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At least 1,621 records · Page 90Linked to original sources
PubMed2004

A scalable high-content cytotoxicity assay insensitive to changes in mitochondrial metabolic activity.

Quantitative assessments of cell proliferation and cytotoxicity are key components of anticancer drug discovery. Advancements in genomics and combinatorial chemistry have increased the demand for cytotoxicity measurements, and almost all assays for cell proliferation that are compatible with high-throughput screening are single-parameter, homogeneous biochemical assays. Furthermore, the most popular methods currently in use are based on measurements of mitochondrial activity and therefore provide only an indirect measure of cell growth. Here we describe a high-content screening (HCS) methodology that permits the direct quantification of cell numbers as well as multiple measurements of drug activity in individual cells without the ambiguities of previously described homogenous metabolism-based assays. Using two human tumor cell lines, we compared our HCS method with a commonly used homogenous colorimetric assay that detects mitochondrial activity and found the colorimetric assay substantially underrepresented the cytotoxic effects of two mechanistically diverse, clinically used, anticancer drugs: a DNA-damaging agent, bleomycin, and a tubulin stabilizer, paclitaxel. Simultaneous evaluation of cell numbers, mitochondrial mass, and nuclear morphology by HCS provided an explanation for the differential toxicity. Furthermore, we expanded the scope of the HCS assay to include tubulin mass measurements in paclitaxel-treated cells as an example for a specific drug target. The data illustrate the utility of HCS as a powerful analysis tool compatible with the demands of high-throughput screening, and adaptable to include fluorescence-based markers of drug activity.

Antineoplastic Agents↗
PubMed2005

New advances in the DNA damage response network of Fanconi anemia and BRCA proteins. FAAP95 replaces BRCA2 as the true FANCB protein.

Fanconi anemia (FA) proteins function in a DNA damage response pathway that appears to be part of the network including breast cancer susceptibility gene products, BRCA1 and BRCA2. In response to DNA damage or replication signals, a nuclear FA core complex of at least 6 FA proteins (FANCA, FANCC, FANCE, FANCF, FANCG and FANCL) is activated and leads to monoubiquitination of the downstream FA protein, FANCD2. One puzzling question for this pathway is the role of BRCA2. A previous study has proposed that BRCA2 could be identical to two FA proteins: FANCD1, which functions either downstream or in a parallel pathway; and FANCB, which functions upstream of the FANCD2 monoubiquitination. Now, a new study shows that the real FANCB protein is not BRCA2, but a previously uncharacterized component of the FA core complex, FAAP95, suggesting that BRCA2 does not act upstream of the FA pathway. Interestingly, the newly discovered FANCB gene is X-linked and subject to X-inactivation. The presence of a single active copy of FANCB and its essentiality for a functional FA-BRCA pathway make it a potentially vulnerable component of the cellular machinery that maintains genomic integrity.

BRCA1 Protein↗
PubMedDate not supplied

[Highest level of division in the organism classification. 1. Prokaryotes and eukaryotes].

The works on the general classification of all organisms are considered as a convenient opportunity to sum up numerous data obtained in organic world studying. The present stage is characterized by rapid development of the molecular reconstructions that have already caused considerable changes in our classification practice. These changes look especially impressive at studying the organism cellular structure. The great massive of new data allow us to compare Prokaryotes and Eukaryotes on the nucleic acids and especially proteins whose number in Eukaryote cell approaches to several thousands. Basing on the structure of macromolecules one can hypothesize with great certainty about Prokaryote or Eukaryotes origin. The article presents the detailed characteristic of Prokaryotes or Eukaryotes with the emphasis placed on the comparative analysis of biological macromolecules. Among specially considered cellular structures and processes are cell wall, intracellular components, cellular cycle, nucleus, DNA compactness, replication, genome organization, transcription, posttranscriptional modifications, introns, ribosomes and translation, cytoskeleton, mitosis, cytokinesis, cellular organelles, intracellular membranes systems, modes of nutrition, sexual condition. The macromolecular analysis let to carry out the homology of structures and to find out some new connections. It was shown that typology considered as a search for morphological patterns within the biodiversity structure has almost exhausted the subject. It was directed mostly to distinguishing "main" group in contrast with intermediate and aberrant ones, which were considered as minor phenomenon. At present due to macromolecules systematics it is able to estimate the whole diversity of forms including typologically transitive.

Biodiversity↗
PubMed2004

Membrane receptor mapping: the membrane topography of Fc(epsilon)RI signaling.

Ligand binding to membrane receptors initiates cascades of biochemical events leading to physiological responses. Hundreds of proteins and lipids are implicated in signaling networks and programs in genomics and proteomics are continuously adding new components to the signaling "parts lists". Here, we generate high resolution maps of signaling networks using cytoplasmic face-up membrane sheets that can be labeled with immunogold probes (3-10 nm) and imaged in the transmission electron microscope. Our model system is the mast cell and we focus on mapping the topography of the high affinity IgE receptor, Fc(epsilon)RI, its associated tyrosine kinases, Lyn and Syk, and the signaling proteins that propagate signals from these kinases. Crosslinked receptors and their signaling partners segregate during signaling to multiple, dynamic membrane domains, including a transient Fc(epsilon)RI-Lyn domain and at least two other distinct domains, one characterized by the presence of receptor, Syk and multiple signaling proteins, but not Lyn (primary signaling domains), and one characterized by the presence of LAT and PLCgamma1 but not receptor (secondary signaling domains). PI 3-kinase associates with both primary and secondary signaling domains and may help to recruit specific signaling proteins through the local remodeling of inositol phospholipids. The lipid raft markers, GM1 and Thy-1, fail to localize in native membrane sheets either with each other or with signaling domains. We introduce new probes to localize multiple signaling molecules on the same membrane sheet and new computational tools to capture and analyze their topographical relationships. In the future, we expect that high resolution maps of signaling networks will be integrated with chemical kinetic analyses, with cell fractionation data and with a range of real-time fluorescence measurements, into mathematical models with power to predict mechanisms that regulate the efficiency, specificity, amplitude and duration of signaling pathways.

Animals↗
PubMedDate not supplied

[The role of regulatory networks of the cell response to damages in radiation effects].

In view of modern knowledge and concepts about components, function and mechanisms of response of cell molecular structures to damaging effects, response which is generating specialized modules of reactions, it is shown that main components of the mechanism of maintenance of genome constancy at ionizing radiation exposure are checkpoints of cell cycle, DNA repair and apoptosis. They operate under the control of a genetic system at participation of Tp53 gene, corresponding protein and of regulatory networks formed by cascades of mitogen-activated protein kinases (MAPK). At ionizing radiation exposure the MAPK special modules participate in formation of radiation effect: ERK 1/2 (extracellular signal-regulated kinase 1 and 2), JNK/SAPK (c-Jun N-terminal kinase/stress activated protein kinase) and p38 MAPK. Executing physiological functions of maintenance of normal life activity of cells, they do not lose this capacity after exposure to ionizing radiation, participating in formation of radiation effect in a wide range of doses, and are inactivated only by exposure to very high doses. It is concluded that in light of the modern data the main problem is not a problem of mechanisms of biological effect of ionizing radiation but a problem of biological mechanisms of radiation exposure.

Animals↗
PubMedDate not supplied

[Genetic function of the multiploid virions of alphaviruses].

The natural process of infection with multiploid virions of alphaviruses was modeled by inoculation of cells with artificially obtained aggregates. After infection with the aggregates the progeny of attenuated clones of Venezuelan equine encephalomyelitis virus was found to have some new properties. The virus produced larger plaques and had higher reproduction activity. In some cases, changes in the electrophoretic mobility of virion structural proteins was observed, especially a decrease in the molecular weight of the most conservative of them, C-protein. These properties were retained in passages of the virus in cell cultures. The observed phenomenon is associated with genetic interaction between defective virus genomes getting into cells as the aggregate component. Formation of multiploid virions and infection with these particles are considered as the process providing for retention of the biological properties of the virus under conditions of accumulation of DI-particles in the population.

Alphavirus↗
PubMed1998

[The mechanisms of human cytomegalovirus DNA replication].

Human cytomegalovirus (HCMV) is a human herpesvirus with a linear double stranded DNA which have the largest genomes, about 230 kbp in length, of any of the herpesviruses. The replication of HCMV DNA initiated from HCMV DNA replication origin (oriLyt) and formed concatemers by rolling circule-type replication. The genome inversions of the L and S components occur within concatemeric DNA and formed four isomeres. Then, the concatemeric DNA is cleaved to a unit length at the termini with the "a" sequence as a cis-acting signal and packaged into preformed capsids in the nucleus. Six HCMV genes common to herpesvirus group encode essential proteins that constitute the replication fork complex. In addition, five viral genes are required for efficient DNA replication as trans-acting factors. Several HCMV genes are HSV homologs involved in nucleotide metabolism and DNA repair.

Capsid↗
PubMed2002

The generic genome browser: a building block for a model organism system database.

The Generic Model Organism System Database Project (GMOD) seeks to develop reusable software components for model organism system databases. In this paper we describe the Generic Genome Browser (GBrowse), a Web-based application for displaying genomic annotations and other features. For the end user, features of the browser include the ability to scroll and zoom through arbitrary regions of a genome, to enter a region of the genome by searching for a landmark or performing a full text search of all features, and the ability to enable and disable tracks and change their relative order and appearance. The user can upload private annotations to view them in the context of the public ones, and publish those annotations to the community. For the data provider, features of the browser software include reliance on readily available open source components, simple installation, flexible configuration, and easy integration with other components of a model organism system Web site. GBrowse is freely available under an open source license. The software, its documentation, and support are available at http://www.gmod.org.

Animals↗
PubMed2005

Genome-wide analysis of mammalian DNA segment fusion/fission.

As a powerful tool for gene function prediction, gene fusion has been widely studied in prokaryotes and certain groups of eukaryotes, but it has been little applied in studies of mammalian genomes. With the first fully sequenced mammalian genomes (human, mouse, rat) now available, we defined and collected a set of fusion/fission event-linked segments (FFLS) based on structured organized genomic alignment. The statistics of the sequence features highlighted the FFLSs against their random context. We found that there are three groups of FFLSs with different component pairs (i.e. gene-gene, gene-noncoding and noncoding-noncoding) in all three mammalian genomes. The proteins encoded by the components of FFLSs in the first group shown a strong tendency to interact with each other. The segmental components in the last two groups which did not contain any protein-coding genes, were found not only to be transcribed to some level, but also more conserved than the random background. Thus, these segments are possibly carrying certain biologically functional elements. We propose that FFLS may be a potential tool for prediction and analysis of function and functional interaction of genetic elements, including both genes and noncoding elements, in mammalian genomes. The full list of the FFLSs in the genomes of the three mammals is available as supporting information at doi:10.1016/j.jtbi.2005.09.016.

Animals↗
PubMed2005

Implications of a genomic search for autophagy-related genes in trypanosomatids.

Autophagy is the process by which cellular components are directed to and degraded in the vacuole or lysosome and has been studied largely in yeasts. We present here an in silico genomic analysis of trypanosomatid autophagy aimed at highlighting similarities and differences with autophagy in other organisms. Less than half of the yeast autophagy-related proteins examined have certain putative orthologues in trypanosomatids. A cytosol-to-vacuole transport system is clearly lacking in these organisms. Other absences are even more unexpected and have implications for our understanding of the molecular mechanisms of autophagy. The results are consistent with taxon-specific addition of components to a core autophagy machinery during evolution.

Animals↗
PubMed1996

Characterization of an adherence and antigenic determinant of the ArgI protease of Porphyromonas gingivalis which is present on multiple gene products.

This study was performed to characterize the antigen(s) recognized by a panel of monoclonal antibodies (MAbs) produced to be specific for Porphyromonas gingivalis whole cells which we had previously shown to bind to epitopes recognized by sera from periodontitis patients. Preliminary data had suggested that the arginine-specific proteases of P. gingivalis (ArgI, ArgIA, and ArgIB) contained the antigenic determinants of four of these antibodies (MAbs 1A1, 2B/H9, 7D5, and 3B1). The location of the binding sites was examined with purified P. gingivalis enzymes and recombinant regions of the ArgI polyprotein expressed by subclones of the prpR1 gene in Escherichia coli XL-1 Blue cells. All four antibodies were reactive with protein determinants within the beta subunit, a hemagglutinin and/or adhesin component, of the ArgI dimer. MAb 1A1 strongly inhibited the agglutination of human erythrocytes by P. gingivalis W50 culture supernatant, suggesting that the binding site for this antibody contains residues which are critical for the interaction with the erythrocyte surface. The determinant for MAb 1A1 was examined further by construction of a set of truncated forms of the beta component expressed as fusion proteins with glutathione S-transferase at the N terminus. Analysis of these constructs mapped the binding site for MAb 1A1 to PrpRI residues G-907 to T-931, GVSPKVCKDV TVEGSNEFAP VQNLT. Western blot (immunoblot) analysis of P. gingivalis whole-cell proteins demonstrated that MAb 1A1 reacts with several proteins in the Mr range of 20,000 to 120,000. Furthermore, an oligonucleotide probe corresponding to the coding sequence for the region of the ArgI beta component containing the MAb 1A1 binding site hybridized to multiple bands on genomic digests of P. gingivalis DNA. These data indicate that the MAb 1A1 epitope may be a component of a binding domain common to multiple gene products of this organism and may thus represent a functionally important target of the host's specific immune response to P. gingivalis in periodontal disease.

Adhesins, Bacterial↗
PubMed2004

Plant nuclear envelope proteins.

Compared to research in the animal field, the plant NE has been clearly under-investigated. The available data so far indicate similarities as well as striking differences that raise interesting questions about the function and evolution of the NE in different kingdoms. Despite a seemingly similar structure and organization of the NE, many of the proteins that are integral components of the animal NE appear to lack homologues in plant cells. The sequencing of the Arabidopsis genome has not led to the identification of homologues of animal NE components, but has indicated that the plant NE must have a distinct protein composition different from that found in metazoan cells. Besides providing a selective barrier between the nucleoplasm and the cytoplasm, the plant NE functions as a scaffold for chromatin but the scaffolding components are not identical to those found in animal cells. The NE comprises an MTOC in higher plant cells, a striking difference to the organization of microtubule nucleation in other eukaryotic cells. Nuclear pores are present in the plant NE, but identifiable orthologues of most animal and yeast nucleoporins are presently lacking. The transport pathway through the nuclear pores via the action of karyopherins and the Ran cycle is conserved in plant cells. Interestingly, RanGAP is sequestered to the NE in plant cells and animal cells, yet the targeting domains and mechanisms of attachment are different between the two kingdoms. At present, only a few proteins localized at the plant NE have been identified molecularly. Future research will have to expand the list of known protein components involved in building a functional plant NE.

Active Transport, Cell Nucleus↗
PubMed2005

Porcine Toll-like receptor 1, 6, and 10 genes: complete sequencing of genomic region and expression analysis.

Toll-like receptors (TLRs) recognize various microbial components and play key roles in activating the innate immune system. Hence, their function is important in swine infectious diseases. We completely determined 173,804 bp of nucleotide sequence of a genomic region including porcine TLR6 and the newly identified porcine TLR homologues TLR1 and TLR10. The porcine genomic structure of these genes was highly conserved in comparison with the corresponding region in humans. Analysis of their expression in porcine tissues showed differences in expression patterns between porcine TLR10 and TLR1 or TLR6. Moreover, phylogenetic analysis of the cytoplasmic regions of TLR genes suggested that the signal transduction pathway of TLR10 was different from those of TLR1 and TLR6. We also developed six polymorphic microsatellite markers within this genomic region; these markers will be valuable for association studies between TLR genes and resistance or susceptibility to infectious diseases in swine.

Amino Acid Sequence↗
PubMed2005

Distribution and intensity of constraint in mammalian genomic sequence.

Comparisons of orthologous genomic DNA sequences can be used to characterize regions that have been subject to purifying selection and are enriched for functional elements. We here present the results of such an analysis on an alignment of sequences from 29 mammalian species. The alignment captures approximately 3.9 neutral substitutions per site and spans approximately 1.9 Mbp of the human genome. We identify constrained elements from 3 bp to over 1 kbp in length, covering approximately 5.5% of the human locus. Our estimate for the total amount of nonexonic constraint experienced by this locus is roughly twice that for exonic constraint. Constrained elements tend to cluster, and we identify large constrained regions that correspond well with known functional elements. While constraint density inversely correlates with mobile element density, we also show the presence of unambiguously constrained elements overlapping mammalian ancestral repeats. In addition, we describe a number of elements in this region that have undergone intense purifying selection throughout mammalian evolution, and we show that these important elements are more numerous than previously thought. These results were obtained with Genomic Evolutionary Rate Profiling (GERP), a statistically rigorous and biologically transparent framework for constrained element identification. GERP identifies regions at high resolution that exhibit nucleotide substitution deficits, and measures these deficits as "rejected substitutions". Rejected substitutions reflect the intensity of past purifying selection and are used to rank and characterize constrained elements. We anticipate that GERP and the types of analyses it facilitates will provide further insights and improved annotation for the human genome as mammalian genome sequence data become richer.

Animals↗
PubMed2002

Perspective: the search for genes for type 2 diabetes in the post-genome era.

Twin and family studies have demonstrated a strong genetic component to type 2 diabetes mellitus (T2DM), but mapping the susceptibility genes that account for this risk has proved difficult. At least seven single gene defects are known to cause T2DM, often with early onset and insulin deficiency, but these causes account for 5% or less of all T2DM. A large number of candidate genes have been evaluated for typical T2DM, but few have been confirmed in multiple studies, and among these, the effect on individual risk is modest. A large number of genome-wide scans have been published in the last few years, and at least four regions show evidence in multiple studies. However, only NIDDM1 has been mapped to a single gene, and that gene (calpain 10) appears to have a major role only in selected populations. Work is ongoing in many laboratories and multiple populations to map additional regions, but T2DM and other complex diseases have proved recalcitrant to current methodology. In addition to the ongoing progress in completing the genome sequence and in developing a comprehensive map of single nucleotide polymorphisms, new statistical models will be needed to incorporate the multiple loci with modest effect and the known environmental interactions that characterize the susceptibility to T2DM.

Animals↗
PubMed1998

Potent inhibition of respiratory syncytial virus replication using a 2-5A-antisense chimera targeted to signals within the virus genomic RNA.

The 2-5A system is a recognized mechanistic component of the antiviral action of interferon. Interferon-induced 2-5A synthetase generates 2-5A, which, in turn, activates the latent constitutive RNase L that degrades viral RNA. Chemical conjugation of 2-5A to an antisense oligonucleotide can target the 2-5A-dependent RNase L to the antisense-specified RNA and effect its selective destruction. Such a 2-5A-antisense chimera (NIH351) has been developed that targets a consensus sequence within the respiratory syncytial virus (RSV) genomic RNA. NIH351 was 50- to 90-fold more potent against RSV strain A2 than was ribavirin, the presently approved drug for clinical management of RSV infection. It was similarly active against a variety of RSV strains of both A and B subgroups and possessed a cell culture selectivity index comparable to ribavirin. In addition, the anti-RSV activity of NIH351 was shown to be virus-specific and a result of a true antisense effect, because a scrambled nucleotide sequence in the antisense domain of NIH351 caused a significant decrease in antiviral activity. The 2-5A system's RNase L was implicated in the mechanism of action of NIH351 because a congener with a disabled 2-5A moiety was of greatly reduced anti-RSV effectiveness. These findings represent an innovative approach to the control of RSV replication.

Adenine Nucleotides↗
PubMed1983

Cloning of a human complement component C4 gene.

Six overlapping cosmid clones having an average insert size of 40 kilobase pairs were identified and isolated from a human genomic library by using a cDNA probe, Alu-7, specific for the amino acid sequence of C4d, a known region of the fourth component of human complement. Analysis of these genomic clones by restriction digestion and Southern blotting shows that all six probably contain the same complete C4 gene. Nucleotide sequence comparison of the genomic clone Cos-A and the cDNA clone Alu-7 shows an identical sequence except for the presence of a 1,500-base-pair intron in the genomic sequence. The amino acid sequence predicted from the nucleotide sequence agrees with the known C4d region amino acid sequence with one exception.

Amino Acid Sequence↗
PubMed2005

Gene finding in the chicken genome.

BACKGROUND: Despite the continuous production of genome sequence for a number of organisms, reliable, comprehensive, and cost effective gene prediction remains problematic. This is particularly true for genomes for which there is not a large collection of known gene sequences, such as the recently published chicken genome. We used the chicken sequence to test comparative and homology-based gene-finding methods followed by experimental validation as an effective genome annotation method. RESULTS: We performed experimental evaluation by RT-PCR of three different computational gene finders, Ensembl, SGP2 and TWINSCAN, applied to the chicken genome. A Venn diagram was computed and each component of it was evaluated. The results showed that de novo comparative methods can identify up to about 700 chicken genes with no previous evidence of expression, and can correctly extend about 40% of homology-based predictions at the 5' end. CONCLUSIONS: De novo comparative gene prediction followed by experimental verification is effective at enhancing the annotation of the newly sequenced genomes provided by standard homology-based methods.

Animals↗