Aminoethane Sulfonic Acid Magnesium Salt Inhibits Ca2+ Entry Through NMDA Receptor Ion Channel In Vitro.

The effect of a cerebroprotective agent magnesium bis-aminoethanesulfonate (laboratory code FS-LKhT-317) on intracellular calcium concentration was studied by the fluorescent imaging technique on neuroglial cell culture from Spraque-Dawley rat hippocampus. The substance produced a pronounced inhibitory effect and suppressed NMDA receptor activity in concentrations of ≥50 µM. The observed effects were reversible or partially reversible and were detected by a decrease in Ca2+ signal amplitude in neurons in response to NMDA applications in a Mg2+-free medium and by inhibition of Ca2+ pulses in magnesium-free medium (elimination of magnesium block).

[Hygienic regulation of neonicotinoids derivative in the soil].

For the assurance of the safe regulations for the use in agriculture persistent in soil insecticide neonicotinoids derivative, studies have been executed to investigate its impact on the soil microbiocenosis and migration to the neighboring environment of the active ingredient of this pesticide and the preparation based on it. On the ground of obtained experimental data there were determined threshold doses for the following indices of the harmfulness: migration-water, translocation (transmission into plants) and general sanitary indices. There were established limiting indices of the harmfulness: translocation and migration-water indices. The maximum permissible concentration (MPC) of neonicotinoids derivative in the soil accounted for 0.5 mg/kg of the soil. This content of the pesticide in the soil prevents its accumulation in plants in concentrations exceeding the maximum permissible levels (MPLs), in food products, it fails both to give rise in its transition in groundwater above the MPL for water reservoirs and influence on the soil microbiocenosis and self-purification processes.

A novel member of the glycosyltransferase family, beta 3 Gn-T2, highly downregulated in invasive human bladder transitional cell carcinomas.

Differential display reverse transcription (DDRT)-polymerase chain reaction (PCR) was used to compare the transcriptomes of invasive and noninvasive fresh human bladder transitional cell carcinomas. A differentially expressed novel gene sharing structural similarity with the human beta 3-galactosyltransferase family, beta-1,3-N-acetylglucosaminyltransferase-T2 (beta 3Gn-T2), was identified. The full-length beta 3Gn-T2 cDNA, containing a complete open reading frame of 1193 bp, was cloned and sequenced. beta 3Gn-T2 exhibited 29-41% homology to the multigene beta 3-galactosyltransferase family. Expression of the full-length beta 3Gn-T2 cDNA in an in vitro coupled transcription/translation assay yielded a primary translation product with an apparent Mr of 46 kDa, which is in agreement with the predicted 397-amino-acid protein encoded by beta 3Gn-T2. Multiple peptide alignment showed several sequence motifs corresponding to putative catalytic domains that are conserved throughout all members of the beta 3-galactosyltransferase family, namely, a type II transmembrane domain, a conserved DxD motif, an N-glycosylation site, and five conserved cysteins. By RT-PCR strong downregulation of beta 3Gn-T2 expression was noted in invasive human bladder transitional cell carcinomas (16 fresh biopsy samples: grade III, T2-T4) compared with their noninvasive counterparts (15 fresh biopsies: grade II, Ta), suggesting that beta 3Gn-T2 may be involved in cancer progression.

Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics.

Novel and powerful technologies such as DNA microarrays and proteomics have made possible the analysis of the expression levels of multiple genes simultaneously both in health and disease. In combination, these technologies promise to revolutionize biology, in particular in the area of molecular medicine as they are expected to reveal gene regulation events involved in disease progression as well as to pinpoint potential targets for drug discovery and diagnostics. Here, we review the current status of these technologies and highlight some studies in which they have been applied in concert to the analysis of biopsy specimens.

[Rapid indication of microbiological damage and biodegradation of structural materials in space vehicles]. / Ekspressnaia indikatsiia mikrobiologicheskogo povrezhdeniia i biodegradatsii konstruktsionnykh materialov kosmicheskikh ob''ektov.

The study was undertaken to design a kit of indicators for rapid detection and group identification of microbes causing biological damages to the materials. Indication is accomplished by dropwise colorimetric reaction for which modified chromatographic materials serve as substrates. Reactions are made by the indicators whose design allows reagents and samples to be measured on board space vehicles. The designed kit of indicators detects microorganisms, biodegrade products, and identifies them by groups: vegetative or spore-forming bacteria, and lower fungi. The paper considers the characteristics of colorimetric reactions and an algorithm of group identification of microbes by the respective combination of positive and negative results of the reactions. It also presents data on the sensitivity levels of tests provided by the kit, which were obtained from laboratory and field ground tests.

Identification of true differentially expressed mRNAs in a pair of human bladder transitional cell carcinomas using an improved differential display procedure.

Differential display in combination with arbitrarily primed polymerase chain reaction (PCR) fingerprinting has become one of the most powerful techniques to identify and isolate mRNAs that are differentially expressed in pairs of biological samples. However, in many cases the cDNA band corresponding to the differentially amplified product contains several cDNA species that comigrate with the cDNA of interest due to the poor resolution of the fingerprinting gels, thus hampering further analysis and identification of the desirable cDNA. To improve the electrophoretic resolution of differentially amplified cDNAs, we have utilized Resolver Gold agarose gel electrophoresis (Ingenius) as an additional step to overcome downstream problems encountered during RNA fingerprinting experiments. To illustrate the power of the modified differential display procedure we present a detailed analysis of the cDNA products differentially displayed in tumor biopsies obtained from a noninvasive (grade II, Ta) and an invasive (grade III, T2-T4) human bladder transitional cell carcinoma (TCC). Several genes that were differentially expressed in this tumor pair were identified. These included: tropomyosin 4, the protein disulfide isomerase precursor (PDI), MRP14, signal transducer CD24, keratins 8 and 13, cytochrome oxidase subunit IV (COXIV), putative transcription factor HOX-1.3, as well as two novel genes of yet unknown function. All of the identified cDNAs were shown to be truly differentially expressed by Northern blotting, reverse transcriptase-PCR (RT-PCR), and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of the corresponding lesions.

A comprehensive protein resource for the study of bladder cancer: http://biobase.dk/cgi-bin/celis.

In our laboratories we are exploring the possibility of using proteome expression profiles of fresh bladder tumors (transitional cell carcinomas, TCCs; squamous cell carcinomas, SCCs) and random biopsies as fingerprints to subclassify histopathological types and as a starting point to search for protein markers that may form the basis for diagnosis, prognosis, and treatment. Ultimately, the goal of these studies is to identify signaling pathways and components that are affected at various stages of bladder cancer progression and that may provide novel leads in drug discovery. Here we present our ongoing efforts to establish comprehensive two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) databases of TCCs and SCCs which are being constructed based on the proteomic and immunohistochemical analysis of hundreds of fresh tumors, random biopsies and cystectomies received shortly after operation (http://biobase.dk/cgi-bin/celis).

Characterization of DNA topoisomerase II alpha/beta heterodimers in HeLa cells.

In mammalian cells, DNA topoisomerase II is the product of two distinct genes encoding the alpha and beta isoforms of the enzyme. Besides homodimeric topoisomerase IIalpha and IIbeta, we have recently shown that alpha/beta heterodimers constitute a third population of topoisomerase II in HeLa cells. We found that topoisomerase II heterodimers are not restricted to HeLa cells but exist in different mammalian cell types, and up to 25% of the total topoisomerase IIbeta population is involved in heterodimer formation. Studies of topoisomerase II phosphorylation in HeLa cells show that heterodimers are phosphorylated in vivo to a significantly lower level compared to homodimeric alpha enzymes, but in contrast to the latter neither heterodimers nor topoisomerase IIbeta homodimers coprecipitate together with a kinase activity that is able to mediate their phosphorylation. However, both enzymes can still be phosphorylated by exogenously added casein kinase II. The differential phosphorylation of topoisomerase II heterodimers suggests an alternative regulation of this topoisomerase II subclass compared to the homodimeric topoisomerase IIalpha counterparts.

Human and mouse proteomic databases: novel resources in the protein universe.

Proteomics is an emerging area of research of the post-genomic era that deals with the global analysis of gene expression using a plethora of techniques to resolve (high resolution two-dimensional polyacrylamide gel electrophoresis, 2D PAGE), identify (peptide sequencing by Edman degradation, mass spectrometry, Western immunoblotting, etc.), quantitate and characterize proteins, as well as to store (comprehensive 2D PAGE databases), communicate and interlink protein and DNA sequence and mapping information from genome projects. Here we review the current status as well as applications of human and mouse proteomic 2D PAGE databases that are being systematically constructed for the global analysis of gene expression in both health and disease (http://biobase.dk/cgi-bin/celis). Furthermore, we discuss the problems one faces when using powerful proteomic technology to study heterogeneous tissue and tumor biopsies, and emphasize the importance of building comprehensive databases that contain a critical mass of information for both known and novel proteins in normal and disease conditions.

Protein abundancy and mRNA levels of the adipocyte-type fatty acid binding protein correlate in non-invasive and invasive bladder transitional cell carcinomas.

The adipocyte type fatty acid-binding protein (A-FABP) is a small molecular weight fatty acid-binding protein whose expression correlates both with the grade of atypia and the stage of bladder transitional cell carcinomas (TCCs). To determine if the protein abundancy correlates with the mRNA levels in non-invasive and invasive lesions, we have analysed fresh TCCs (grade II, Ta; grade III, T2-4) by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and measured the mRNA levels using the reverse transcription linked polymerase chain reaction (RT-PCR). Overall, the results showed a good correlation between protein abundancy and mRNA levels, indicating that the lack of expression of the protein observed in some lesions reflects low levels of transcription of the A-FABP gene rather than translational regulation. In addition, our studies showed that the loss of A-FABP protein observed in some tumors is not compensated by an increase in the skin fatty acid-binding protein PA-FABP, as is the case in the A-FABP knockout mice.

Human rab11a: transcription, chromosome mapping and effect on the expression levels of host GTP-binding proteins.

Rab11a is a member of the rab-branch of the ras-like small GTP-binding protein superfamily that is associated with both constitutive and regulated secretory pathways. Using a direct procedure for cDNA cloning of small ras-related GTPases, that is based on the screening of eukaryotic cDNA expression libraries using [alpha-32P]GTP as a probe, we have isolated two cDNA clones encoding rab11a. Both clones share identical coding sequences, but differ in the length and sequence of their 3' untranslated regions (3'-UTR). Northern blot hybridisation analysis of various human tissues revealed indeed two mRNA species with lengths of 1.0 and 2.3 kb, respectively. Sequence analysis of the cDNAs identified two different putative polyadenylation signals (AATAAA) at positions 927 and 2302 of the larger transcript. In addition, the 3'-UTR of the larger transcript exhibited several AU-rich elements (ARE) that are believed to control gene expression by regulating the rate of mRNA degradation. Southern blots of human DNA digested with several rare restriction enzymes, and separated by pulse-field gel electrophoresis, yielded the same macro-restriction fragment pattern when hybridised with probes that discriminate between the two transcripts. Taken together, these findings imply that the two mRNA species originate from a single gene, which we have mapped to 15q21.3-q22.31, by the use of different polyadenylation sites. As expected, both rab11a-cDNAs yielded the same protein product when transiently expressed in COS-1 cells, and surprisingly, upregulated the proteome expression profile (de novo synthesis or posttranslational modification of preexisting proteins) of a few other, yet unknown GTP-binding proteins.

Human 2-D PAGE databases for proteome analysis in health and disease: http://biobase.dk/cgi-bin/celis.

Human 2-D PAGE Databases established at the Danish Centre for Human Genome Research are now available on the World Wide Web (http://biobase.dk/cgi-bin/celis). The databanks, which offer a comprehensive approach to the analysis of the human proteome both in health and disease, contain data on known and unknown proteins recorded in various IEF and NEPHGE 2-D PAGE reference maps (non-cultured keratinocytes, non-cultured transitional cell carcinomas, MRC-5 fibroblasts and urine). One can display names and information on specific protein spots by clicking on the image of the gel representing the 2-D gel map in which one is interested. In addition, the database can be searched by protein name, keywords or organelle or cellular component. The entry files contain links to other databases such as Medline, Swiss-Prot, PIR, PDB, CySPID, OMIM, Methabolic pathways, etc. The on-line information is updated regularly.

Active heterodimers are formed from human DNA topoisomerase II alpha and II beta isoforms.

DNA topoisomerase II is a nuclear enzyme essential for chromosome dynamics and DNA metabolism. In mammalian cells, two genetically and biochemically distinct topoisomerase II forms exist, which are designated topoisomerase II alpha and topoisomerase II beta. In our studies of human topoisomerase II, we have found that a substantial fraction of the enzyme exists as alpha/beta heterodimers in HeLa cells. The ability to form heterodimers was verified when human topoisomerases II alpha and II beta were coexpressed in yeast and investigated in a dimerization assay. Analysis of purified heterodimers shows that these enzymes maintain topoisomerase II specific catalytic activities. The natural existence of an active heterodimeric subclass of topoisomerase II merits attention whenever topoisomerases II alpha and II beta function, localization, and cell cycle regulation are investigated.

Long-range fragmentation of the eukaryotic genome by exogenous and endogenous nucleases proceeds in a specific fashion via preferential DNA cleavage at matrix attachment sites.

Small cell lung cancer cells (OC-NYH-VM) were permeabilized and treated with different nucleases. The long-range distribution of DNA cleavage sites in the amplified c-myc gene locus was then analyzed by pulsed field gel electrophoretic separation of the released 50-kilobase to 1-megabase DNA fragments followed by indirect end labeling. Exogenous DNase I and nucleases specific for the single-stranded DNA were found to generate similar nonrandom patterns of large DNA fragments. The cleavage sites were located close to or even colocalized with matrix attachment regions, which were mapped independently using a recently developed procedure for DNA loop excision by DNA topoisomerase II-mediated DNA cleavage. Endogenous acidic nuclease with the properties of DNase II also digested DNA preferentially in proximity to the matrix attachment regions, generating characteristic patterns of excised DNA loops and their oligomers. A similar, although less specific, pattern of DNA fragmentation was observed after incubation of permeabilized cells under conditions favoring the activity of endogenous neutral Ca(2+)- and Mg(2+)-dependent nucleases. These findings are discussed in the context of the current model of the spatial domain organization of eukaryotic genome.

The channels model of nuclear matrix structure.

The specificity of eukaryotic DNA organization into loops fixed to the nuclear matrix/chromosomal scaffold has been studied for more than fifteen years. The results and conclusions of different authors remain, however, controversial. Recently, we have elaborated a new approach to the study of chromosomal DNA loops. Instead of characterizing loop basements (nuclear matrix DNA), we have concentrated our efforts on the characterization of individual loops after their excision by DNA topoisomerase II-mediated DNA cleavage at matrix attachment sites. In this review the results of applying this mapping approach are compared with the results and conclusions from studies of nuclear matrix DNA. An attempt is also made to reconsider all data about the specificity of DNA interactions with the nuclear matrix and to suggest a model of spatial organization of the eukaryotic genome which resolves apparent contradictions between these data.

Different topoisomerase II antitumor drugs direct similar specific long-range fragmentation of an amplified c-MYC gene locus in living cells and in high-salt-extracted nuclei.

We have analyzed the long-range distribution of topoisomerase II-mediated cleavages induced in an amplified human c-MYC gene locus in the presence of several antitumor agents. The long-range cleavage patterns were found to be nonrandom and similar for all antitumor drugs tested. Cleavages occurred within several kilobase-long areas (approximately 5 kb) highly accessible to topoisomerase II and separated by extended regions (approximately 70-100 kb) of less accessibility, possibly reflecting the mode of DNA organization into loops along the chromosome. Within the cleavage areas, the patterns of cleavage sites showed a certain dependence on the type of drug used for entrapment of topoisomerase II-DNA complexes. Importantly, distribution of cleavage areas in native chromatin and histone-depleted nuclei was very similar, if not identical, suggesting that the primary target of antitumor agents in vivo is topoisomerase II associated with the high-salt-insoluble nuclear matrix. These data show that matrix-attached DNA is preferentially damaged by topoisomerase II-targeting agents, which may be an important cellular event contributing to drug-induced cell death.

Specificity and functional significance of DNA interaction with the nuclear matrix: new approaches to clarify the old questions.

In this chapter the specificity of chromosomal DNA partitioning into topological loops is discussed. Different experimental approaches used for the analysis of the above problem are critically reviewed. This discussion is followed by presentation of a novel approach for mapping the DNA loop anchorage sites that we have developed. This approach, based on the excision of the whole DNA loops by topoisomerase II-mediated DNA cleavage at matrix attachment sites, seems to constitute a unique tool for the analysis of topological organization of chromosomal DNA in living cells. We also discuss experimental results indicating that the DNA-loop anchorage sites form "weak points" in chromosomes that are preferentially sensitive to cleavage with both endogenous and exogenous nucleases. In connection with this discussion, rationales for the supposition that DNA loops constitute basic units of eukaryotic genome organization and evolution are considered. The chapter concludes by suggesting a new model of spatial organization of eukaryotic genome within the cell nucleus that resolves apparent contradictions between different data on the specificity of DNA interaction with the nuclear matrix.

[Evaluation of immunoreactivity of class 2 HLA-determinant using mixed lymphocyte culture]. / Otsenka immunoreaktivnosti HLA-determinant klassa 2 metodom smeshannoi kul'tury limfotsitov.

Immunoreactivity (proliferative capacity) of normal donor peripheral blood mononuclears expressing HLA-DR antigens in MLC test was studied. HLA-DR cellular phenotype was found to influence the intensity of proliferative response. HLA-DR highly reactive (HLA-DR5, HLA-DR7, HLA-DR4) and low-reactive (HLA-DR1, HLA-DR2, HLA-DR6) determinants were distinguished.

Characterization of an altered DNA catalysis of a camptothecin-resistant eukaryotic topoisomerase I.

We investigated topoisomerase I activity at a specific camptothecin-enhanced cleavage site by use of a partly double-stranded DNA substrate. The cleavage site belongs to a group of DNA topoisomerase I sites which is only efficiently cleaved by wild-type topoisomerase I (topo I-wt) in the presence of camptothecin. With a mutated camptothecin-resistant form of topoisomerase I (topo I-K5) previous attempts to reveal cleavage activity at this site have failed. On this basis it was questioned whether the mutant enzyme has an altered DNA sequence recognition or a changed rate of catalysis at the site. Utilizing a newly developed assay system we demonstrate that topo I-K5 not only recognizes and binds to the strongly camptothecin-enhanced cleavage site but also has considerable cleavage/religation activity at this particular DNA site. Thus, topo I-K5 has a 10-fold higher rate of catalysis and a 10-fold higher affinity for DNA relative to topo I-wt. Our data indicate that the higher cleavage/religation activity of topo I-K5 is a result of improved DNA binding and a concomitant shift in the equilibrium between cleavage and religation towards the religation step. Thus, a recently identified point mutation which characterizes the camptothecin-resistant topo I-K5 has altered the enzymatic catalysis without disturbing the DNA sequence specificity of the enzyme.