Identification of Novel Interaction Partners of AIF Protein on the Outer Mitochondrial Membrane.

In response to the wide variety of external and internal signals, mammalian cells undergo apoptosis, programmed cell death. Dysregulation of apoptosis is involved in multiple human diseases, including cancer, autoimmunity, and ischemic injuries. Two types of apoptosis have been described: the caspase-dependent one, leading to digestion of cellular proteins, and caspase-independent apoptosis, resulting in DNA fragmentation. The latter type of apoptosis is executed by AIF protein and is believed to have appeared first during evolution. The key step in the caspase-independent apoptosis program is the dissociation of AIF from the outer mitochondrial membrane (OMM). However, the molecular mechanism of interaction between AIF and OMM remains poorly understood. In this study, we demonstrated that AIF can bind to OMM via mortalin protein. We confirmed interaction between AIF and mortalin both in vitro and in vivo and mapped the amino acid sequences that are important for the binding of these proteins. Next, we showed that apoptosis induction by chemotherapy leads to downregulation of AIF-mortalin interaction and dissociation of AIF from the OMM. Finally, a bioinformatic analysis demonstrated that a high level of mortalin expression correlates with a worse survival prognosis for glioma patients. Altogether, our data revealed that mortalin plays an important role in the regulation of the caspase-independent apoptotic pathway and allowed us to speculate that inhibition of AIF-mortalin interaction may induce a dissociation of AIF from the OMM and subsequent apoptosis of cancer cells.

Modern Approaches for Identification of Modified Nucleotides in RNA.

This review considers approaches for detection of modified monomers in the RNA structure of living organisms. Recently, some data on dynamic alterations in the pool of modifications of the key RNA species that depend on external factors affecting the cells and physiological conditions of the whole organism have been accumulated. The recent studies have presented experimental data on relationship between the mechanisms of formation of modified/minor nucleotides of RNA in mammalian cells and the development of various pathologies. The development of novel methods for detection of chemical modifications of RNA nucleotides in the cells of living organisms and accumulation of knowledge on the contribution of modified monomers to metabolism and functioning of individual RNA species establish the basis for creation of novel diagnostic and therapeutic approaches. This review includes a short description of routine methods for determination of modified nucleotides in RNA and considers in detail modern approaches that enable not only detection but also quantitative assessment of the modification level of various nucleotides in individual RNA species.

Modified Method of rRNA Structure Analysis Reveals Novel Characteristics of Box C/D RNA Analogues.

Ribosomal RNA (rRNA) maturation is a complex process that involves chemical modifications of the bases or sugar residues of specific nucleotides. One of the most abundant types of rRNA modifications, ribose 2'-O-methylation, is guided by ribonucleoprotein complexes containing small nucleolar box C/D RNAs. Since the majority of 2'-O-methylated nucleotides are located in the most conserved regions of rRNA that comprise functionally important centers of the ribosome, an alteration in a 2'-O-methylation profile can affect ribosome assembly and function. One of the key approaches for localization of 2'-O-methylated nucleotides in long RNAs is a method based on the termination of reverse transcription. The current study presents an adaptation of this method for the use of fluorescently labeled primers and analysis of termination products by capillary gel electrophoresis on an automated genetic analyzer. The developed approach allowed us to analyze the influence of the synthetic analogues of box C/D RNAs on post-transcriptional modifications of human 28S rRNA in MCF-7 cells. It has been established that the transfection of MCF-7 cells with a box C/D RNA analogue leads to an enhanced modification level of certain native sites of 2'-O-methylation in the target rRNA. The observed effect of synthetic RNAs on the 2'-O-methylation of rRNA in human cells demonstrates a path towards targeted regulation of rRNA post-transcriptional maturation. The described approach can be applied in the development of novel diagnostic methods for detecting diseases in humans.

[The influence of recombinant nucleophosmin 1 on artificial RNA internalization into human adenocarcinoma MCF-7 cells].

In this study we obtained and characterized the recombinant analogue of multifunctional nucleolar phosphoprotein nucleophosmin 1 (NPM1) involved in crucial cellular processes such as transcription, reparation and mitosis. The influence ofnucleophosmin 1 on extrcellular RNAs accumulation in human adenocarcinoma cells MCF-7 was analyzed. It was found that incubation of AluY RNA (n > 300 nt), U24 snoRNA analogues (n ~ 80 nt) with Npm1-His6 resulted in RNA-protein non-covalent complexes formation, but not in case of the short oligoribonucleotide (n = 22 nt). It was shown that interaction of AluY RNA analogue with Npm1-His6 significantly increases transfection efficacy of the RNA into MCF-7 human cells. Altogether, these data allow us to conclude, that nucleophosmin 1 not only binds RNA with complex secondary structure, but also promotes uptake and internalization of such RNA by human cells.

Analogues of Artificial Human Box C/D Small Nucleolar RNA As Regulators of Alternative Splicing of a pre-mRNA Target.

Small nucleolar RNAs (snoRNAs) play a key role in ribosomal RNA (rRNA) biogenesis. Box C/D snoRNAs guide the site-specific 2'-O-ribose methylation of nucleotides in rRNAs and small nuclear RNAs (snRNAs). A number of box C/D snoRNAs and their fragments have recently been reported to regulate post-transcriptional modifications and the alternative splicing of pre-mRNA. Artificial analogues of U24 snoRNAs directed to nucleotides in 28S and 18S rRNAs, as well as pre-mRNAs and mature mRNAs of human heat shock cognate protein (hsc70), were designed and synthesized in this study. It was found that after the transfection of MCF-7 human cells with artificial box C/D RNAs in complex with lipofectamine, snoRNA analogues penetrated into cells and accumulated in the cytoplasm and nucleus. It was demonstrated that the transfection of cultured human cells with artificial box C/D snoRNA targeted to pre-mRNAs induce partial splicing impairments. It was found that transfection with artificial snoRNAs directed to 18S and 28S rRNA nucleotides, significant for ribosome functioning, induce a decrease in MCF-7 cell viability.

[New conceptions in complex management of spine trauma patients including reconstructive microsurgery and succeeding rehabilitation].

This work is been based upon the experience of performing 100 reconstructive microsurgical operations with transplantation of combined vascular-neural autografts, in 24 cases--with introduction cellular material into zone of transplantation. The complex approach aimed to creating optimal conditions for the functional regeneration of the spinal cord (SC), including neurosurgical methods restoring anatomical integrity of the organ and also multicomponent and staging rehabilitation of patients has been developed. On the base of complex approach during a postoperative period there has been put synchronous multifactorial action, consisting of functional multichannel electrical pacing of muscles simultaneously participating in tumble. The patient concentrates all his attention on the trial to tumble with paralized extremities by himself. At that the series and the site of application of these electrical impulses forms the algorithm of this tumble. Synchronism of produced afferent and efferent impulses creates new possibilities in activating of regenerating processes in the damaged area of the spinal cord. As a result of treatment within the bounds of the developed program the authors get clinically and electrophysiologically proved certain improvement that they can not only predict but guarantee patients of this group. Presented here results of the complex treatment of patients with trauma of the spinal cord within the framework of research international program confidently testify advantages of the new approach to solving of this problem. For the first time in history of medicine there is created close cooperation and regular continuity between molecular biology, neurosurgery and rehabilitation. This scientific alliance permits in proper time to keep a close watch, quickly correct and improve every stage of treatment. The effect of implement of innovations is been summarized at the each next stage of proposed complex treatment and eventually significantly elevates an ultimate result of medical rehabilitation of invalids with trauma of the spinal cord.

Human fetal neural stem cells in rat brain: effects of preculturing and transplantation.

The fate of human fetal stem/progenitor cells transplanted into rat brain depends on conditions of preculturing (long or short) and state and site of transplantation. Human nestin-positive stem cells cultured according to the short protocol did not migrate into hypoxic and normal brain after transplantation, but actively migrated in damaged spinal cord. After transplantation of long-cultured cells into the brain mainly committed neuroblasts and solitary nestin-positive cells migrated from the site of transplantation into the brain.

Immunohistochemical study of fetal stem/progenitor cells from human brain transplanted into traumatized spinal cord of adult rats.

Neural stem/progenitor cells from human fetal brain were grown in a tissue culture and transplanted into traumatized spinal cord of adult rats. The behavior and differentiation of transplanted cells were studied morphologically by means of histological and immunohistochemical methods and confocal microscopy. Human neural stem/progenitor cells were viable for not less than 3 months. They migrated and differentiated into neurons and glia in the traumatized spinal cord of adult rats.

Xenotransplantation of stem/progenitor cells from human fetal brain to adult rats with spinal trauma.

In vitro grown neural stem cells from human fetal brain were transplanted to adult rats with spinal trauma. The spinal cord was examined morphologically using histological and immunohistochemical methods on days 5, 15, 30, and 110. Human neural stem/progenitor cells were viable, migrated, and differentiated into neurons and glia in the traumatized spinal cord in adult rats.

[Methods of detection and characteristics of clinical manifestations of tuberculosis in children and adolescents]. / Metody vyiavleniia i kharakteristika klinicheskikh proiavlenii tuberkuleznoi infektsii u detei i podrostkov.

Methods of detection of tuberculous infection in 127 children and adolescents treated in special hospitals and sanatoria have seen analyzed. The diagnosis was made using tuberculin in 62.2%, epidemiological evidence in 12.6%, fluorographic findings in 7.8%, survey of risk groups in 7.1%. 10.2% of the patients sought medical advice. A favourable course of the disease was seen in cases detected at tuberculin diagnosis and risk group examination. Complications occurred primarily in those who sought medical advice.