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At least 829 records · Page 46Linked to original sources
PubMed2006

Parafibromin is a nuclear protein with a functional monopartite nuclear localization signal.

Parafibromin is a nuclear protein with a tumour suppressor role in the development of non-hereditary and hereditary parathyroid carcinomas, and the hyperparathyroidism-jaw tumour (HPT-JT) syndrome, which is associated with renal and uterine tumours. Nuclear localization signal(s), (NLS(s)), of the 61 kDa parafibromin remain to be defined. Utilization of computer-prediction programmes, identified five NLSs (three bipartite (BP) and two monopartite (MP)). To investigate their functionality, wild-type (WT) and mutant parafibromin constructs tagged with enhanced green fluorescent protein or cMyc were transiently expressed in COS-7 cells, or human embryonic kidney 293 (HEK293) cells, and their subcellular locations determined by confocal fluorescence microscopy. Western blot analyses of nuclear and cytoplasmic fractions from the transfected cells were also performed. WT parafibromin localized to the nucleus and deletions or mutations of the three predicted BP and one of the predicted MP NLSs did not affect this localization. In contrast, deletions or mutations of a MP NLS, at residues 136-139, resulted in loss of nuclear localization. Furthermore, the critical basic residues, KKXR, of this MP NLS were found to be evolutionarily conserved, and over 60% of all parafibromin mutations lead to a loss of this NLS. Thus, an important functional domain of parafibromin, consisting of an evolutionarily conserved MP NLS, has been identified.

Amino Acid Sequence↗
PubMedDate not supplied

Comparative genomics analysis of human sequence variation in the UGT1A gene cluster.

Common polymorphisms within the human UGT1A gene locus are associated with irinotecan and tranilast toxicity. To uncover additional functional variation across this gene cluster, cross-species sequence comparisons were performed. Evolutionarily conserved segments (a total of 47.1 kb) were re-sequenced in 24 African-American, 24 European-American, and 24 Asian individuals, and 381 segregating sites (including 123 singletons) were identified. Highly conserved coding sites were less likely to be polymorphic than diverged sites (P<0.0001) but this pattern was not observed at non-coding sites (P=0.1025). Among coding variants, the distribution of those computationally predicted to affect function was skewed toward low frequencies. Some alleles occurred at similar frequencies in each population; others had wide disparities. Although strong linkage disequilibrium was detected among the hepatically expressed genes, the degree of linkage disequilibrium varied among populations. These results suggest that rare functional gene variants and inter-population variability must be considered in the interpretation of association studies between UGT1A and drug metabolism/toxicity phenotypes.

Animals↗
PubMed2004

[Rh(I)L]-catalyzed cyclotetramerization of 1,3-butadiene: a theoretical investigation of alternative mechanistic paths for the generation of the [Rh(III)(octadienediyl)(PR3)]+ complex.

A detailed theoretical investigation of alternative mechanistic paths for the formation of the [Rh(III)(octadienediyl)(PiPr3)]+ complex is presented, employing a gradient-corrected density functional theory (DFT) method (BP86). This process represents most likely the first step in the recently reported [Rh(I)L]-catalyzed cyclotetramerization of butadiene (M. Bosch, M. S. Brookhart, K. Ilg and H. Werner, Angew. Chem., Int. Ed., 2000, 39, 2304). The favorable route for oxidative addition under C-C-bond formation starts from the prevalent [Rh(I)(butadiene)2(PiPr3)]+ form of the active catalyst through oxidative coupling between two cis-eta4-butadienes. This affords the [Rh(III)(bis-eta3-anti-octadienediyl)(PiPr3)]+ compound as the kinetic coupling product that consecutively undergoes transformation into the thermodynamically favorable bis-eta3-syn-octadienediyl-Rh(III) isomer via facile allylic conversions occurring in the octadienediyl framework. The computationally predicted energy profile is almost in quantitative agreement with the experimentally determined kinetics and allows a consistent rationalization of the experimental observations.

Journal Article↗
PubMed2005

An AU-rich stem-loop structure is a critical feature of the perinuclear localization signal of c-myc mRNA.

In eukaryotic cells, several mRNAs including those of c-myc and c-fos are localized to the perinuclear cytoplasm and associated with the cytoskeleton. The localization element of c-myc mRNA is present within its 3'UTR (3'-untranslated region) but the precise nature of this signal has remained unidentified. Chemical/enzymatic cleavage with RNases (ribonucleases) and lead have identified single-stranded and double-stranded regions in RNA transcripts of nucleotides 194-280 of the c-myc 3'UTR. Combined with computer predicted structure these results indicate that this region folds so that part of it forms a stem-loop structure. A mutation, that has been previously shown to prevent localization, leads to a different secondary RNA structure in this region as indicated by altered cleavage patterns. Competitive gel-retardation assays, using labelled transcripts corresponding to nucleotides 205-280 of c-myc 3'UTR, and fibroblast extracts revealed that the stem-loop region was sufficient for RNA-protein complex formation. In situ hybridization studies in cells transfected with reporter constructs, in which all or parts of the region corresponding to this stem-loop were linked to beta-globin, indicated that this region was sufficient for localization and that deletion of the nucleotides corresponding to the proposed upper-stem or terminal loop prevented localization. Our hypothesis is that an AU-rich stem-loop structure within nt 222-267 in the c-myc 3'UTR forms the perinuclear localization signal. Bioinformatic analysis suggests that this signal shares features with 3'UTRs of other localized mRNAs and that these features may represent a conserved form of signal in mRNA localization mechanisms.

3' Untranslated Regions↗
PubMed1985

A conformational study of a glutamine- and proline-rich cereal seed protein, C hordein.

A combination of c.d. spectroscopy and computer prediction is used to show that C hordein has an unusual secondary structure with an absence of alpha-helix and beta-sheet, but the presence of regularly repeated beta-turns. This is associated with a repetitive primary structure based mainly on blocks of eight residues. Similar spectral changes occurred when the protein was heated from 6 to 86 degrees C in aq. 70% (v/v) ethanol or dissolved in increasing concentrations (50-100%, v/v) of trifluoroethanol in water. The studies indicated that the conformation is stabilized by strong hydrophobic interactions and by extensive hydrogen-bonding.

Amino Acid Sequence↗
PubMed2001

Identification of antigenic domains on the human sodium-iodide symporter which are recognized by autoantibodies from patients with autoimmune thyroid disease.

The sodium-iodide symporter (NIS) is a novel autoantigen in autoimmune thyroid disease. In the present study we have characterized the antigenic domains on the human symporter which are recognized by autoantibodies from patients with either Graves' disease (GD) or autoimmune hypothyroidism (AH). Deletion derivatives of complementary DNA (cDNA) encoding the Na(+)/I(-) symporter were constructed using polymerase chain reaction (PCR) amplification. These deletion constructs were translated in vitro with the concomitant incorporation of [(35)S]methionine into the protein products. The reactivity of seven GD and six AH sera, which were known to contain symporter-binding antibodies, to each of the radiolabelled modified symporters was then determined in immunoprecipitation experiments. Analyses of the results obtained in the radiobinding assays suggest the existence of multiple antibody binding sites on human NIS (hNIS), including regions between amino acids (aa) 1--134, 191--286, 290--411, 411--520 and 520--588. Computer prediction of the potential B cell epitopes on the symporter revealed that, apart from aa 134--191, all the epitope domains identified overlapped, at least in part, with areas predicted to be highly antigenic. Interestingly, the antigenic domains represented by aa 191--286, 290--411 and 411--520 include regions of the polypeptide which form putative extracellular domains in the secondary structure model of the rat symporter. No correlation between the recognition of specific epitopes on the human symporter and the type of autoimmune thyroid disease was demonstrated.

Adult↗
PubMed2003

Autoantibodies in vitiligo patients recognize multiple domains of the melanin-concentrating hormone receptor.

Previously, we reported the identification of autoantibodies against the melanin-concentrating hormone receptor 1 in patients with vitiligo. In this study, the B cell epitopes on melanin-concentrating hormone receptor 1 that are recognized by these autoantibodies have been identified. Deletion derivatives of melanin-concentrating hormone receptor 1 complementary DNA were constructed and then translated in vitro with the concomitant incorporation of [35S]-methionine into the protein products. The [35S]-labeled melanin-concentrating hormone receptor 1 derivatives were subsequently used in radio-binding assays to investigate the reactivity of sera from nine vitiligo patients that were known to contain antibodies to the receptor. Analysis of the results obtained in the radio-binding assays suggested the existence of multiple antibody binding sites on melanin-concentrating hormone receptor 1, including regions between amino acids 1 to 138 and 139 to 298. Several patients exhibited autoantibodies to more than one melanin-concentrating hormone receptor 1 epitope indicating a heterogeneous humoral response to the receptor. Computer prediction of the potential B cell epitopes on melanin-concentrating hormone receptor 1 revealed that the epitope domains identified overlapped, at least in part, with regions predicted to be highly antigenic.

Adult↗
PubMed1985

[In vivo dosimetry in whole body irradiation].

The results of measurements on an Alderson phantom, as well as in-vivo-measurements, on five patients during total body irradiation are described. They were compared with computer-predicted values using the lateral A.P./P.A. irradiation, as used in Tübingen; measurements were carried out by a TLD method. Particular attention was paid to the critical areas such as the head, the neck and the lungs. The results in general agreed to within 5% and were in good accord with international findings.

Humans↗
PubMed2005

Optical and structural modeling of disclination lattices in carbonaceous mesophases.

An integrated microstructural and optical model for carbonaceous mesophases is developed and used to explain the principles that govern the formation and stability of experimentally observed disclination lattices. The model is able to capture the orientation features of disclination lattices, including the type and location of disclination lines, and the orientation field in the mesophase matrix. The optical model based on reflection polarized optical microscopy is able to replicate all the details observed in actual observations. The typical brush figures have the proper distribution, orientation, and intensity. The computational predictions offer science-based routes to create and control desirable material architectures based on carbonaceous mesophase-carbon fiber composites.

Journal Article↗
PubMed2005

A data integration methodology for systems biology: experimental verification.

The integration of data from multiple global assays is essential to understanding dynamic spatiotemporal interactions within cells. In a companion paper, we reported a data integration methodology, designated Pointillist, that can handle multiple data types from technologies with different noise characteristics. Here we demonstrate its application to the integration of 18 data sets relating to galactose utilization in yeast. These data include global changes in mRNA and protein abundance, genome-wide protein-DNA interaction data, database information, and computational predictions of protein-DNA and protein-protein interactions. We divided the integration task to determine three network components: key system elements (genes and proteins), protein-protein interactions, and protein-DNA interactions. Results indicate that the reconstructed network efficiently focuses on and recapitulates the known biology of galactose utilization. It also provided new insights, some of which were verified experimentally. The methodology described here, addresses a critical need across all domains of molecular and cell biology, to effectively integrate large and disparate data sets.

Chromatin Immunoprecipitation↗
PubMed2006

Flexibly varying folding mechanism of a nearly symmetrical protein: B domain of protein A.

The folding pathway of the B domain of protein A is the pathway most intensively studied by computer simulations. Recent systematic measurement of Phi values by Sato et al., however, has shown that none of the published computational predictions is consistent with the detailed features of the experimentally observed folding mechanism. In this article we use a statistical mechanical model of folding to show that sensitive dependence of multiple transition state ensembles on temperature and the denaturant concentration is the key to resolving the inconsistency among simulations and the experiment. Such sensitivity in multiple transition state ensembles is a natural consequence of symmetry-breaking in a nearly symmetrical protein.

Dimerization↗
PubMed2006

Genes for the peptidoglycan synthesis pathway are essential for chloroplast division in moss.

The general consensus is that a cyanobacterium phagocytosed by a host cell evolved into the plastids of red and green algae, land plants, and glaucophytes. In contrast to the plastids of glaucophytes, which retain a cyanobacterial-type peptidoglycan layer, no wall-like structures have been detected in plastids from other sources. Although the genome of Arabidopsis thaliana contains five genes that are essential for peptidoglycan synthesis, MurE, MurG, two genes for D-Ala-D-Ala ligase (Ddl), and the gene for translocase I (MraY), their functions have not been determined. We report that the moss Physcomitrella patens has nine homologous genes related to peptidoglycan biosynthesis: MurA, B, C, D, E, and F, Ddl, genes for the penicillin-binding protein Pbp, and dd-carboxypeptidase (Dac). Corroborating a computer prediction, analysis of the GFP fusion proteins with the N terminus of PpMurE or of PpPbp suggests that these proteins are located in the chloroplasts. Gene disruption of the PpMurE gene in P. patens resulted in the appearance of macrochloroplasts both in protonema and in leaf cells. Moreover, gene knockout of the P. patens Pbp gene showed inhibition of chloroplast division in this moss; however, no Pbp gene was found in A. thaliana.

Arabidopsis↗
PubMed2003

The conformation of neurotensin bound to its G protein-coupled receptor.

G protein-coupled receptors (GPCRs) mediate the perception of smell, light, taste, and pain. They are involved in signal recognition and cell communication and are some of the most important targets for drug development. Because currently no direct structural information on high-affinity ligands bound to GPCRs is available, rational drug design is limited to computational prediction combined with mutagenesis experiments. Here, we present the conformation of a high-affinity peptide agonist (neurotensin, NT) bound to its GPCR NTS-1, determined by direct structural methods. Functional receptors were expressed in Escherichia coli, purified in milligram amounts by using optimized procedures, and subsequently reconstituted into lipid vesicles. Solid-state NMR experiments were tailored to allow for the unequivocal detection of microgram quantities of 13C,15N-labeled NT(8-13) in complex with functional NTS-1. The NMR data are consistent with a disordered state of the ligand in the absence of receptor. Upon receptor binding, the peptide undergoes a linear rearrangement, adopting a beta-strand conformation. Our results provide a viable structural template for further pharmacological investigations.

GTP-Binding Proteins↗
PubMed1985

Human apolipoprotein B-100: cloning, analysis of liver mRNA, and assignment of the gene to chromosome 2.

Human apolipoprotein B-100 (apo B-100) is the major apolipoprotein of low density lipoproteins and the principal ligand for interaction with the low density lipoprotein receptor. The human apo B-100 gene has been inserted into a lambda gt-11 expression vector, and the apo B-100 cDNA clones have been identified by screening with a monospecific apo B-100 antiserum, by screening with synthetic oligonucleotides based on the amino acid sequence of peptides isolated from apo B-100, and by immunoblot analysis of the expressed protein with a monoclonal antibody to apo B-100. The complete nucleotide and derived-amino acid sequence of a 1.7-kilobase cDNA clone of apo B-100 was determined. The 560-amino acid residues of apo B-100 contain no unique linear or repeating sequences of amino acids. The computer-predicted conformation of the apo B-100 protein contains segments of helical structure; however, a large portion of the protein is organized into beta-structure. The beta-structure may be important in lipid-apo B-100 interactions in low density lipoprotein and may contribute to the insolubility of delipidated apo B-100 in aqueous buffers. RNA blot hybridization analysis of liver mRNA utilizing a Nco I/HindIII apo B-100 cDNA probe revealed that the apo B-100 mRNA is 15-18 kilobases long, which is of sufficient size to code for a 250-387 kDa apolipoprotein, the proposed molecular size of delipidated plasma apo B-100. The gene for human apo B-100 has been localized to chromosome 2 by filter hybridization of human-mouse somatic cell hybrids utilizing a 400-base-pair Nco I/HindIII apo B-100 cDNA probe. This location is in contrast to the low density lipoprotein receptor that has been localized to chromosome 19. The cloning of human apo B-100 has provided new insights into the structure and physicochemical properties of apo B-100 and will facilitate studies on the factors modulating apo B-100 biosynthesis and the expression of the apo B-100 gene in patients with dyslipoproteinemias.

Amino Acid Sequence↗
PubMed1993

Molecular and biological changes in the cold-adapted "master strain" A/AA/6/60 (H2N2) influenza virus.

The live cold-adapted (ca) A/AA/6/60 influenza vaccine is being commercially developed for worldwide use in children and is being used as a model for other live vaccines. Although it has been proven safe and immunogenic, the molecular basis of cold adaptation has never been determined. To identify sequence changes responsible for cold adaptation, we have compared the sequence of the master ca vaccine strain to its progenitor wild-type virus, wt A/AA/6/60 E2 (wt2). Only 4 nt differences encoding 2 aa differences were found in three gene segments. Computer-predicted RNA folds project different secondary structures between the ca and wt2 molecules based on the two silent differences between them. Genes coding for the acidic polymerase, matrix, and nonstructural proteins are identical between the two viruses. The few differences found in the ca A/AA/6/60 virus after its long stepwise passage at 25 degrees C in primary chicken kidney cells suggest that cold adaptation resulted in greater genetic stability for the highly variable RNA genome.

Adaptation, Physiological↗
PubMed1999

Mapping the molecular determinant of pathogenicity in a hammerhead viroid: a tetraloop within the in vivo branched RNA conformation.

Chrysanthemum chlorotic mottle viroid (CChMVd) is an RNA of 398-399 nt that can adopt hammerhead structures in both polarity strands. We have identified by Northern-blot hybridization a nonsymptomatic strain (CChMVd-NS) that protects against challenge inoculation with the symptomatic strain (CChMVd-S). Analysis of CChMVd-NS cDNA clones has revealed a size and sequence very similar to those of the CChMVd-S strain. Some of the mutations observed in CChMVd-NS molecular variants were previously identified in CChMVd-S RNA, but others were never found in this RNA. When bioassayed in chrysanthemum, cDNA clones containing the CChMVd-NS specific mutations were infectious but nonsymptomatic. Site-directed mutagenesis showed that one of the CChMVd-NS-specific mutations, a UUUC --> GAAA substitution, was sufficient to change the symptomatic phenotype into the nonsymptomatic one without altering the final accumulation level of the viroid RNA. The pathogenicity determinant-to our knowledge, a determinant of this class has not been described previously in hammerhead viroids-is located in a tetraloop of the computer-predicted branched conformation for CChMVd RNA. Analysis of the sequence heterogeneity found in CChMVd-S and -NS variants strongly supports the existence of such a conformation in vivo, showing that the rod-like or quasi-rod-like secondary structure is not a universal paradigm for viroids.

Asteraceae↗
PubMed1999

A model-based approach for assessing in vivo combination therapy interactions.

We present an approach for evaluating the efficacy of combination antitumor agent schedules that accounts for order and timing of drug administration. Our model-based approach compares in vivo tumor volume data over a time course and offers a quantitative definition for additivity of drug effects, relative to which synergism and antagonism are interpreted. We begin by fitting data from individual mice receiving at most one drug to a differential equation tumor growth/drug effect model and combine individual parameter estimates to obtain population statistics. Using two null hypotheses: (i) combination therapy is consistent with additivity or (ii) combination therapy is equivalent to treating with the more effective single agent alone, we compute predicted tumor growth trajectories and their distribution for combination treated animals. We illustrate this approach by comparing entire observed and expected tumor volume trajectories for a data set in which HER-2/neu-overexpressing MCF-7 human breast cancer xenografts are treated with a humanized, anti-HER-2 monoclonal antibody (rhuMAb HER-2), doxorubicin, or one of five proposed combination therapy schedules.

Animals↗
PubMed1999

HSP70 homolog functions in cell-to-cell movement of a plant virus.

Plant closteroviruses encode a homolog of the HSP70 (heat shock protein, 70 kDa) family of cellular proteins. To facilitate studies of the function of HSP70 homolog (HSP70h) in viral infection, the beet yellows closterovirus (BYV) was modified to express green fluorescent protein. This tagged virus was competent in cell-to-cell movement, producing multicellular infection foci similar to those formed by the wild-type BYV. Inactivation of the HSP70h gene by replacement of the start codon or by deletion of 493 codons resulted in complete arrest of BYV translocation from cell to cell. Identical movement-deficient phenotypes were observed in BYV variants possessing HSP70h that lacked the computer-predicted ATPase domain or the C-terminal domain, or that harbored point mutations in the putative catalytic site of the ATPase. These results demonstrate that the virus-specific member of the HSP70 family of molecular chaperones functions in intercellular translocation and represents an additional type of a plant viral-movement protein.

Adenosine Triphosphatases↗