[Expression of genes involved in retinoic acid biosynthesis in human gastric cancer].

All-trans-retinoic acid (ATRA) is the main biologically active metabolite of retinol (vitamin A) that is required for the regulation of such processes as embryogenesis, tissue differentiation, proliferation, and others. Multiple alcohol, retinol and retinaldehyde dehydrogenases (ADHs, RDHs and RALDHs) as well as aldo-keto reductases (AKRs) catalyze the biosynthesis of retinoic acid in humans. For many normal and neoplastic tissues, the key ATRA-synthesizing enzymes remain unknown. We identified ATRA-generating genes that are expressed in normal and malignant gastric tissues using the transcriptomic database analysis. Quantitative changes in the expression levels of these genes in gastric cancer were determined by semi-quantitative RT-PCR and real-time PCR. Significant decreases in the mRNA levels of genes encoding enzymes that catalyze the reversible oxidation/reduction of retinol and retinaldehyde (ADH4, ADH1B, ADH1C, RDHL, AKR1B10, AKR1B1, and RDH12), as well as the oxidation of retinaldehyde (RALDH1) were revealed in most of the tumor samples. The sharp reduction in the expression levels of genes encoding the key enzymes that convert retinol and retinaldehyde to retinoic acid could lead to a significant decrease in the content of ATRA--the transcriptional regulator of many genes, which in turn can lead to a dysregulation of cell proliferation/differentiation and initiate cancer development.

[Genetic diversity and evolution of the influenza C virus].

The influenza C virus is spread worldwide and causes diseases of the upper and (less frequently) lower respiratory tract in human. The virus is not pandemic, but it circulates together with pandemic influenza A and B viruses during winter months and has quite similar clinical manifestations. The influenza C virus is also encountered in animals (pigs and dogs) and is known to override the interspecific barriers oftransmssion. The immune system of mammals often fails to recognize new antigenic variants of influenza C virus, which invariably arise in nature, resulting in outbreaks of diseases, although the structure of antigens in influenza C virus in general is much more stable than those of influenza viruses A and B. Variability of genetic information in natural isolates of viruses is determined by mutations, reassortment, and recombination. However, recombination events very rarely occur in genomes of negative-strand RNA viruses, including those of influenza, and virtually have no effect on their evolution. Unambiguous explanations for this phenomenon have thus far not been proposed. There is no proof of recombination processes in the influenza C virus genome. On the contrary, reassortant viruses derived from different strains of influenza C virus frequently appear in vitro and are likely to be common in nature. The genome of influenza C virus comprises seven segments. Based on the comparison of sequences in one of its genes (HEF), six genetic or antigenic lineages of this virus can be distinguished (Yamagata/26/81, Aichi/1/81, Mississippi/80, Taylor/1233/47, Sao Paulo/378/82, and Kanagawa/1/76). However, the available genetic data show that all the seven segments of the influenza C virus genome evolve independently.

[Increase in NETO2 gene expression is a potential molecular genetic marker in renal and lung cancers].

Multiple changes in the genome, transcriptome, and proteome are frequent in cancer cells. A search for molecular markers based on DNA, mRNA, or proteins is a main method to develop early specific diagnostics for cancer. While universal markers are still unavailable, similar trends are known for the expression patterns of particular genes in certain epithelial tumors. A bioinformatic screening of transcriptomic databases identified the NETO2 gene as a new potential promising marker of renal cancer. A substantial increase in NETO2 mRNA level was detected in 90% clear-cell renal cell carcinomas, 70% of non-small cell lung cancers, and 50% of papillary renal cancers by real-time PCR. The NETO2 mRNA level was increased to a lesser extent in cervical carcinoma and colon cancer and tended to decrease in cancer of the stomach. The NETO2 gene, which codes for a membrane glycoprotein with an unclear function, was assumed to provide a new promising marker for early diagnosis in renal cancer and non-small cell lung cancer.

[Novel reference gene RPN1 for normalization of quantitative data in lung and kidney cancer].

Quantitative methods of gene expression analysis in tumors require accurate data normalization, which allows comparison of different mRNA/cDNA samples with unknown concentration. For this purpose reference genes with stable expression level (such as GAPDH, ACTB, HPRT1, TBP) are used. The choice of appropriate reference genes is still actual because well-known reference genes are not suitable for certain cancer types frequently and their unreasonable use without additional tests lead to wrong conclusions. We have developed the bioinformatic approach and selected a new potential reference gene RPN1 for lung and kidney tumors. This gene is located at the long arm of chromosome 3. Our method includes mining of the dbEST and Oncomine databases and functional analysis of genes. The RPN1 was selected from 1500 candidate housekeeping genes. Using comparative genomic hybridization with NotI-microarrays we found no methylation, deletions and/or amplifications at the RPN1-containing locus in 56 non-small cell lung and 42 clear cell renal cancer samples. Using RT-qPCR we showed low variability of RPN1 mRNA level comparable to those of reference genes GAPDH and GUSB in lung and kidney cancer. The mRNA levels of two target genes coding hyalouronidases--HYAL1 and HYAL2--were estimated and normalized relative to pair RPN1--GAPDH genes for lung cancer and RPN1--GUSB for kidney cancer. These combinations were shown to be optimal for obtaining accurate and reproducible data. All obtained results allow us to suggest RPN1 as novel reference gene for quantitative data normalization in gene expression studies for lung and kidney cancers.

[Downregulation of AKR1B10 gene expression in colorectal cancer].

Colorectal cancer is one of the most common cancers in the world. In our work changes of AKR1B1 and AKR1B10 gene expression levels in colorectal tumors were studied. Their potential diagnostic value was previously shown for several other cancer types. These genes encode aldoso reductases, which belong to the aldo-keto reductases superfamily consisting of enzymes capable to reduce numerous aromatic and aliphatic aldehydes and ketones. They are also involved into retinoid metabolism and cancerogenesis. We have carried out comparative analysis of mRNA levels of AKR1B1 and AKR1B10 genes in paired samples of normal and colorectal tumor tissues using RT-PCR and quantitative PCR. We have shown for the first time the decrease of activity of these genes in colorectal carcinomas. Significant reduction of AKR1B10 mRNA level was detected in the most of tumor samples (88%, 65/74) even at the early stages of malignancy, and in more than 60% of cases this downregulation was much higher than 10 folds. The decrease of AKR1B1 mRNA level was shown in 10% of tumors only. Therefore, we have detected quite different mRNA expression patterns in colorectal cancer for these two structurally similar genes. These data could indicate different functional roles of these two genes in colorectum. The significant decrease of AKR1B10 mRNA in most samples of colorectal cancer could be considered as potential diagnostic marker of this type of cancer.

[Asymmetry of the GC content in vicinity of transcription starts (with participation of polymerase PoII) and its correlation with location of adsorption sites of protein SP1 on DNA].

Nucleotide DNA sequences within the clusters of transcription starts, determined by the method of cap analysis of gene expression, have some distinguishing features. The sequences of these clusters are rich in nucleotides C and G, and there is an asymmetry of the nucleotide content, which correlates with the choice of chain from which the transcription in the cluster occurs. On the coding chain, the concentration of guanine exceeds the concentration of cytosine. In the nucleotide sequence of the cluster on the coding chain, the frequency of the polynucleotide tracts of the avoided nucleotide (cytosine), normalized to the frequency expected based on the content of this nucleotide in the cluster, is significantly higher compared with the normalized frequency of the polynucleotide tracts of the other nucleotide (guanine). Similarly, the statistical significance of the C-rich variant of the site of specific binding of the transcription initiation factor Sp1 in the coding chain is higher than that of the G-rich variant. However, the assumption can hardly be confirmed that the choice of the variant of the binding region of protein Sp1 correlates with the choice of the transcription chain. It is more likely that both variants are more or less equally probable, and the binding region of protein Sp1 acts in this case as a factor of selection, which counteracts the mutations inducing the shift of the nucleotide content.

[Proteomic expression analysis of human colorectal cancer: of soluble overexpressed proteins].

Colon cancer is one of the leading causes of cancer deaths in developed countries due to the absence of tumor specific markers for early diagnosis of the disease, providing adequate sensitivity. Search for diagnostic markers of various types of cancer by proteomic approaches has been limited by large differences in protein centration. We used preliminary extraction of major cellular proteins by 0.2 M sodium chloride in presence of nonionic detergent NP-40 in order to raise the sensitivity of the 2D PAGE detection of low-abundant soluble proteins, some of which may penetrate in blood circulation during carcinogenesis. Application of this procedure prior to 2D comparative analysis of proteomes of normal tissues and matched colon cancer specimens led to selection of ten proteins, which are frequently overexpressed in colon adenocarcinomas. Mass-spectrometric identification of selected proteins led to discovery of two novel protein markers of colon tumors--TAF9 and CISH. Low level of CISH expression in various tissues suggests that it is a novel prospective marker for diagnosis of colon cancer.

[Colorectal cancer 2D-proteomics: identification of altered protein expression].

Modern proteomic techniques make it possible to identify numerous changes in protein expression in tumor in comparison to normal tissues. Despite the wide application of proteomics in current studies, identification of proteins with stable concentration differences in normal and cancer cells remains rather difficult. The current study was directed to the search of new potential protein colorectal cancer markers using comparative proteomics of protein extracts obtained from primary tumors and adjacent normal tissues. This widespread neoplasm is characterized by lack of evident symptoms at early stages of cancerogenesis. It is highly important to develop fast and sensitive methods of molecular diagnostics. We studied paired cancerous and normal clinical tissue samples from 11 patients with colorectal adenocarcinomas by comparative 2-D PAGE and MALDI-TOF mass-spectrometry identification. Sixteen proteins with stable differential expression were selected and identified, including 13 overexpressed and 3 downregulated proteins. In summary, we describe the discovery overexpression of GPD1 and RRBP1 proteins and lack of expression for HNRNPH1 and SERPINB6 proteins which are new candidate biomarkers of colon cancer.

[Expression of FTL and FTH genes encoding ferretin subunits in lung and renal carcinomas].

The level of ferritin in serum is known to be increased frequently in most human cancers. Ferritin consists of the heavy and light chains, encoded by FTL and FTH genes. The analysis of the EST database showed that the level of FTL and FTH mRNA is decreased in lung squamous cell carcinomas as compared to the normal tissues, no change in the mRNA level was observed in clear cell renal cell carcinoma. Using real-time PCR we estimated the mRNA level of these genes in primary tumors. It was shown significant and frequent decrease of FTL and FTH mRNA level in lung squamous cell carcinoma: on the average by 11 and 9 times in 83% (33/40) and 73% (11/15) of cases, respectively. In clear cell renal cell carcinoma the changes were not so marked both with respect to the level of decrease (on the average 6 and 3 times) and to its frequency (58 and 27%). In the present work it has been shown for the first time that the FTL mRNA is frequently down-regulated even at the early stages of lung squamous cell carcinoma in all studied samples. This fact permits to consider this gene as potential oncomarker of early diagnosis. The FTL mRNA content may be quantified by non-concurrent hybridization on expression DNA microarrays. The possible causes of a serum ferritin increase in lung cancer and renal cancer are discussed.

[Repetitive sequences of the tree shrew genome (Mammalia, Scandentia)].

Copies of two repetitive elements of the genome of common tree shrew (Tupaia glis) were cloned and sequenced. The first element, Tu III, is a approximately 260 bp long short interspersed element (SINE) with the 5'-end derived from glycine RNA. Tu III carries long polypurine- and polypyrimidine-rich tracts, which may contribute to the specific secondary structure of Tu III RNA. This SINE was also found in the genome of smooth-tailed tree shrew of another genus (Dendrogale). Tu III seems to be confined to the order Scandentia (tree shrews) since it was not found in DNA of other tested mammals. The second element Tu-SAT1 is a tandem repeat with a monomer length of 365 bp. Some properties of its nucleotide sequence suggest that Tu-SAT1 is a centromeric satellite.

[Thermal denaturation of eukaryotic class 1 translation termination factor eRF1. Relationship between stability of the eRF1 molecule and alteration of functional activity of its mutants].

Thermal denaturation of eukaryotic class-1 translation termination factor eRF1 and its mutants was examined using differential scanning microcalorimetry (DSK). Changes of free energy caused by mutants in the N domain of human eRF1 were calculated. Melting of eRF1 molecule composed of three individual domains is cooperative. Some amino acid substitutions did not affect protein thermostability and in some other cases even slightly stabilize the protein globule. These imply that these amino acid residues are not involved in maintenance of the 3D structure of human eRF1. Thus, in Glu55Asp, Tyr125Phe, Asn61Ser, Glu55Arg, Glu55A1a, Asn61Ser + Ser64Asp, Cys127Ala and Ser64Asp mutants selective inactivation of release activity is not caused by a destabilization of protein 3D structure and, most likely, is associated with local stereochemical changes introduced by substitutions of amino acid side chains in the functionally essential sites of N-domain molecule. Some residues (Asn129, Phe131) as shown by calorimetric measurements are essential for preservation of stable protein structure, but at the same time they affect selective stop codon recognition probably via their neighboring amino acids. Recognition of UAG and UAA stop codons in vitro is more sensitive to preservation of protein stability than the UGA recognition.

[Transcription TIMP3, DAPk1 and AKR1B10 genes in squamous cell lung cancer].

Lung cancer is one of the most frequent neoplasia in the Russia, the United States and Europe. This cancer is associated with functional activity changes of many genes. In the present study TIMP3, DAPK1 and AKR1B10 genes transcription analysis of squamous cell lung cancer specimens was carried out using reverse transcription-PCR. Substantial increasing of AKR1B10 transcription level is revealed in 80% tumor samples. TIMP3 and DAPK1 transcription level is considerably decreased in 76 and 72% tumor specimens, accordingly. These results may point out that all three genes are important for squamous cell lung cancer tumorogenesis while AKR1B10 is potential oncogene whereas TIMP3 and DAPK1 are potential tumor suppressor genes. We suggest that revealed substantial transcription level-changes of investigated genes may be used for oncodiagnostics.

[Segment duplications in the human genome]. / Segmentnye duplikatsii v genome cheloveka.

The review considers the structure, evolution, and possible mechanisms of spreading of intrachromosomal and interchromosomal segment duplications (SD), which account for more than 5% of the human genome. Most SD are mosaic and consist of multiple modules, which occur in several copies in different genome regions. SD are preferentially located in pericentric and subtelomeric regions, which are least studied on the human chromosomes. Homologous recombination between SD results in various chromosome rearrangements, contributing to the genome instability and the origin of several human hereditary disorders.

[Tandem and interspersed repeats contribute to the mosaic structure of segmented duplications in the human genome]. / Mozaichnaia struktura segmentnykh duplikatsii v genome cheloveka obrazuetsia pri uchastii tandemnykh i dispergirovannykh povtorov.

Intrachromosomal and interchromosomal segmental duplications account for more than 5% of the human genome. To analyze the processes resulting in the complex mosaic structure of duplicons, a draft human genome sequence was searched for duplicated segments of a genomic fragment of the pericentric region of the chromosome 21 short arm. The duplicons found consist of modules having paralogs in various genome regions. Module ends are flanked with various tandem or interspersed repeats, which are more unstable as compared with unique sequences. In most cases, the boundaries of duplicated segments exactly coincide with or are in close proximity to hot spots of various rearrangements within repeats or boundaries between repeats and unique sequences or between two different repeats. Homologous recombination between repetitive elements was assumed to be the major mechanism contributing to the mosaic structure of duplicons.

[Structural organization of the human genome: distribution of nucleotides, Alu-repeats and exons in chromosomes 21 and 22]. / Strukturnaia organizatsiia genoma cheloveka: raspredelenie nukleotidov, Alu-povtorov i ékzonov v khromosomakh 21 i 22.

Analysis of DNA sequences of the human chromosomes 21 and 22 performed using a specially designed MegaGene software allowed us to obtain the following results. Purine and pyrimidine nucleotide residues are unevenly distributed along both chromosomes, displaying maxima and minima (Y waves phi) with a period of about 3 Mbp. Distribution of G + C along both chromosomes has no distinct maxima and minima, however, chromosome 21 contains considerably less G + C than chromosome 22. Both exons and Alu repeats are unevenly distributed along chromosome 21: they are scarce in its left part and abundant in the right part, while MIR elements are quite monotonously spread along this chromosome. The Alu repeats show a wave-like distribution pattern similar for both repeat orientations. The number of the Alu repeats of opposite orientations was equal for both studied chromosomes, and this may be considered a new property of the human genome. The positive correlation between the exon and Alu distribution patterns along the chromosome, the concurrent distribution of Alu repeats in both orientations along the chromosome, and the equal copy numbers for Alu in direct and inverted orientations within an individual chromosome point to their important role in the human genome, and do not fit the notion that Alu repeats belong to parasitic (junk) DNA.

[Electrical response of inner membrane structures of corynebacteria during electrotransformation]. / Elektricheskii otvet vnutrennikh membrannykh struktur kletok korinebakterii pri élektrotransformatsii.

The efficiency of the electrotransformation of intact cells of corynebacteria by a solitary impulse with a complex shape amounted to 10(6) transformants/microgram of plasmid pNV1 DNA at an electric field strength of 14.2 kW/cm; the voltage-current curve of the cell samples was nonlinear. Under these conditions, the structure of the electric current impulse passing intact cells or protoplasts included oscillations characterized by increasing amplitude and a duration of 170 microseconds, which were not detected in the structure of the electric current impulses at field strengths insufficient for obtaining transformants. These changes in the impulse shape suggest the involvement of internal closed membrane structures in the electrical response of cells to the exogenous electric impulse. Most probably, under conditions of electrical treatment optimal for transformation, electropores are formed in the intracellular membranes of corynebacteria.

[Organization of ribosomal DNA from the phytoflagellates Astasia longa and Euglena gracilis: comparison of the structure of 19S and 28S rRNA genes]. / Organizatsiia ribosomnoi DNK fitoflagelliat Astasia longa i Euglena gracilis: sravnenie struktury genov 19S i 28S rRNK.

Restriction maps of Astasia longa and Euglena gracilis var. bacillaris were built and localization of 5.8S, 19S and 28S rRNA genes was established on them by blot-hybridization method. In the A. longa and E. gracilis plasmid rDNA three intergeneous regions were found, two of which were intergeneous transcribed spacers flanking 5.8S RNA gene, and the third region seems to be an untranscribed spacer. Localization of 9 primers from E. gracilis was established on A. longa 19S rRNA gene by PCR; it was similar to E. gracilis. Using amplified A. longa 19S rRNA gene (2300 bp) we have determined the sequence of its 3'-region, which showed 87% homology with the same region of E. gracilis. Using plasmid pA1 S3-H1 containing S3-H1 fragment of A. longa rDNA the sequence of 28S rRNA gene 3'-region was determined. This sequence includes regions homologous to corresponding regions of E. gracilis Z: 6 nucleotides of 12/13 internal spacer, complete 13/14 internal spacer (86 bp), genes for 13th and 14th 28S rRNA components (56 and 86 bp) with approximately 90% of homology with E. gracilis Z, and fragment of untranscribed spacer (136 bp) with approximately 70% homology. It was shown that 28S rRNA genes of A. longa and E. gracilis have similar structure. Our data allow to conclude that these phytoflagellats are closely related.