Glucosylceramide,a neutral glycosphingolipid anticoagulant cofactor,enhances the interaction of human- and bovine-activated protein C with negatively charged phospholipid vesicles

The effect of glucosylceramide (GlcCer) on activated protein C (APC)-phospholipid interactions was examined using fluorescence resonance energy transfer. Human APC, labeled with either fluorescein (Fl-APC) or dansyl (DEGR-APC) donor, bound to phosphatidylcholine/phosphatidylserine (PC/PS, 9:1 w/w) vesicles containing octadecylrhodamine (OR) acceptor with a K(d) (app) = 16 micro g/ml, whereas Fl-APC (or DEGR-APC) bound to PC/PS/GlcCer(OR) (8:1:1) vesicles with a K(d) (app) = 3 micro g/ml. This 5-fold increase in apparent affinity was not species-specific since bovine DEGR-APC also showed a similar GlcCer-dependent enhancement of binding of APC to PC/PS vesicles. From the efficiency of fluorescence resonance energy transfer, distances of closest approach of approximately 63 and approximately 64 A were estimated between the dansyl on DEGR-APC and rhodamine in PC/PS/GlcCer(OR) and PC/PS(OR), respectively, assuming kappa(2) = 2/3. DEGR-APC bound to short chain C8-GlcCer with an apparent K(d) of 460 nm. The presence of C8-GlcCer selectively enhanced the binding of C16,6-NBD-phosphatidylserine but not C16,6-7-nitrobenz-2-oxa-1,3-diazole (NBD)-phosphatidylcholine to coumarin-labeled APC. These data suggest that APC binds to GlcCer, that PC/PS/GlcCer vesicles like PC/PS vesicles bind to the N-terminal gamma-carboxyglutamic acid domain of APC, and that one mechanism by which GlcCer enhances the activity of APC is by increasing its affinity for membrane surfaces containing negatively charged phospholipids.     

The central domain is required to target and anchor perilipin A to lipid droplets

The perilipins are the most abundant proteins coating the surfaces of lipid droplets in adipocytes and are found at lower levels surrounding lipid droplets in steroidogenic cells. Perilipins drive triacylglycerol storage in adipocytes by regulating the rate of basal lipolysis and are also required to maximize hormonally stimulated lipolysis. To map the domains that target and anchor perilipin A to lipid droplets, we stably expressed fragments of perilipin A in 3T3-L1 fibroblasts. Immunofluorescence microscopy and immunoblotting of proteins from isolated lipid droplets revealed that neither the amino nor the carboxyl terminus is required to target perilipin A to lipid droplets; however, there are multiple, partially redundant targeting signals within a central domain including 25% of the primary amino acid sequence. A peptide composed of the central domain of perilipin A directed a fused green fluorescent protein to the surfaces of lipid droplets. Full-length perilipin A associates with lipid droplets via hydrophobic interactions, as shown by the persistence of perilipins on lipid droplets after centrifugation through an alkaline carbonate solution. Results of the mutagenesis studies indicate that the sequences responsible for anchoring perilipin A to lipid droplets are most likely domains of moderately hydrophobic amino acids located within the central 25% of the protein. Thus, we conclude that the central 25% of the perilipin A sequence contains all of the amino acids necessary to target and anchor the protein to lipid droplets.     

Differentiation potential of intestinal mesenchyme and its interaction with epithelial cells: a study using beta-galactosidase-expressing fibroblast lines

Rat intestinal fibroblast lines (F1:G9 and A1:F1) differing in their potential to support intestinal mucosal development were marked with reporter genes to investigate their differentiation potential. The fibroblasts were transfected with plasmids expressing either beta-galactosidase (with or without a nuclear localisation signal) or green fluorescent protein (GFP). Transfection using Tfx50 or Fugene was more efficient than electroporation. The expression of beta-galactosidase was more stable and stronger than GFP. Cells were optimally labelled using the plasmid pL27B-GAL, and sub-clones with a strong and uniform nuclear expression of beta-galactosidase were isolated. These clones expressed beta-galactosidase even after prolonged passage in the absence of selection. The beta-galactosidase tagged lines (F1:G9gal and A1:F1gal) retained the morphological characteristics, viability and differentiation properties of the parental non-transfected lines. In co-culture with a colorectal tumour cell line Caco-2, the F1:G9gal and A1:F1gal cells differed in their morphological organisation but this did not change their expression of smooth muscle alpha-actin.     

Requirement of BAX for efficient adenovirus-induced apoptosis

Infection of human epithelial cells with adenoviruses induces an apoptosis paradigm that is efficiently suppressed by the expression of viral E1B-19K protein, which is a functional homolog of the cellular antiapoptosis protein BCL-2. The mechanisms of adenovirus (Ad)-induced apoptosis appear to involve the cellular BCL-2 family proapoptotic proteins. Recent genetic studies with fibroblasts derived from mutant mouse embryos indicate that a class of the BCL-2 family proapoptotic proteins (designated BH-123 or multidomain proteins) such as BAX and BAK constitutes an essential component of the core apoptosis machinery in animal cells. We have examined the role of BAX in Ad-induced apoptosis in human epithelial cells using two colon cancer cell lines, HCT116Bax (Bax(+/-)) and HCT116BaxKO (Bax(-/-)) (L. Zhang, J. Yu, B. H. Park, K. W. Kinzler, and B. Vogelstein, Science 290:989-992, 2000). Infection of Bax(+/-) cells with an Ad type 2 mutant (dl250) defective in expression of the E1B-19K protein resulted in enhanced cytopathic effect, large plaques on cell monolayers, fragmentation of cellular DNA, and enhanced cell death. These mutant phenotypes were not efficiently expressed in Bax(-/-) cells, suggesting that BAX is essential for Ad-induced apoptosis. Infection of Bax(+/-) cells with dl250 induced increased levels of an N-terminally processed form of BAX. Cells infected with the 19K mutant also contained enhanced levels of truncated BAX in membrane-inserted form. Our results suggest that at least a part of the mechanism utilized by E1B-19K to suppress apoptosis during Ad infection may involve modulation of the activities of BAX.     

MRD: a microsatellite repeats database for prokaryotic and eukaryotic genomes

MRD is a database system to access the microsatellite repeats information of genomes such as archea, eubacteria, and other eukaryotic genomes whose sequence information is available in public domains. MRD stores information about simple tandemly repeated k-mer sequences where k= 1 to 6, i.e. monomer to hexamer. The web interface allows the users to search for the repeat of their interest and to know about the association of the repeat with genes and genomic regions in the specific organism. The data contains the abundance and distribution of microsatellites in the coding and non-coding regions of the genome. The exact location of repeats with respect to genomic regions of interest (such as UTR, exon, intron or intergenic regions) whichever is applicable to organism is highlighted. MRD is available on the World Wide Web at and/or . The database is designed as an open-ended system to accommodate the microsatellite repeats information of other genomes whose complete sequences will be available in future through public domain.     

Ab initio structure of human seminal plasma prostatic inhibin gives significant insight into its biological functions

Human seminal plasma prostatic inhibin (HSPI) is a protein isolated from the human prostate gland. Despite its profound biomedical and biotechnological importance, the 3D structure of this protein of 94 amino acids remains undeciphered. The difficulties in extracting it in pure form and crystallizing it have restrained the determination of its structure experimentally. The homology-based computational methods are also not applicable, as HSPI lacks sufficient sequence homology with known structures in the protein data banks. We have predicted the structure of HSPI by a knowledge-based method using nonparametric multivariate statistical techniques. Stereochemical and other standard validation tests confirm this to be a well-refined structure. The biophysical properties exhibited by this structure are in good agreement with the NMR experimental observations. Docking and other computational studies on this structure provide significant explanation and insight into its binding activities and related biological and immunogenic functions and offer new directions for its potential applications.     

Harlequin (hlq) and short blue root (sbr), two Arabidopsis mutants that ectopically express an abscisic acid- and auxin-inducible transgenic carrot promoter and have pleiotropic effects on morphogenesis

Plant growth and development is regulated by complex interactions among different hormonal, developmental and environmental signalling pathways. Isolation of mutants in these processes is a powerful approach to dissect unknown mechanisms in regulatory networks. The plant hormones abscisic acid (ABA) and auxin are involved in vegetative, developmental and environmental growth responses, including cell division and elongation, vascular tissue differentiation and stress adaptation. The uidA (-glucuronidase; GUS) reporter gene driven by the carrot (Daucus carota) late embryogenesis-abundant Dc3 promoter in transgenic Arabidopsis thaliana seedlings is ABA-inducible in the root zone of elongation and vasculature. We show here that the ABA-insensitive2-1 mutation (abi2) reduces ABA-inducible Dc3-GUS expression in these root tissues. Dc3-GUS expression is also induced in root cortex cells by indole-3-acetic acid. We mutagenized, with ethyl methane sulfonate, 5100 M₁ abi2/abi2 homozygous plants of a line that carries two independent Dc3-GUS reporter genes and screened M₂ clonal lines for ABA-inducible Dc3-GUS expression in roots. We isolated two novel single-gene nuclear mutants, harlequin (hlq) and short blue root (sbr), that ectopically express Dc3-GUS in roots and have pleiotropic effects on morphogenesis. The hlq mutant expresses Dc3-GUS in a checkered pattern in epidermis of roots and hypocotyls, accumulates callose and has deformed and collapsed epidermal cells and abnormal and reduced root hairs and leaf trichomes. It (hlq) is also dwarfed, skotomorphogenic and sterile. The sbr mutant is a seedling-lethal dwarf that over-expresses Dc3-GUS in the root and has radially swollen epidermal cells in the root and hypocotyl, supernumerary cell number in the root cortex and epidermis, abnormal vasculature, and abnormal epidermal cell patterning in cotyledons and leaves. It (sbr) also exhibits a semidominant root phenotype of reduced growth and lateral root initiation. The hlq and sbr mutants are not rescued by exogenous application of plant growth regulators. The hlqand sbr mutants do not require the abi2-1 mutant gene for their phenotypes and map to chromosome III and I, respectively. Further characterization of the hlq and sbr phenotypes and genes may provide insights into the relationship of hormone- and stress-regulated gene expression to morphogenesis and plant growth.     

Identification of key concepts in biomedical literature using a modified Markov heuristic

MOTIVATION: The recent explosion of interest in mining the biomedical literature for associations between defined entities such as genes, diseases and drugs has made apparent the need for robust methods of identifying occurrences of these entities in biomedical text. Such concept-based indexing is strongly dependent on the availability of a comprehensive ontology or lexicon of biomedical terms. However, such ontologies are very difficult and expensive to construct, and often require extensive manual curation to render them suitable for use by automatic indexing programs. Furthermore, the use of statistically salient noun phrases as surrogates for curated terminology is not without difficulties, due to the lack of high-quality part-of-speech taggers specific to medical nomenclature. RESULTS: We describe a method of improving the quality of automatically extracted noun phrases by employing prior knowledge during the HMM training procedure for the tagger. This enhancement, when combined with appropriate training data, can greatly improve the quality and relevance of the extracted phrases, thereby enabling greater accuracy in downstream literature mining tasks.     

Patterns of transitional mutation biases within and among mammalian genomes

Significant transition/transversion mutation bias is a well-appreciated aspect of mammalian nuclear genomes; however, patterns of bias among genes within a genome and among species remain largely uncharacterized. Understanding these patterns is important for understanding similarities and differences in mutational patterns among genomes and genomic regions. Therefore, we have conducted an analysis of 7,587 pairs of sequences of 4,347 mammalian protein-coding genes from seven species (human, mouse, rat, cow, sheep, pig, and macaque) and from the introns of 51 gene pairs and multiple intergenic regions (37 kbp, 52 kbp and 65 kbp) from the human, chimpanzee, and baboon genomes. Our analyses show that genes and regions with widely varying base composition exhibit uniformity of transition mutation rate both within and among mammalian lineages, as long as the transitional mutations caused by CpG hypermutability are excluded. The estimates show no relationship to potential intrachromosomal or interchromosomal effects. This uniformity points to similarity in point mutation processes in genomic regions with substantially different GC-content biases.