[Prospective assessment of a training effect in correction of endothelial dysfunction and atherosclerosis risk factors in students (results of examination of Tomsk male students)].

AIM: To assess a training effect correcting endothelial dysfunction (ED), neurovegetative status and atherosclerosis risk factors (ARF) in male students. MATERIAL AND METHODS: Ninety healthy students aged 18-25 years with ARF and ED were followed up after bicycle exercise (BE) 3 times a week: 30 males did BE for 1 month, 30--for 2 months and 30--for 3 months. Blood lipids, exercise tolerance (ET), parameters of cardiointervalography, dopplerangiography of the brachial artery and rheovasography of the forearm at rest and in tests with reactive hyperemia, hyperventilation were estimated immediately after, 1 and 3 months after BE. RESULTS: Two-month BE forms adequate structural trace the effect of which can be partially found 3 months later. Three-month BE tells on adaptation as it produces proatherogenic shifts in blood lipids. After the two-month cycle of BE positive effects regress in the following order: normalization of blood lipids, improvement of vegetovascular reactivity, high exercise tolerance and endothelium-related vasodilation. CONCLUSION: Two-month exercise is effective and safe, forms adaptive reserve of the body, structural effect of which partially persists for 3 months.

[Preclinical assessment of disturbances of vascular and vegetative reactivity of peripheral blood circulation in young men (students) with risk factors of atherosclerosis].

Hemodynamics of forearm and neurovegetative status were studied in 142 clinically healthy male students with risk factors of atherosclerosis and in 54 male students without risk factors of atherosclerosis at rest and during test with reactive hyperemia. Rheovasography of forearm, cardiointervalography, Doppler examination of brachial artery, and test with reactive hyperemia were undertaken in all subjects. The results testify to the fact that endothelial dysfunction leads to spastic state of regional hemodynamics and hyperactivity of sympathetic nervous system at rest as well as during reactive hyperemia which has a "delayed" character.

[The influence of hyperventilation in combination with active orthostasis on the condition of vegetative homeostasis in young men with atherosclerosis risk factors and endothelial dysfunction].

The aim of the study was to evaluate the effects of hyperventilation test (HVT), active orthostatic position test (AOPT), and their combination on the vegetative homeostasis in male students with risk factors of atherosclerosis (RFA) and endothelial dysfunction (ED). The mentioned tests and their combination were performed in 142 male students with RFA and ED, and 54 male students without them during brachial arterial rheovasography and cardiointervalography. Hyperactivation of the central regulatory circuit and a spastic hemodynamic condition at rest were found in subjects with FRA and ED. The sympathetic functional reserve was overstrained according to AOPT, distinctly decreased according to HVT, and exhausted when both tests were performed. The combination of HVT and AOPT is an informative stress test for pre-clinical evaluation of disturbances in adaptation mechanisms and vegetative homeostasis tensity in young men with RFA and ED. A "high value" of adaptation and the vegetative homeostasis overstrain in male students with FRA and ED indicate a premorbid condition in them.

Drosha in primary microRNA processing.

MicroRNA (miRNA)-mediated gene silencing is one of the major regulatory pathways in eukaryotes. Much effort has been made to identify the factors involved in the pathway, and our understanding of RNA silencing has significantly advanced in recent years. Our group has been working on some of the issues regarding miRNA biogenesis and, in this paper, we summarize what we and other workers in the field have learned thus far. The focus remains on the role of Drosha and DGCR8 in the early events of miRNA biogenesis in animals.

[Comparison of the results of functional tests used in non-invasive examination of endothelial function]. / Sopostavlenie rezul'tatov funktsional'nykh prob, ispol'zuiushchikhsia v neinvasivnoi otsenke funktsii éndoteliia.

AIM: To analyze results of two tests used in non-invasive assessment of endothelial function: D. Celermajer test (T1) and reactive hyperemia test (T2). MATERIAL AND METHODS: Ultrasound investigation (7.5 MHz) was employed to measure brachial artery (BA) diameter, blood flow rate and its volume at rest above the site of occlusion (T1) and under it (T2). Control group consisted of 30 men with risk factors for atherosclerosis. RESULTS: Peak value of the diameter dilation was within 10% in both tests, the difference being insignificant. The same dilatation of BA can be explained by different shift effect on the endothelium: in T1 the shift disturbance was longer while in T2 it was more intensive. CONCLUSION: To reveal endothelial dysfunction, the site of occlusion and location of the artery are not very important. However, because the shift disturbance time is different it is desirable to use the same test modification in one trial protocol.

Magoh, a human homolog of Drosophila mago nashi protein, is a component of the splicing-dependent exon-exon junction complex.

The RNA-binding protein Y14 binds preferentially to mRNAs produced by splicing and is a component of a multiprotein complex that assembles approximately 20 nucleotides upstream of exon-exon junctions. This complex probably has important functions in post-splicing events including nuclear export and nonsense-mediated decay of mRNA. We show that Y14 binds to two previously reported components, Aly/REF and RNPS1, and to the mRNA export factor TAP. Moreover, we identified magoh, a human homolog of the Drosophila mago nashi gene product, as a novel component of the complex. Magoh binds avidly and directly to Y14 and TAP, but not to other known components of the complex, and is found in Y14-containing mRNPs in vivo. Importantly, magoh also binds to mRNAs produced by splicing upstream (approximately 20 nucleotides) of exon- exon junctions and its binding to mRNA persists after export. These experiments thus reveal specific protein-protein interactions among the proteins of the splicing-dependent mRNP complex and suggest an important role for the highly evolutionarily conserved magoh protein in this complex.

Role of the nonsense-mediated decay factor hUpf3 in the splicing-dependent exon-exon junction complex.

Nonsense-mediated messenger RNA (mRNA) decay, or NMD, is a critical process of selective degradation of mRNAs that contain premature stop codons. NMD depends on both pre-mRNA splicing and translation, and it requires recognition of the position of stop codons relative to exon-exon junctions. A key factor in NMD is hUpf3, a mostly nuclear protein that shuttles between the nucleus and cytoplasm and interacts specifically with spliced mRNAs. We found that hUpf3 interacts with Y14, a component of post-splicing mRNA-protein (mRNP) complexes, and that hUpf3 is enriched in Y14-containing mRNP complexes. The mRNA export factors Aly/REF and TAP are also associated with nuclear hUpf3, indicating that hUpf3 is in mRNP complexes that are poised for nuclear export. Like Y14 and Aly/REF, hUpf3 binds to spliced mRNAs specifically ( approximately 20 nucleotides) upstream of exon-exon junctions. The splicing-dependent binding of hUpf3 to mRNAs before export, as part of the complex that assembles near exon-exon junctions, allows it to serve as a link between splicing and NMD in the cytoplasm.

Nuclear mRNA binding proteins couple pre-mRNA splicing and post-splicing events.

New information about the pathway of eukaryotic gene expression indicates that many of the steps in this pathway are functionally interconnected. An important link has recently emerged between pre-mRNA splicing and the post-splicing events such as mRNA export and mRNA decay. Recent results reveal that the coupling is mediated by a novel group of nuclear mRNA-binding proteins that are recruited to the mRNAs by spiceosome. These proteins, including Y14, Aly/REF, RNPS1, SRm160, and DEK, are assembled into a stable complex near exon-exon junctions of spliced mRNAs. Several of them persist in their attachment to mRNAs in the cytoplasm thus communicating the history of splicing to the downstream events. The detailed mechanism of coupling and the factors that mediate these processes remain to be determined in the coming years.

The Y14 protein communicates to the cytoplasm the position of exon-exon junctions.

We recently described an RNA-binding protein, Y14, that binds preferentially to spliced mRNAs and persists in the cytoplasm. Y14 is part of a multi-protein complex that also contains the mRNA export factor TAP. This suggests that splicing imprints the mRNA with a unique set of proteins that communicate the history of the transcript to the cytoplasm. Here, using microinjection of pre-mRNAs into Xenopus oocyte nuclei followed by immunoprecipitation of RNase-fragmented mRNAs from the cytoplasm, we show that Y14 is stably bound to sequences immediately upstream of exon-exon junctions. This feature appears to be unique to Y14. Using monoclonal antibodies that we produced against Aly/REF, another component recently reported to be an mRNA export factor, we show that Aly/REF is associated with spliced mRNAs in the nucleus but is not detectable on mRNAs in the cytoplasm. Thus, we propose that the splicing- dependent binding of Y14 provides a position-specific molecular memory that communicates to the cytoplasm the location of exon and intron boundaries. This novel mechanism may play an important role in post-splicing events.

Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm.

We describe a novel RNA binding protein, Y14, a predominantly nuclear nucleocytoplasmic shuttling protein. Interestingly, Y14 associates preferentially with mRNAs produced by splicing but not with pre-mRNAs, introns, or mRNAs produced from intronless cDNAs. Y14 associates with both nuclear mRNAs and newly exported cytoplasmic mRNAs. Splicing of a single intron is sufficient for Y14 association. Y14-containing nuclear complexes are different from general hnRNP complexes. They contain hnRNP proteins and several unique proteins including the mRNA export factor TAP. Thus, Y14 defines novel intermediates in the pathway of gene expression, postsplicing nuclear preexport mRNPs, and newly exported cytoplasmic mRNPs, whose composition is established by splicing. These findings suggest that pre-mRNA splicing imprints mRNA with a unique set of proteins that persists in the cytoplasm and thereby communicates the history of the transcript.

Growth factor enhanced retroviral gene transfer to the adult central nervous system.

The use of viral vectors for gene delivery into mammalian cells provides a new approach in the treatment of many human diseases. The first viral vector approved for human clinical trials was murine leukemia virus (MLV), which remains the most commonly used vector in clinical trials to date. However, the application of MLV vectors is limited since MLV requires cells to be actively dividing in order for transduction and therefore gene delivery to occur. This limitation precludes the use of MLV for delivering genes to the adult CNS, where very little cell division is occurring. However, we speculated that this inherent limitation of ML V may be overcome by utilizing the known mitogenic effect of growth factors on cells of the CNS. Specifically, an in vivo application of growth factor to the adult brain, if able to induce cell division, could enhance MLV-based gene transfer to the adult brain. We now show that an exogenous application of basic fibroblast growth factor induces cell division in vivo. Under these conditions, where cells of the adult brain are stimulated to divide, MLV-based gene transfer is significantly enhanced. This novel approach precludes any vector modifications and provides a simple and effective way of delivering genes to cells of the adult brain utilizing MLV-based retroviral vectors.

A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codon-optimized HIV-1 gag-pol gene.

The human immunodeficiency virus (HIV) genome is AU rich, and this imparts a codon bias that is quite different from the one used by human genes. The codon usage is particularly marked for the gag, pol, and env genes. Interestingly, the expression of these genes is dependent on the presence of the Rev/Rev-responsive element (RRE) regulatory system, even in contexts other than the HIV genome. The Rev dependency has been explained in part by the presence of RNA instability sequences residing in these coding regions. The requirement for Rev also places a limitation on the development of HIV-based vectors, because of the requirement to provide an accessory factor. We have now synthesized a complete codon-optimized HIV-1 gag-pol gene. We show that expression levels are high and that expression is Rev independent. This effect is due to an increase in the amount of gag-pol mRNA. Provision of the RRE in cis did not lower protein or RNA levels or stimulate a Rev response. Furthermore we have used this synthetic gag-pol gene to produce HIV vectors that now lack all of the accessory proteins. These vectors should now be safer than murine leukemia virus-based vectors.

Heterologous gene expression in avian cells: potential as a producer of recombinant proteins.

We have explored the possibility of using avian cells for the expression of human proteins. We found that various avian cells including quail fibrosarcoma cells (QF), duck embryo cells (DE) and primary chicken embryo fibroblasts (CE) could efficiently be transfected with DNA by calcium phosphate coprecipitation, and that promoters which are transcriptionally active in mammalian cells also functioned well in these avian cells. Among the promoters we tested, the major immediate early promoter of human cytomegalovirus drove the highest level of chloramphenicol acetyl transferase (CAT) expression, outperforming the SV40 early promoter and the RSV LTR. Using the bacterial CAT gene as a reporter, we found that levels of CAT activity were higher in QF and DE cells than in mammalian cells such as CHO, HeLa, Vero and 293T cells. We further cloned a sequence encoding human erythropoietin (EPO) and compared its expression in QF and mammalian cells. Consistent with the CAT data, in transient transfection assays, QF cells produced higher levels of EPO than the mammalian cell lines tested. QF cells which can be passaged permanently were stably transfected with an EPO expression vector. The subcloned QF line was able to produce up to 1,700 U/ml EPO from 3 x 10(6) cells in 72 h. Purified QF-produced EPO showed a broad but discrete protein band, ranging from 33 to 41 kD and was as biologically active as CHO-produced EPO. Although a number of factors still remain to be optimized, our results demonstrate the potential of avian cells such as QF as producers of heterologous proteins.

Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1.

The use of human immunodeficiency virus vectors for gene therapy is hampered by concern over their safety. This concern might be ameliorated, in part, if the viral accessory genes and proteins could be eliminated from the vector genomes and particles. Here we describe a minimal vector system that is capable of transducing nondividing cells and which does not contain tat, vif, vpr, vpu, and nef.

Essential role of NF-kappa B in transactivation of the human immunodeficiency virus long terminal repeat by the human cytomegalovirus 1E1 protein.

The 72 kDa 1E1 protein of human cytomegalovirus (HCMV) is one of a few viral regulatory proteins expressed immediately after infection of a host cell. Although it is now well-established that 1E1 is a potent transcriptional activator of the human immunodeficiency virus (HIV) long terminal repeat (LTR), the identity of the nucleotide sequence responsive to 1E1 remains elusive and the molecular mechanism of this interaction is not well-understood. We have constructed various LTR mutants and tested them for their ability to be activated by 1E1 using transient transfection assays. Mutations in the NF-kappa B sites, of either a few changes in the nucleotide sequence or a deletion of the entire region, abrogated 1E1-driven transactivation. Deletion of the Tat-responsive element (TAR) had no significant effect on reporter expression. Mutations in the Sp1 sites or the TATA box significantly lowered LTR activity, but this is probably due to an effect on the general transcription system, as these elements are also required for the transactivation of the LTR by many stimulators including Tat, tumour necrosis factor alpha (TNF-alpha). E1A/E1B and phorbol myristate acetate (PMA). In addition, gel retardation analysis demonstrated that NF- kappa B activity was significantly increased in human T lymphoid H9 and monocytic U937 cell lines constitutively expressing 1E1. Taken together, these data suggest that NF- kappa B plays a central role in the 1E1 transactivation of the HIV LTR.