD9 or a decrease in Arf6 activity is sufficient to restore MMP14 activity and the invasive properties of tumor cells. Importantly, NEDD9 inhibition by Vivo-Morpholinos, an antisense therapy, decreases primary tumor growth and metastasis in xenograft models of breast cancer. Collectively, our findings uncover a novel mechanism to control tumor-cell dissemination through NEDD9/Arf6-dependent regulation of MMP14/TIMP2 trafficking, and validate NEDD9 as a clinically relevant therapeutic target to treat metastatic cancer.

NORTHERN LEAF BLIGHT HELMINTHOSPORIUM TURCICUM ON MAIZE IN LATVIA.

In Latvia, climatic factors are influential in spreading of the Northern leaf blight of maize caused by Setosphaeria turcica (SETOTU, anamorph Exserohilum turcicum, Helminthosporium turcicum). The field experiments with the aim to investigate the effect of strobilurines to control of SETOTU in maize and the possibility to have a greening effect on yield and the silage quality parameters there were conducted in Latvian Plant Protection Research Centre in 2010. The effect of fungicides under natural and artificial infection with SETOTU on yield of maize was evaluated in 2012 and 2013. Trials designed with four replicates using randomized blocks, a plot size of 30 m2. Plots were inoculated at beginning of anthesis stage of maize by conidium of local population of H. turcicum propagated on PDA. Application of fungicides has been done in two times. Weather conditions were favourable for infestation of Northern leaf blight in maize. Disease severity was recorded according to the EPPO Guideline PP 1/272(1) on 10 plants from two central rows by 5 layers of leaves. Yield was recorded from two central separately harvested rows of each plot. The silage quality parameters in 2010 were analysed by Blgg BV Company, Netherlands. After the artificial inoculation an increase of the disease pressure in maize was observed. A good effect of the fungicides to control SETOTU was observed in all trials. No significant differences in efficacy were found between the treatments of Propulse (fluopyram 125 g L(-1), prothioconazole 125 g L(-1)), Opera (epoxiconazole 75, pyraclostrobin 199, 5 g L(-1) and Opera N (epoxiconazole 75, pyraclostrobin 102 g L(-1)). The effect of application time at the BBCH 55-59 was higher compared with application time at the BBCH 30-37. Two applications of Propulse compared with the single showed higher effect on SETOTU. The prolonged effect of Propulse on SETOTU was similar to Opera and Opera N. Greening effect was significant for all treatments compared with the untreated. In all treatments the increase of yield of fresh and dry mass was significant to the untreated. The positive influence of Opera N on the feed milk unit (VEM), protein (DVE, VOS, RP), digest OM (VCOS), starch, FAT content, NEL, energy metabolite (ME), nXP, UDP was recorded. The treatment of Opera N showed a tendency to decrease the silage quality parameters such as fatty acid vola (FOS), crude fiber (RF), crude ASH (RA), sugar content, acid det. fiber (ADF), acid det. lignin (ADL).

[Efficacy of the complex drug cytoflavin in the treatment of consequences of mild brain injury].

Sixty outpatients, aged 18-50 years, with mild cranial-brain trauma (brain concussion, mild brain injury), occurred 21-180 days before the enrollment in the study, were examined. Patients of the main group received cytoflavin in dose 425 mg, 2 tablets twice a day during 25 days, patients of the control group received aminalon in dose 500 mg, 2 tablets 3 times a day during 25 days. The therapeutic efficacy was assessed on days 1, 30 and 60 with the battery of neuropsychological scales. The efficacy and safety of cytoflavin in the monotherapy of patients with remote consequences of mild cranial-brain trauma was shown. The effect of cytoflavin was developed significantly more rapidly compared to aminalon. There were positive changes on scales of pain severity, psychoemotional disorders (anxiety, depression, asthenia), sleep quality, autonomic dysfunctions as well as in the performance on neurocognitive tests assessing memory, sustained attention, information processing speed, productivity. The duration of using analgesics and sedatives as add-on drugs was reduced significantly. The drug remained effective till the 60th day after the 30 day withdrawal. Side-effects of cytoflavin (the short-term rise of arterial pressure, insomnia and abdominalgia) did not last long and no additional treatment, withdrawal or reduction of cytoflavin dose was needed.

HEF1 is a necessary and specific downstream effector of FAK that promotes the migration of glioblastoma cells.

The highly invasive behavior of glioblastoma cells contributes to the morbidity and mortality associated with these tumors. The integrin-mediated adhesion and migration of glioblastoma cells on brain matrix proteins is enhanced by stimulation with growth factors, including platelet-derived growth factor (PDGF). As focal adhesion kinase (FAK), a nonreceptor cytoplasmic tyrosine kinase, has been shown to promote cell migration in various other cell types, we analysed its role in glioblastoma cell migration. Forced overexpression of FAK in serum-starved glioblastoma cells plated on recombinant (rec)-osteopontin resulted in a twofold enhancement of basal migration and a ninefold enhancement of PDGF-BB-stimulated migration. Both expression of mutant FAK(397F) and the downregulation of FAK with small interfering (si) RNA inhibited basal and PDGF-stimulated migration. FAK overexpression and PDGF stimulation was found to increase the phosphorylation of the Crk-associated substrate (CAS) family member human enhancer of filamentation 1 (HEF1), but not p130CAS or Src-interacting protein (Sin)/Efs, although the levels of expression of these proteins was similar. Moreover downregulation of HEF1 with siRNA, but not p130CAS, inhibited basal and PDGF-stimulated migration. The phosphorylated HEF1 colocalized with vinculin and was associated almost exclusively with 0.1% Triton X-100 insoluble material, consistent with its signaling at focal adhesions. FAK overexpression promoted invasion through normal brain homogenate and siHEF1 inhibited this invasion. Results presented here suggest that HEF1 acts as a necessary and specific downstream effector of FAK in the invasive behavior of glioblastoma cells and may be an effective target for treatment of these tumors.

[Influence of smoking on the cerebral hemodynamics and biochemical blood indices in chronic insufficiency of brain circulation]. / Vliianie kureniia na tserebral'nuiu gemodinamiku i biokhimicheskie pokazateli krovi pri khronicheskoi nedostatochnosti mozgovogo krovoobrashcheniia.

The influence of smoking on cerebral hemodynamics and biochemical blood indices has been studied in 50 male patients with chronic insufficiency of brain circulation (CIBC), aged 40-50 years, divided into 2 groups: smoking (n=26) and nonsmoking (n=24). Smoking was shown to play a substantial role in the development of discirculatory encephalopathy with atrophic brain changes, causing metabolic disturbances (a shift of acid-basic balance towards acidosis) and microcirculation disorders due to altered cerebrovascular reactivity. These alterations develop previously to hemodynamically significant atherosclerotic arteries lesion and emerge irrespective of the presence of atherosclerotic vascular changes, atherogenic shifts of lipid metabolism, disturbances of free-radical processes and platelet aggregation.

Cell growth inhibition by the multifunctional multivalent zinc-finger factor CTCF.

The 11-zinc finger protein CCTC-binding factor (CTCF) employs different sets of zinc fingers to form distinct complexes with varying CTCF- target sequences (CTSs) that mediate the repression or activation of gene expression and the creation of hormone-responsive gene silencers and of diverse vertebrate enhancer-blocking elements (chromatin insulators). To determine how these varying effects would integrate in vivo, we engineered a variety of expression systems to study effects of CTCF on cell growth. Here we show that ectopic expression of CTCF in many cell types inhibits cell clonogenicity by causing profound growth retardation without apoptosis. In asynchronous cultures, the cell-cycle profile of CTCF-expressing cells remained unaltered, which suggested that progression through the cycle was slowed at multiple points. Although conditionally induced CTCF caused the S-phase block, CTCF can also arrest cell division. Viable CTCF-expressing cells could be maintained without dividing for several days. While MYC is the well-characterized CTCF target, the inhibitory effects of CTCF on cell growth could not be ascribed solely to repression of MYC, suggesting that additional CTS-driven genes involved in growth-regulatory circuits, such as p19ARF, are likely to contribute to CTCF-induced growth arrest. These findings indicate that CTCF may regulate cell-cycle progression at multiple steps within the cycle, and add to the growing evidence for the function of CTCF as a tumor suppressor gene.

Functional phosphorylation sites in the C-terminal region of the multivalent multifunctional transcriptional factor CTCF.

CTCF is a widely expressed and highly conserved multi-Zn-finger (ZF) nuclear factor. Binding to various CTCF target sites (CTSs) is mediated by combinatorial contributions of different ZFs. Different CTSs mediate distinct CTCF functions in transcriptional regulation, including promoter repression or activation and hormone-responsive gene silencing. In addition, the necessary and sufficient core sequences of diverse enhancer-blocking (insulator) elements, including CpG methylation-sensitive ones, have recently been pinpointed to CTSs. To determine whether a posttranslational modification may modulate CTCF functions, we studied CTCF phosphorylation. We demonstrated that most of the modifications that occur at the carboxy terminus in vivo can be reproduced in vitro with casein kinase II (CKII). Major modification sites map to four serines within the S(604)KKEDS(609)S(610)DS(612)E motif that is highly conserved in vertebrates. Specific mutations of these serines abrogate phosphorylation of CTCF in vivo and CKII-induced phosphorylation in vitro. In addition, we showed that completely preventing phosphorylation by substituting all serines within this site resulted in markedly enhanced repression of the CTS-bearing vertebrate c-myc promoters, but did not alter CTCF nuclear localization or in vitro DNA-binding characteristics assayed with c-myc CTSs. Moreover, these substitutions manifested a profound effect on negative cell growth regulation by wild-type CTCF. CKII may thus be responsible for attenuation of CTCF activity, either acting on its own or by providing the signal for phosphorylation by other kinases and for CTCF-interacting protein partners.

Signal transduction driving technology driving signal transduction: factors in the design of targeted therapies.

A significant number of human diseases can be attributed to defects in cellular signal transduction pathways. Large-scale proteomics projects now in progress seek to better define critical components of signal transduction networks, to enable more intelligent design of therapeutic agents that can specifically correct disease-specific signaling alterations by targeting individual proteins. A complicating factor in this endeavor is the fact that intracellular signaling involves many diverse mechanisms that in sum finely modulate the activity of individual proteins in response to different biological inputs. Ability to develop reagents that selectively correct disease-associated signaling activities, while leaving intact benign or essential activities, encompassed within a single protein requires an intimate knowledge of pathway-specific control mechanisms. To illustrate these points, we provide examples of some of the complex control mechanisms regulating the Cas proteins, which contribute to integrin-dependent biological response. We then discuss issues involved in systematically incorporating information related to complex control mechanisms in proteomic databases. Finally, we describe some recent instances in which protein interaction technologies have been specifically adapted to identify small molecule agents that regulate protein response in physiologically desirable ways, and discuss issues relevant to future drug discovery efforts.

Functional association of CTCF with the insulator upstream of the H19 gene is parent of origin-specific and methylation-sensitive.

In mammals, a subset of genes inherit gametic marks that establish parent of origin-dependent expression patterns in the soma ([1] and references therein). The currently most extensively studied examples of this phenomenon, termed genomic imprinting, are the physically linked Igf2 (insulin-like growth factor II) and H19 genes, which are expressed mono-allelically from opposite parental alleles [1] [2]. The repressed status of the maternal Igf2 allele is due to cis elements that prevent the H19 enhancers [3] from accessing the Igf2 promoters on the maternal chromosome [4] [5]. A differentially methylated domain (DMD) in the 5' flank of H19 is maintained paternally methylated and maternally unmethylated [6] [7]. We show here by gel-shift and chromatin immunopurification analyses that binding of the highly conserved multivalent factor CTCF ([8] [9] and references therein) to the H19 DMD is methylation-sensitive and parent of origin-dependent. Selectively mutating CTCF-contacting nucleotides, which were identified by methylation interference within the extended binding sites initially revealed by nuclease footprinting, abrogated the H19 DMD enhancer-blocking property. These observations suggest that molecular mechanisms of genomic imprinting may use an unusual ability of CTCF to interact with a diverse spectrum of variant target sites, some of which include CpGs that are responsible for methylation-sensitive CTCF binding in vitro and in vivo.

Comparative allelotyping of the short arm of human chromosome 3 in epithelial tumors of four different types.

Comparative allelotyping of the short arm of human chromosome 3 (3p) in four types of epithelial carcinomas was performed using an identical set of polymorphic markers. In total, 117 samples of non-papillary renal cell carcinoma (RCC), non-small cell lung carcinoma (NSCLC), carcinoma of uterine cervix (CC), and breast carcinoma (BC) were screened for loss of heterozygosity (LOH) with 10 di-, tri- and tetrameric markers covering nine bands of 3p. High LOH frequencies were detected in at least one locus: RCC (36/43, 84%), BC (20/26, 77%), NSCLC (16/24, 67%), and CC (15/24, 62%). Small interstitial deletions prevailed in BC and CC whereas large continuous and discontinuous deletions were mainly found in RCC and NSCLC. Different epithelial tumors displayed unique LOH profiles with partial overlaps in 3p26.1, 3p21.31, and 3p13. The overlap around D3S2409 (3p21.31) appeared common for RCC, BC and CC.

[p53 with a mutation in codon 273 increases the probability of amplifying the dhfr gene in Rat-1 and LIM1215 cells]. / p53 s mutatsiei v kodone 273 uvelichivaet veroiatnost' amplifikatsii gena dhfr v kletkakh Rat-1 i LIM1215.

The effect of the expression of the exogenous human mutant p53 (Arg-->His in codon 273) on the amplification rate of the gene dhfr in permissive Rat-1 and LIM1215 cells was studied. It was shown that injection of a retroviral construction with p53His273 resulted in the accumulation of methotrexate-resistant variants with an increased number of dhfr copies in populations of recipient cells. Luria-Delbruck fluctuation analysis revealed a four- to six-fold increase in the rate of appearance of new methotrexate-resistant cells. Chromosomal analysis demonstrated an extrachromosomal location of amplified DNA in cells containing p53His273, as was the case for control sublines. The data obtained indicate that modifications of p53 may induce gene amplification not only via removing the proliferation block of cells with amplified genes in selective medium, but also via some other mechanisms, that seem to increase the chromosomal recombination rate.

Influence of exogenous ras and p53 on P-glycoprotein function in immortalized rodent fibroblasts.

The ability of ras oncogenes and mutant p53 to activate reporter gene expression from human and rodent mdr1 gene promoters was described, although functional significance of this finding was unclear. We analyzed the influence of various forms of recombinant human ras and p53 on the mdr1 gene expression and P-glycoprotein (Pgp) function in rodent immortalized fibroblasts. The ras genes, in addition to activation of exogenous human mdr1 gene promoter, caused an increase in (i) expression of endogenous mdr1 mRNA, (ii) Pgp activity as determined by flow cytometry analysis of Rhodamine 123 exclusion, and (iii) resistance of cells to the cytotoxic action of colchicine and some other drugs. To elucidate whether the same signalling pathway is responsible for multidrug resistance induced by various oncogenes and protein kinase C (PKC), we tested the effects of v-mos and the PKC agonist 12-O-tetradecanoylphorbol-13-acetate. Similarly to cells transformed by ras, a Rat1 subline transformed by the v-mos oncogene was characterized by decreased drug sensitivity. On the contrary, Rat1 cells treated with the protein kinase C agonist 12-O-tetradecanoylphorbol-13-acetate showed neither increased mdr1 mRNA expression nor stimulation of Pgp function. Introduction by retrovirus-mediated gene transfer of wild-type p53 into Rat1 cells or into murine p53-deficient 10(1) and 10(3) cells did not change the Pgp function significantly, whereas in Rat1 cells transformed by activated N-ras or v-mos, expression of wild-type p53 caused partial reversion of oncogene-induced drug resistance.(ABSTRACT TRUNCATED AT 250 WORDS)