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PubMed1993

Cooperative assembly of the bovine papilloma virus E1 and E2 proteins on the replication origin requires an intact E2 binding site.

Using quantitative gel retardation assays the properties of the bovine papilloma virus (BPV) origin recognition protein E1 and the effect of the viral E2 protein on the binding of E1 to BPV origin DNA were examined. As reported previously (Seo, Y.S., Mueller, F., Lusky, M., Gibbs, E., Kim, H.-Y., Phillips, B. and J. Hurwitz (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2865-2869), the E1 protein binds specifically to DNA sequences within the BPV origin (ori+) of replication. We also show that the presence of MgCl2 and ATP could stabilize the E1 ori+ DNA complex. At low levels of E1, ori+ DNA binding was greatly stimulated by the viral E2 protein when the intact E2 binding site 12 was present on the DNA. In addition DNA-protein complexes formed in the presence of both E1 and E2 were more stable than those formed with E1 alone. In the absence of an E2 binding site the E2 protein inhibited the binding of E1 to the BPV origin. Spacing of 0 or 9 base pairs between the E1 binding site and the E2 binding site 12 abolished the stimulation of E1-DNA binding by E2, whereas spacing of 6 base pairs between the two binding sites allowed for efficient stimulation. The data presented account for a direct role of E2 in BPV DNA replication. We propose that the cooperative binding of both the E1 and E2 proteins to BPV ori+ DNA is mediated by protein-protein interactions and by protein-DNA interactions, which include the formation of specific contacts of E2 with DNA.

Adenosine Triphosphate↗

PubMed1998

Perceptual learning.

Perceptual learning involves relatively long-lasting changes to an organism's perceptual system that improve its ability to respond to its environment. Four mechanisms of perceptual learning are discussed: attention weighting, imprinting, differentiation, and unitization. By attention weighting, perception becomes adapted to tasks and environments by increasing the attention paid to important dimensions and features. By imprinting, receptors are developed that are specialized for stimuli or parts of stimuli. By differentiation, stimuli that were once indistinguishable become psychologically separated. By unitization, tasks that originally required detection of several parts are accomplished by detecting a single constructed unit representing a complex configuration. Research from cognitive psychology, psychophysics, neuroscience, expert/novice differences, development, computer science, and cross-cultural differences is described that relates to these mechanisms. The locus, limits, and applications of perceptual learning are also discussed.

Attention↗

PubMed1998

The high-potential flavin and heme of nitric oxide synthase are not magnetically linked: implications for electron transfer.

BACKGROUND: The homodimeric nitric oxide synthase (NOS) catalyzes conversion of L-arginine to L-citrulline and nitric oxide. Each subunit contains two flavins and one protoporphyrin IX heme. A key component of the reaction is the transfer of electrons from the flavins to the heme. The NOS gene encodes two domains linked by a short helix containing a calmodulin-recognition sequence. The reductase domain binds the flavin cofactors, while the oxygenase domain binds heme and L-arginine and additionally mediates the dimerization of the NOS subunits. We investigated the origin of the unusual magnetic properties (rapid-spin relaxation) of an air-stable free radical localized to a reductase domain flavin cofactor. RESULTS: We characterized the air-stable flavin in wild-type NOS, both in the presence and absence of calcium and calmodulin, the imidazole-bound heme complex of wild-type NOS, the NOS Cys415-->Ala mutant, and the isolated reductase domain. All preparations of NOS had the same flavin electron-spin relaxation behavior. No half-field transitions or temperature-dependent changes in the linewidth of the radical spin signal were detected. CONCLUSIONS: These data suggest that the observed relaxation enhancement of the NOS flavin radical is caused by the environment provided by the reductase domain. No magnetic interaction between the heme and flavin cofactors was detected, suggesting that the flavin and heme centers are probably separated by more than 15 A.

Animals↗

PubMed2003

Recognition of the clinical signs and symptoms of Joubert syndrome.

Joubert Syndrome is a rare, autosomal recessive disorder that affects the cerebellum and brain stem. It presents with a distinct respiratory pattern and profound tachypnea in the newborn period. This article provides an overview of the condition and discusses the embryologic origins of this syndrome. A focused history and systematic physical assessment provide a step-by-step guide to enhance the early recognition of clinical signs and symptoms of this disorder. A series of clinical photographs and a brief case report offer insight into the classic presentation of this uncommon disorder. The diagnosis of Joubert syndrome is confirmed with magnetic resonance imaging, which reveals a classic neuroradiologic finding, characterized as the molar tooth sign. A discussion of the range of developmental outcomes and complex multispecialty care and intensive support that these infants and their families require is also provided.

Abnormalities, Multiple↗

PubMed1995

The tissue-specific nuclear matrix protein, NMP-2, is a member of the AML/CBF/PEBP2/runt domain transcription factor family: interactions with the osteocalcin gene promoter.

The nuclear matrix protein, NMP-2, was originally identified as an osteoblast-specific DNA-binding complex localized exclusively to the nuclear matrix. NMP-2 was shown to recognize two binding sites, site A (nt-605 to -599) and site B (nt -441 to -435), in the rat bone-specific osteocalcin gene promoter. This study shows that the NMP-2 binding sites A and B as well as a third NMP-2 binding site (nt -135 to -130) constitute a consensus sequence, ATGCTGGT, and represent an AML-1 recognition motif. AML-1 is a member of the AML transcription factor family which is associated with acute myelogenous leukemia and binds to the sequence TGCTGGT via its DNA-binding runt domain. Electrophoretic mobility shift assays reveal that a component of NMP-2 is a member of the AML/PEBP2/runt domain transcription factor family based on cross-competition with AML-1 consensus oligonucleotide. Limited immunoreactivity of NMP-2 with a polyclonal N-terminal AML-1 antibody and inability of the AML-1 partner protein CBF-beta to form complexes with NMP-2 indicate that NMP-2 is not identical to AML-1 but represents a variant AML/PEBP2/runt domain protein. Western and Northern blots reveal the presence of multiple AML-related proteins and AML-1 transcripts in several osseous cell lines. Furthermore, our results indicate that AML family members may selectively partition between nuclear matrix and nonmatrix compartments. Because proteins that contain a runt domain are implicated in tissue-specific transcriptional regulation, our results support the concept that the nuclear matrix mediates osteoblast-specific expression of the osteocalcin gene.

Animals↗

PubMed2000

A concise genetic and clinical guide to multiple endocrine neoplasias and related syndromes.

Several familial neoplastic syndromes are associated with endocrine gland oncogenesis. The main ones are: multiple endocrine neoplasia type 1 (MEN 1), which affects primarily the pituitary, pancreas, and parathyroid glands; MEN 2A and MEN 2B, which involve mainly the thyroid and parathyroid glands and the adrenal medulla; familial medullary thyroid carcinoma (FMTC), which affects only the thyroid gland; and, finally, Carney complex, which affects the adrenal cortex, pituitary, thyroid gland, and the gonads. Carney complex is also associated with pigmentation abnormalities and myxoid and other neoplasms of mesenchymal origin. Thus, this syndrome also belongs to another group of genetic disorders, those associated with pigmentation defects and multiple tumors, including tumors of the endocrine glands. Peutz-Jeghers syndrome and Cowden disease are just two of these disorders that have recently been elucidated at the molecular level. von Hippel-Lindau disease is another condition that affects the pancreas and adrenal medulla and its gene is also known. The inheritance of the MENs, Carney complex, and related syndromes is autosomal dominant. Clinical recognition of these syndromes at a young age improves clinical outcome and prognosis of the various tumors and decreases associated morbidity and mortality. This review considers a wider, more inclusive view of the MEN syndromes, summarizes their clinical features and presents the newest information on their molecular elucidation.

Humans↗

PubMed2003

Direct and specific recognition of glycosaminoglycans by antibodies after their separation by agarose gel electrophoresis and blotting on cetylpyridinium chloride-treated nitrocellulose membranes.

A method for the immunodetection of several natural complex polysaccharides (glycosaminoglycans) after their separation by conventional agarose gel electrophoresis, blotting and immobilizing on nitrocellulose membranes derivatized with the cationic detergent cetylpyridinium chloride (CPC), and direct and specific immunodetection by antibodies is described. This new approach is based on the principles that were used to develop the Western blot, and is applied to the separation of the glycosaminoglycans purified from normal human urine. After migration in agarose gel electrophoresis, chondroitin sulfate samples of different origin were blotted and transferred onto nitrocellulose membranes treated with CPC. Immunodetection was performed using the anti-chondroitin-6-sulfate antibody that specifically recognizes intact chondroitin-6-sulfate. By calculating the ratio between the antibody staining (epitope) and alcian blue staining (mass), the epitope density expressed as a percentage, i.e., the number of repetitive epitopes per mass, was obtained. These values were in agreement with the quantitation of 6-sulfated groups of chondroitin sulfate performed by the evaluation of unsatured disaccharide-6-sulfate (DeltaDi6S) produced after treatment with chondroitinase ABC and separated by high-performance liquid chromatography (HPLC). Furthermore, immunodetection of heparan sulfate was performed using the anti-heparan sulfate antibody.

Antibodies↗

PubMed1983

Epitope specificity and Ia restriction of T cell responses to insulin in a system of complementing Ir genes: analysis with primed lymph node T cells and a long-term cultured T cell line.

The antibody response of (H-2b X H-2k)F1 mice to pig insulin (PI) has previously been shown to be under the control of H-2-linked, complementing Ir genes. In addition, this response was reported to depend on the genetic background of the parental strains (Keck, K., Eur. J. Immunol. 1977. 7: 811). Here it is demonstrated that the secondary in vitro response of proliferating T cells shows the same dependence on H-2-linked Ir genes yet an influence of the background genes could not be detected. The complementing genes were mapped to the Kb, I-Ab and Kk, I-Ak regions. For restimulation of F1 T cells by PI, the Ir genes of both parental chromosomes have to be expressed in the same antigen-presenting cell, suggesting complementation at the molecular rather than at a cell interaction level. With a long-term cultured, PI-specific T cell line (ST2) of (B10 X B10.BR)F1 origin the complementation data could be confirmed by mapping the Ia restriction elements to Kb, I-Ab and I-Ak. The reactivity pattern of this line towards species variants of insulin and the isolated A and B polypeptide chains in the presence of syngeneic accessory cells suggests that the glutamic acid residue in position 4 of the A polypeptide chain (Asp in mouse insulin) is essential for recognition in conjunction with an (I-Ab X I-Ak)F1 hybrid Ia complex. I-Ab-encoded molecules carrying specificity Ia. W39 which, according to Rosenwasser, L. J. and Huber, B. T. are essential for the presentation of BI to (CBA/N X C57BL/6)F1 T cells, are not required as components of the F1-unique restriction element recognized by the F1 T cells of the ST2 line in conjunction with PI. This is indicated by the fact that accessory cells of (CBA/N X B10)F1 hybrids, regardless of their sex, could present PI as well as beef, sheep and horse insulin to the F1-restricted ST2 cells.

Animals↗

PubMed1998

The how and why of protein-carbohydrate interaction: a primer to the theoretical concept and a guide to application in drug design.

The common principles of molecular recognition with cooperative or bidentate hydrogen bonds, dispersion forces and hydrophobic packing govern the specificity of protein-carbohydrate interaction. Enthalpy/entropy-compensation is also valid, maintaining KD-values in the range of 30 mM to 200 nM. The individual contributions of the enthalpic and entropic factors which originate from the receptor, the ligand and/or the solvent to the overall free energy change can at least be estimated by a combination of computer-assisted molecular modeling, NMR spectroscopy of the reactants before and after complex formation and thermodynamic measurements. The delineation of adaptable parameters such as ligand or receptor side chain flexibility points to a route to practicable guidelines for a rational design of mimetics in glycosciences.

Carbohydrates↗

PubMed2005

GENETAG: a tagged corpus for gene/protein named entity recognition.

BACKGROUND: Named entity recognition (NER) is an important first step for text mining the biomedical literature. Evaluating the performance of biomedical NER systems is impossible without a standardized test corpus. The annotation of such a corpus for gene/protein name NER is a difficult process due to the complexity of gene/protein names. We describe the construction and annotation of GENETAG, a corpus of 20K MEDLINE sentences for gene/protein NER. 15K GENETAG sentences were used for the BioCreAtIvE Task 1A Competition. RESULTS: To ensure heterogeneity of the corpus, MEDLINE sentences were first scored for term similarity to documents with known gene names, and 10K high- and 10K low-scoring sentences were chosen at random. The original 20K sentences were run through a gene/protein name tagger, and the results were modified manually to reflect a wide definition of gene/protein names subject to a specificity constraint, a rule that required the tagged entities to refer to specific entities. Each sentence in GENETAG was annotated with acceptable alternatives to the gene/protein names it contained, allowing for partial matching with semantic constraints. Semantic constraints are rules requiring the tagged entity to contain its true meaning in the sentence context. Application of these constraints results in a more meaningful measure of the performance of an NER system than unrestricted partial matching. CONCLUSION: The annotation of GENETAG required intricate manual judgments by annotators which hindered tagging consistency. The data were pre-segmented into words, to provide indices supporting comparison of system responses to the "gold standard". However, character-based indices would have been more robust than word-based indices. GENETAG Train, Test and Round1 data and ancillary programs are freely available at ftp://ftp.ncbi.nlm.nih.gov/pub/tanabe/GENETAG.tar.gz. A newer version of GENETAG-05, will be released later this year.

Animals↗

PubMed1988

High titer anti-HIV antibody reactivity associated with a paraprotein spike in a homosexual male with AIDS related complex.

We observed a human immunodeficiency virus (HIV)-infected homosexual male with AIDS related complex (ARC) who had a serum globulin level of 80 g/L. Serum protein electrophoresis revealed a gamma globulin fraction of 40 g/L, of which 50% (20 g/L) was contained within a paraprotein spike, comprised predominantly of IgG kappa. This patient also had high titer anti-HIV antibodies in his serum, which were Western blot reactive at a final dilution of 1:500,000, and recognized gp120env, p66pol, p55gag, p53pol, p41gag, and p24gag. Because paraproteins in the past have been shown to be directed against specific antigens, we purified this patient's paraprotein using a modified high performance liquid chromatography (HPLC)-hydroxylapatite procedure and tested the purified paraprotein for anti-HIV antibody activity. The purified paraprotein retained anti-HIV antibody activity to a final dilution of 1:100,000, and recognized p66pol, p55gag, p53pol, p41gag, and p24gag. The recognition of both "gag" and "pol" gene products suggested that the purified paraprotein might not be monoclonal in origin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified paraprotein contained at least two immunoglobulin light chain species (Mol wt 30 to 33 Kd). Affinity chromatography of the purified paraprotein using a p24-Sepharose 4B matrix separated the "gag" and "pol" antibody activities. Immunoglobulin gene rearrangement analysis of a bone marrow aspirate (which contained 15% plasma cells) failed to reveal a clonal population of immunoglobulin producing cells. We conclude that this patient's paraprotein accounted for most of the anti-HIV activity present in whole serum, and that this paraprotein was not monoclonal in origin.

AIDS-Related Complex↗

PubMed2006

Synthesis of minus-strand copies of a viral transgene during viral infections of transgenic plants.

Viral transgenes designed to provide resistance to specific plant viruses frequently consist of the coat protein gene and a contiguous 3' untranslated region (3'UTR) of viral origin. In many RNA viruses the viral 3'UTR establishes a recognition and initiation site for viral RNA replication. Thus the transgenic transcript may contain a functional virus replication site. Experiments were designed to determine if a challenging virus would recognize this replication site on a nuclear derived transcript and synthesize the complementary RNA. These data demonstrate that upon infection by a virus that recognizes the viral replication site, a full-length complement of the transgenic transcript is produced. In these experiments the replication complex of Brome Mosaic bromovirus recognized the transgenic transcript derived from a Cowpea Chlorotic Mottle bromovirus transgene. The resulting RNA may contribute to RNA recombination events.

Bromovirus↗

PubMed1982

Phagocyte recognition by lectin receptors.

A "lectin-like" receptor for bacterial cell wall sugars, originally described in the membrane of mouse peritoneal macrophages, which facilitates binding of various bacteria to the macrophage surface, has now been shown to be present on most phagocytes from a variety of species. The role of such a receptor in the inflammatory response has been investigated by examining changes in its expression in response to chemoattractants. The chemoattractants, whilst enhancing or not altering expression of C3 and Fc receptors, respectively, appeared to decrease lectin receptor expression. The possibility of a relationship between I-region gene products of the major histocompatibility complex (MHC) and the bacterial binding lectin receptor was investigated. It was found that various conventional and monoclonal antibodies directed against I-region determinants inhibited binding of bacteria to mouse peritoneal macrophages. These findings suggest the need to examine the possibility that, in addition to phagocytosis and elimination of bacteria, the lectin receptor may also be involved in antigen presentation and macrophage-T-lymphocyte cooperative events leading to an immune response.

Animals↗

PubMed2005

Identifying critical variables of principal components for unsupervised feature selection.

Principal components analysis (PCA) is probably the best-known approach to unsupervised dimensionality reduction. However, axes of the lower-dimensional space, ie., principal components (PCs), are a set of new variables carrying no clear physical meanings. Thus, interpretation of results obtained in the lower-dimensional PCA space and data acquisition for test samples still involve all of the original measurements. To deal with this problem, we develop two algorithms to link the physically meaningless PCs back to a subset of original measurements. The main idea of the algorithms is to evaluate and select feature subsets based on their capacities to reproduce sample projections on principal axes. The strength of the new algorithms is that the computaion complexity involved is significantly reduced, compared with the data structural similarity-based feature evaluation.

Algorithms↗

PubMed2005

Role of papillomavirus E1 initiator dimerization in DNA replication.

Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.

Amino Acid Substitution↗

PubMed1987

Increased fucosylation of glycolipids in a human leukaemia cell line (K562-Clone I) with decreased sensitivity to NK-mediated lysis.

A subpopulation of human lymphoid cells called natural killers is able to lyse certain normal and neoplastic targets in an in vitro cytotoxicity assay. The molecules which enable them to recognize sensitive cells, or permit tumour cells to escape remain unknown. In the studies described here we have compared some of the plasma membrane characteristics of a NK-sensitive human leukaemia cell line (K562) with those of a partially resistant subclone derived from it (K562-Clone I). Gel electrophoresis of cell-surface proteins radiolabelled by lactoperoxidase-catalysed iodination, periodate-borohydride tritiation, or biosynthetically by incubation with [3H]fucose did not reveal any reproducible differences between the sensitive and resistant lines. However, analysis of glycolipids showed that Clone I incorporated significantly more fucose than did the parental line, and that it synthesized a minor population of complex structures not found in the original K562. A subclone of Clone I (Clone I-Con Ar1), made resistant to the toxic effects of concanavalin A, became sensitive once again to NK, and showed the parental glycolipid profile. These results suggest that the Clone I line, selected for resistance to NK, may have altered one or more of its intermediate oligosaccharides or pathways of fucose incorporation into glycolipid, and points to one process by which a tumor cell might modulate its surface to escape recognition by natural killers.

Antigens, Neoplasm↗

PubMed1998

C1q receptors: regulating specific functions of phagocytic cells.

A C1q receptor that upregulates the phagocytic capacity of professional phagocytes, C1qRp, has been identified, and its primary structure determined by cDNA cloning and sequencing. Monoclonal antibodies that immunoprecipitate this 126,000 Mr polypeptide inhibit the enhancement of phagocytosis triggered not only by C1q but also by mannose binding lectin (MBL) and pulmonary surfactant protein A (SPA) providing critical evidence that this polypeptide is a functional receptor or component of the receptor that mediates this enhancement of phagocytosis. The amino acid sequence, deduced from the cloned cDNA coding for this receptor, indicates that this surface glycoprotein receptor is a novel type I membrane protein of 631 amino acid containing a region homologous to C-type lectin carbohydrate recognition domains, 5 EGF-like domains, a single transmembrane domain and a 47 amino acid intracellular domain. Expression of this receptor is limited to cells of myeloid origin, platelets and endothelial cells, consistent with a relatively selective function, and making it an attractive candidate for therapeutic modulation of function. A distinct C1q receptor that triggers superoxide in polymorphonuclear leukocytes has been functionally characterized and designated as C1qRO2-. Thus, the accumulated data that will be summarized here demonstrate that there are at least two C1q receptor/receptor complexes (C1qRp and C1qRO2-), each triggering distinct cellular responses, that multiple C1q receptors can be expressed on the same, as well as on different, cell types, and that at least one C1q receptor, C1qRp, is capable of responding to multiple ligands.

Animals↗

PubMed2004

Bound peptide-dependent thermal stability of major histocompatibility complex class II molecule I-Ek.

We used differential scanning calorimetry to study the thermal denaturation of murine major histocompatibility complex class II, I-E(k), accommodating hemoglobin (Hb) peptide mutants possessing a single amino acid substitution of the chemically conserved amino acids buried in the I-Ek pocket (positions 71 and 73) and exposed to the solvent (position 72). All of the I-Ek-Hb(mut) molecules exhibited greater thermal stability at pH 5.5 than at pH 7.4, as for the I-Ek-Hb(wt) molecule, which can explain the peptide exchange function of MHC II. The thermal stability was strongly dependent on the bound peptide sequences; the I-Ek-Hb(mut) molecules were less stable than the I-Ek-Hb(wt) molecules, in good correlation with the relative affinity of each peptide for I-Ek. This supports the notion that the bound peptide is part of the completely folded MHC II molecule. The thermodynamic parameters for I-Ek-Hb(mut) folding can explain the thermodynamic origin of the stability difference, in correlation with the crystal structural analysis, and the limited contributions of the residues to the overall conformation of the I-Ek-peptide complex. We found a linear relationship between the denaturation temperature and the calorimetric enthalpy change. Thus, although the MHC II-peptide complex could have a diverse thermal stability spectrum, depending on the amino acid sequences of the bound peptides, the conformational perturbations are limited. The variations in the MHC II-peptide complex stability would function in antigen recognition by the T cell receptor by affecting the stability of the MHC II-peptide-T cell receptor ternary complex.

Animals↗