bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluid shear stress induces a biphasic response of human monocyte chemotactic protein 1 gene expression in vascular endothelium.
Authors: Shyy Y J , Hsieh H J , Usami S , Chien S
Published: May 2006

The focal distribution of atherosclerotic lesions in the arterial tree is related to the local shear stress generated by blood flow, but the molecular basis of the atherogenic response of endothelial cells in these lesion-prone areas is still unclear. We report that shear stress mediates a biphasic response of monocyte chemotactic protein 1 (MCP-1) gene expression in vascular endothelial cells (EC). Northern blot analysis indicated that the level of MCP-1 mRNA in human umbilical vein EC (HUVEC) subjected to a shear stress of 16 dynes/cm2 (1 dyne = 10 microN) for 1.5 hr increased by 2- to 3-fold when compared with static cells. The MCP-1 gene expression decreased to the basal level at 4 hr and then declined further to become completely quiescent at 5 hr after the onset of shear. Once the gene expression was fully suppressed, it remained quiescent even after static incubation for 1.5 hr and would not respond to reshearing after this static incubation. However, if the postshearing incubation extended from 1.5 to 24 hr, the MCP-1 mRNA returned to the basal level and was then able to increase after the reapplication of shear stress. Nuclear run-on experiments showed that the shear-induced increased MCP-1 mRNA in HUVEC was regulated at the transcriptional level. By using cycloheximide, it was shown that de novo protein synthesis was not necessary for the induction of MCP-1 by shear stress. The biphasic response of MCP-1 gene expression was found in experiments in which the applied shear stress was 6, 16, or 32 dynes/cm2, and it was observed not only in HUVEC but also in HeLa cells, glioma cell lines, and skin fibroblasts. This in vitro study demonstrates that the response of MCP-1 gene to shear stress represents an immediate early gene activation and suggests that this gene is probably suppressed in EC that have been exposed to a constant shear stress.
Specificity of protein kinase inhibitor peptides and induction of long-term potentiation.
Authors: Hvalby O , Hemmings H C , Paulsen O , Czernik A J , Nairn A C , Godfraind J M , Jensen V , Raastad M , Storm J F , Andersen P
Published: May 2006

Previous studies have used synthetic peptide analogs, corresponding to sequences within the pseudosubstrate domain of protein kinase C (PKC) or the autoregulatory domain of Ca2+/calmodulin-dependent protein kinase II (CaMKII), in attempts to define the contribution of each of these protein kinases to induction of long-term potentiation (LTP). However, the specificity of these inhibitor peptides is not absolute. Using intracellular delivery to rat CA1 hippocampal neurons, we have determined the relative potency of two protein kinase inhibitor peptides, PKC-(19-36) and [Ala286]CaMKII-(281-302), as inhibitors of the induction of LTP. Both peptides blocked the induction of LTP; however, PKC-(19-36) was 30-fold more potent than [Ala286]CaMKII-(281-302). The relative specificity of PKC-(19-36), [Ala286]CaMKII-(281-302), and several other CaMKII peptide analogs for protein kinase inhibition in vitro was also determined. A comparison of the potencies of PKC-(19-36) and [Ala286]CaMKII-(281-302) in the physiological assay with their Ki values for protein kinase inhibition in vitro indicates that the blockade of induction of LTP observed for each peptide is attributable to inhibition of PKC.
Cell size predicts morphological complexity in the brains of frogs and salamanders.
Authors: Roth G , Blanke J , Wake D B
Published: May 2006

The morphological organization of the brain of frogs and salamanders varies greatly in the degree to which it is subdivided and differentiated. Members of these taxa are visually oriented predators, but the morphological complexity of the visual centers in the brain varies interspecifically. We give evidence that the morphological complexity of the amphibian tectum mesencephali, the main visual center, can be predicted from knowledge of cell size, which varies greatly among these taxa. Further, cell size is highly correlated with genome size. Frogs with small cells have more complex morphologies of the tectum than do those with large cells independent of body and brain size. In contrast, in salamanders brain-body size relationships also are correlated with morphological complexity of the brain. Small salamanders with large cells have the simplest tecta, whereas large salamanders with small cells exhibit the most complex tectal morphologies. Increases in genome, and consequently cell size, are associated with a decrease in the differentiation rate of nervous tissue, which leads to the observed differences in brain morphology. On the basis of these findings we hypothesize that important features of the structure of the brain can arise independently of functional demands, from changes at a lower level of organismal organization--in this case increase in genome size, which induces simplification of brain morphology.
Double-stranded DNA templates can induce alpha-helical conformation in peptides containing lysine and alanine: functional implications for leucine zipper and helix-loop-helix transcription factors.
Authors: Johnson N P , Lindstrom J , Baase W A , von Hippel P H
Published: May 2006

Transcription factors of the basic-leucine zipper and basic-helix-loop-helix families specifically recognize DNA by means of intrinsically flexible peptide domains that assume an alpha-helical conformation upon binding to target DNA sequences. We have investigated the nonspecific interactions that underlie specific DNA recognition. Circular dichroism measurements showed that 20-bp double-stranded DNA oligonucleotides can act as templates to promote random coil-->alpha-helix transitions in short peptides containing alanine and lysine. This conformational change takes place without altering the structure of the DNA, and neither specific peptide-DNA contacts nor cooperative interactions between peptides are necessary. The conformational change does require (i) double-stranded (but not single-stranded) oligodeoxynucleotides in either the B or the B' conformation and (ii) peptides that can form positively charged amphipathic alpha-helices. In 10 mM Na2HPO4 (pH 7.5; 10 degrees C), the excess free-energy contribution of the DNA template to the stability of the alpha-helical form of the oligopeptides tested was delta Gex = -0.15 (+/- 0.07) kcal/mol per lysine residue. The implications of these results for the thermodynamics and kinetics of DNA target site selection by basic-leucine zipper and basic-helix-loop-helix regulatory proteins are discussed.
Biochemical and pharmacological profile of a potent and selective endothelin B-receptor antagonist, BQ-788.
Authors: Ishikawa K , Ihara M , Noguchi K , Mase T , Mino N , Saeki T , Fukuroda T , Fukami T , Ozaki S , Nagase T
Published: May 2006

We describe the characteristics of a potent and selective endothelin (ET) B-receptor antagonist, BQ-788 [N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D -1- methoxycarbonyltryptophanyl-D-norleucine]. In vitro, this compound potently and competitively inhibits 125I-labeled endothelin 1 (ET-1) binding to ETB receptors on human Girardi heart cells (IC50, 1.2 nM) but only poorly inhibits the binding to ETA receptors on human neuroblastoma cell line SK-N-MC cells (IC50, 1300 nM). In isolated rabbit pulmonary arteries, BQ-788 shows no agonist activity up to 10 microM and competitively antagonizes the vasoconstriction induced by an ETB-selective agonist, BQ-3020 (pA2, 8.4). In rat, an ETA-selective antagonist, BQ-123 (1 mg/kg, i.v.), does not affect transient depressor response to ET-1 (0.3 nmol/kg, i.v.) but potently inhibits following sustained pressor response; vice versa, BQ-788 (1 mg/kg, i.v.) abolishes the depressor response, resulting in a rapid onset of apparently enhanced pressor response. Thus, being a potent and selective ETB receptor antagonist, BQ-788 may be considered as a powerful tool for investigating the role of ET in physiological and pathological processes.
X-ray structure of a cyclophilin B/cyclosporin complex: comparison with cyclophilin A and delineation of its calcineurin-binding domain.
Authors: Mikol V , Kallen J , Walkinshaw M D
Published: May 2006

The crystal structure of a complex between recombinant human cyclophilin B (CypB) and a cyclosporin A (CsA) analog has been determined and refined at 1.85-A resolution to a crystallographic R factor of 16.0%. The overall structures of CypB and of cyclophilin A (CypA) are similar; however, significant differences occur in two loops and at the N and C termini. The CsA-binding pocket in CypB has the same structure as in CypA and cyclosporin shows a similar bound conformation and network of interactions in both CypB and CypA complexes. The network of the water-mediated contacts is also essentially conserved. The higher potency of the CypB/CsA complex versus CypA/CsA in inhibiting the Ca(2+)- and calmodulin-dependent protein phosphatase calcineurin is discussed in terms of the structural differences between the two complexes. The three residues Arg90, Lys113, and Ala128 and the loop containing Arg158 on the surface of CypB are likely to modulate the differences in calcineurin inhibition between CypA and CypB.
Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence.
Authors: Ullman E F , Kirakossian H , Singh S , Wu Z P , Irvin B R , Pease J S , Switchenko A C , Irvine J D , Dafforn A , Skold C N
Published: May 2006

A method for monitoring formation of latex particle pairs by chemiluminescence is described. Molecular oxygen is excited by a photosensitizer and an antenna dye that are dissolved in one of the particles. 1 delta gO2 diffuses to the second particle and initiates a high quantum yield chemiluminescent reaction of an olefin that is dissolved in it. The efficiency of 1 delta gO2 transfer between particles is approximately 3.5%. The technique permits real-time measurement of particle binding kinetics. Second-order rate constants increase with the number of receptor binding sites on the particles and approach diffusion control. By using antibody-coated particles, a homogeneous immunoassay capable of detecting approximately 4 amol of thyroid-stimulating hormone in 12 min was demonstrated. Single molecules of analyte produce particle heterodimers that are detected even when no larger aggregates are formed.
Structure and cell cycle-regulated transcription of the human cyclin A gene.
Authors: Henglein B , Chenivesse X , Wang J , Eick D , Bréchot C
Published: May 2006

Cyclin A is a cell cycle regulatory protein that functions in mitotic and S-phase control in mammalian somatic cells. Its deregulated expression may have a role in cellular transformation. We have cloned and sequenced the human cyclin A gene and cDNAs representing its mRNAs and have characterized its promoter. Using synchronized cultures of NIH 3T3 cells stably transfected with cyclin A promoter/luciferase constructs, we show that the promoter is repressed during the G1 phase of the cell cycle and is activated at S-phase entry. Cell cycle regulation of the cyclin A gene promoter is mediated by sequences extending from -79 to +100 relative to the predominant transcription start site. It does not require the presence of a functional retinoblastoma protein.
Heterogeneity of T-cell receptor alpha-chain complementarity-determining region 3 in myelin basic protein-specific T cells increases with severity of multiple sclerosis.
Authors: Utz U , Brooks J A , McFarland H F , Martin R , Biddison W E
Published: May 2006

The pathogenesis of multiple sclerosis (MS) is thought to involve a T-cell-mediated autoimmune process. Experimental allergic encephalomyelitis (EAE), an animal model resembling MS, can be induced by immunization with myelin antigens such as myelin basic protein. The T-cell antigen receptor (TCR) usage in EAE is highly restricted in some strains of animals and experimental treatments targeting the TCR have been successful in EAE. Examination of the TCR beta-chain variable-region (V beta) usage of MBP-specific T-cell lines in MS patients has produced conflicting results. Our previous studies of TCR alpha-chain variable-region usage in monozygotic twins demonstrated a general skewing of the TCR repertoire in individuals with MS. This skewing became apparent only after stimulation with antigens; in peripheral blood lymphocyte preparations from individuals with MS V alpha 8-bearing T cells were preferentially selected by stimulation with myelin basic protein. In the present study we examined complementarity-determining region 3 of those V alpha 8-positive TCRs. Marked sequence heterogeneity was found in all individuals with severe MS. In contrast, restricted areas of complementarity-determining region 3 were found in healthy control individuals and in individuals with a mild form of MS. Sequences from tetanus toxoid-specific V alpha 8-positive T cells generated from the same individuals were relatively homogeneous within individuals regardless of disease activity and were distinct from the sequences of complementarity-determining region 3 in myelin basic protein-stimulated lines. These findings suggest that disease severity may be associated with increased heterogeneity of myelin antigen-specific T cells and could reflect an impaired ability of the immune system to down-regulate these anti-self responses.
Molecular cloning of fibroblast activation protein alpha, a member of the serine protease family selectively expressed in stromal fibroblasts of epithelial cancers.
Authors: Scanlan M J , Raj B K , Calvo B , Garin-Chesa P , Sanz-Moncasi M P , Healey J H , Old L J , Rettig W J
Published: May 2006

The human fibroblast activation protein alpha (FAP alpha) is a M(r) 95,000 cell surface antigen selectively expressed in reactive stromal fibroblasts of epithelial cancers, granulation tissue of healing wounds, and malignant cells of bone and soft tissue sarcomas. Normal adult tissues are generally FAP alpha-, but some fetal mesenchymal tissues transiently express the molecule. Because of its restricted normal tissue distribution and abundant expression in the stroma of over 90% of breast, colorectal, and lung carcinomas, FAP alpha is under clinical evaluation as a target for immunodetection and immunotherapy of epithelial cancers. In the present study, we have isolated a full-length cDNA for FAP alpha through expression cloning in COS-1 cells. The FAP alpha cDNA codes for a type II integral membrane protein with a large extracellular domain, transmembrane segment, and short cytoplasmic tail. FAP alpha shows 48% amino acid sequence identity to the T-cell activation antigen CD26, a membrane-bound protein with dipeptidyl peptidase IV (DPPIV) activity; however, unlike FAP alpha, CD26 is widely expressed in normal tissues. Three catalytic domains shared by DPPIV homologues in different species and by other serine proteases are conserved in FAP alpha. Immunochemical analysis of COS-1 cells coexpressing FAP alpha and CD26 revealed that the two molecules form heteromeric cell surface complexes, suggesting that a previously identified FAP alpha-associated M(r) 105,000 protein of cultured fibroblasts and growth factor-stimulated melanocytes, FAP beta, is identical to CD26. In vivo coexpression of FAP alpha and CD26 is found in reactive fibroblasts of healing wounds but not in tumor stromal fibroblasts or sarcomas (FAP alpha +/CD26-). The putative serine protease activity of FAP alpha and its in vivo induction pattern may indicate a role for this molecule in the control of fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair, and epithelial carcinogenesis.
Combined intra- and extracellular immunization against human immunodeficiency virus type 1 infection with a human anti-gp120 antibody.
Authors: Chen S Y , Khouri Y , Bagley J , Marasco W A
Published: May 2006

In this study, a human CD4+ T lymphocyte line was transduced to secrete Fab fragments of a broadly neutralizing human monoclonal antibody F105 that reacts with the CD4-binding site of human immunodeficiency virus type 1 (HIV-1) envelope protein. In the transduced cells infected with HIV-1, the nascent Fab fragments bind intracellularly to the HIV-1 envelope protein and inhibit HIV-1 production. The secreted Fab fragments are able to neutralize cell-free HIV-1. In addition, the nascent Fab fragments can inhibit HIV-1 production by binding intracellularly to envelope mutants that escape neutralization by extracellular F105 antibody. The combined intra- and extracellular binding activities of the expressed Fab fragments result in the efficient blocking of cytopathic syncytium formation and infectious virus production. Thus, these antibody-producing T lymphocytes are not only resistant to HIV-1 infection but also can protect surrounding lymphocytes by secreting neutralizing antibodies. This novel strategy of combining intracellular and extracellular immunization may be useful for gene therapy of AIDS and other diseases.
The E2A and tal-1 helix-loop-helix proteins associate in vivo and are modulated by Id proteins during interleukin 6-induced myeloid differentiation.
Authors: Voronova A F , Lee F
Published: May 2006

The immunoglobulin enhancer-binding proteins, E12 and E47, encoded by the E2A gene belong to the basic helix-loop-helix (bHLH) family of regulatory proteins and act as transcriptional activators. In addition to their critical role in B-lymphocyte development, the E12 and E47 proteins have been implicated in the induction of myogenesis as heterodimeric partners of myogenic bHLH proteins, MyoD and myogenin. Here we demonstrate that the E2A proteins form heterodimers with the bHLH oncoprotein tal-1 in myeloid and erythroid cells and that these heterodimers specifically bind to the CANNTG DNA motif. Heterodimerization with tal-1 represses transactivation by E47 and could function to prevent the expression of immunoglobulin genes in cells other than B lymphocytes. DNA binding by E2A-tal-1 heterodimers in the M1 mouse myeloid cell line is abrogated upon terminal macrophage differentiation induced by the cytokine interleukin 6. The loss of E2A-tal-1 DNA binding is correlated with elevated expression of mRNA encoding the dominant negative HLH proteins, Id1 and particularly Id2. Moreover, recombinant Id proteins inhibit the E2A-tal-1-specific DNA binding activity from undifferentiated M1 cells. These results suggest that E2A-tal-1 heterodimers may play a role in preventing terminal differentiation in the myeloid lineage and provide a possible explanation for oncogenic transformation induced by ectopic tal-1 expression in acute T-cell lymphoblastic leukemias.
Loss of receptors for transforming growth factor beta in human T-cell malignancies.
Authors: Kadin M E , Cavaille-Coll M W , Gertz R , Massagué J , Cheifetz S , George D
Published: May 2006

Ki-1 (CD30)+ cutaneous T-cell lymphomas CTCLs) are slowly progressive lymphomas in which initial spontaneous regression is often observed. To better understand the mechanisms of spontaneous regression and eventual tumor progression in Ki-1+ CTCLs, type beta transforming growth factor (TGF-beta)-mediated growth inhibition of clonally related cell lines derived from two time points, before and after tumor progression, was studied. TGF-beta 1 inhibited colony-forming efficiency (CFE) of a cell line (Mac-1) derived from clinically indolent Ki-1+ CTCLs but failed to inhibit CFE of Mac-2A and -2B cell lines from advanced CTCLs. To determine the basis for TGF-beta 1 resistance in advanced CTCL cells, we looked for possible defects in the expression of cell surface TGF-beta receptors. Mac-1 cells were found to express TGF-beta receptors I and II, which mediate growth inhibition, and the TGF-beta-binding proteoglycan betaglycan. In contrast, receptors I and II were not detected in CTCL lines Mac-2A and -2B even though these cell lines did express betaglycan. Various treatments that unmask or induce TGF-beta receptors in other cells failed to show evidence for these receptors in advanced CTCL cells. Loss of TGF-beta receptor expression in these cells correlated with a marked decrease in TGF-beta receptor II mRNA levels. Loss of cell surface TGF-beta receptors was also found in two of five other patients with T-cell lymphomas including the Sezary syndrome and a noncutaneous T-cell lymphoma, suggesting that loss of TGF-beta receptor expression may be a recurrent feature of human T-cell malignancies.
Mutants of Neurospora crassa that alter gene expression and conidia development.
Authors: Madi L , Ebbole D J , White B T , Yanofsky C
Published: May 2006

Several genes have been identified that are highly expressed during conidiation. Inactivation of these genes has no observable phenotypic effect. Transcripts of two such genes, con-6 and con-10, are normally absent from vegetative mycelia. To identify regulatory genes that affect con-6 and/or con-10 expression, strains were prepared in which the regulatory regions for these genes were fused to a gene conferring hygromycin resistance. Mutants were then selected that were resistant to the drug during mycelial growth. Mutations in several of the isolates had trans effects; they activated transcription of the corresponding intact gene and, in most isolates, one or more of the other con genes. Most interestingly, resistant mutants were obtained that were defective at different stages of conidiation. One mutant conidiated under conditions that do not permit conidiation in wild type.
A second lineage of mammalian major histocompatibility complex class I genes.
Authors: Bahram S , Bresnahan M , Geraghty D E , Spies T
Published: May 2006

Major histocompatibility complex (MHC) class I genes typically encode polymorphic peptide-binding chains which are ubiquitously expressed and mediate the recognition of intracellular antigens by cytotoxic T cells. They constitute diverse gene families in different species and include the numerous so-called nonclassical genes in the mouse H-2 complex, of which some have been adapted to variously modified functions. We have identified a distinct family of five related sequences in the human MHC which are distantly homologous to class I chains. These MIC genes (MHC class I chain-related genes) evolved in parallel with the human class I genes and with those of most if not all mammalian orders. The MICA gene in this family is located near HLA-B and is by far the most divergent mammalian MHC class I gene known. It is further distinguished by its unusual exon-intron organization and preferential expression in fibroblasts and epithelial cells. However, the presence of diagnostic residues in the MICA amino acid sequence translated from cDNA suggests that the putative MICA chain folds similarly to typical class I chains and may have the capacity to bind peptide or other short ligands. These results define a second lineage of evolutionarily conserved MHC class I genes. This implies that MICA and possibly other members in this family have been selected for specialized functions that are either ancient or derived from those of typical MHC class I genes, in analogy to some of the nonclassical mouse H-2 genes.
Interaction of the Dr1 inhibitory factor with the TATA binding protein is disrupted by adenovirus E1A.
Authors: Kraus V B , Inostroza J A , Yeung K , Reinberg D , Nevins J R
Published: May 2006

Past experiments have shown that the adenovirus E1A12S product activates the hsp70 promoter, dependent on the TATA element and dependent on N-terminal E1A sequences. Other experiments have identified a factor termed Dr1 that interacts with and inhibits the transcriptional activity of the TATA-binding protein (TBP). We now find that the E1A12S protein can disrupt the interaction of the Dr1 factor with the TATA-specific TBP factor, allowing the productive interaction of TBP with TFIIA. This E1A-mediated disruption is dependent on N-terminal sequences that are also essential for the TATA-dependent trans-activation of the hsp70 promoter. Moreover, we also find that Dr1 expression in transfected cells can inhibit transcription from the hsp70 promoter and that this can be overcome by coexpression of the wild-type E1A protein, dependent on N-terminal sequences. We conclude that the activation of hsp70 through the TATA element may be mechanistically similar to the activation of the E2 promoter via E2F, in each case involving a release of a transcription factor from an inactive complex.
A requirement of hydrophobic and basic amino acid residues for substrate recognition by Ca2+/calmodulin-dependent protein kinase Ia.
Authors: Lee J C , Kwon Y G , Lawrence D S , Edelman A M
Published: May 2006

The substrate recognition determinants of Ca2+/calmodulin-dependent protein kinase Ia were investigated by using peptide analogues based on the amino acid sequence around Ser-9 of synapsin I. The Km and Vmax for the synthetic peptide Leu-Arg-Arg-Arg-Leu-Ser-Asp-Ala-Asn-Phe are 3.9 microM and 18.5 mumol/(min.mg), respectively. Deletion of Leu at the -5 position lowers the Vmax/Km by 470-fold. The requirement for a hydrophobic residue at -5 was confirmed by the 90- to 2400-fold reduction in Vmax/Km produced by Arg, Ala, or Asp substitutions, but only 2.6-fold decrease after Phe substitution at this position. A hydrophobic residue is similarly required at the +4 position. Deletion of Phe at this position produces a 67-fold reduction, and substitution of Ala for Phe a 43-fold reduction in Vmax/Km. In contrast, substitution with Leu increases Vmax/Km by 1.8-fold. Arg at -3 is also required for recognition as shown by an approximately 240-fold decrease in Vmax/Km after Ala substitution at this position. Positions -2, -4, and +1 appear to play secondary roles in substrate recognition. Arg at -2 and -4 are positive determinants, since Ala substitution at these positions decreases Vmax/Km by 4.7- and 11-fold, respectively. Asp at +1 is a negative influence, since Ala and Leu substitutions at this position increase Vmax/Km by 2.3- and 6.3-fold, respectively. Substitution of Ala for Leu at -1 or Thr for Ser at the 0 position has little effect on phosphorylation kinetics. Thus, Ca2+/calmodulin-dependent protein kinase Ia has the minimal substrate recognition motif of Hyd-Xaa-Arg-Xaa-Xaa-(Ser*/Thr*)-Xaa-Xaa-Xaa-Hyd, where Hyd represents a hydrophobic amino acid residue.
Expression of a glycosylphosphatidylinositol-anchored Trypanosoma brucei transferrin-binding protein complex in insect cells.
Authors: Chaudhri M , Steverding D , Kittelberger D , Tjia S , Overath P
Published: May 2006

The expression site-associated gene ESAG 6 was previously implicated in transferrin binding in the protozoan parasite Trypanosoma brucei. ESAG 6 and the closely related ESAG 7 of T. brucei strain AnTat1.3 have now been expressed in insect cells using the baculovirus expression system. Expression of ESAG 6 alone in insect cells gives rise to a glycosylated protein of approximately 52 kDa, which is cell surface-associated through a glycosylphosphatidylinositol anchor at its C terminus. The ESAG 7 product of about 42 kDa is also glycosylated, but lacks the glycosylphosphatidylinositol modification, and is located intracellularly. No transferrin-binding activity is observed when either ESAG is expressed independently. However, their expression results in a cell surface complex of ESAG 6 and 7 products that specifically binds transferrin. This shows that both ESAG 6 and 7 products are necessary and sufficient for binding to transferrin.
Human immunodeficiency virus 1 Tat binds to dipeptidyl aminopeptidase IV (CD26): a possible mechanism for Tat's immunosuppressive activity.
Authors: Gutheil W G , Subramanyam M , Flentke G R , Sanford D G , Munoz E , Huber B T , Bachovchin W W
Published: May 2006

The human immunodeficiency virus 1 (HIV-1) Tat protein suppresses antigen-induced, but not mitogen-induced, activation of human T cells when added to T-cell cultures [Viscidi, R. P., Mayur, K., Lederman, H. M. & Frankel, A. D. (1989) Science 246, 1606-1608]. This activity is potentially pertinent to the development of AIDS because lymphocytes from HIV-infected individuals exhibit a similar antigen-specific dysfunction. Here we report that Tat binds with high affinity to the T-cell activation molecule dipeptidyl aminopeptidase IV (DP IV), also known as CD26. This molecule occurs on the surface of CD4+ cells responsible for the recall antigen response and appears to play an essential role in this response. Tat binds to both the cell surface and soluble forms of DP IV at physiological salt concentrations without inhibiting the protease activity of DP IV against small chromogenic substrates used to assay activity, but Tat markedly inhibits the activity of DP IV at lower salt concentrations. The kinetics of inhibition indicate the affinity of Tat for DP IV varies from 20 pM to 11 nM, and the activity of the Tat-DP IV complex varies from 13% to 100%, as the NaCl concentration varies from 0 to 140 mM. Cytofluorometry experiments demonstrate that Tat competes with anti-Ta1, a monoclonal antibody (mAb) specific for DP IV, for binding to cell surface DP IV, thus indicating that Tat binds DP IV at or near the Ta1 epitope. Moreover, the anti-Ta1 mAb blocks the immunosuppressive activity of Tat. The high affinity of Tat for DP IV, previous evidence implicating DP IV in antigen-specific T-cell activation events, and the ability of anti-Ta1 mAb to block the immunosuppressive effect of Tat make DP IV a plausible receptor for Tat's immunosuppressive activity.
Induction of anergy or active suppression following oral tolerance is determined by antigen dosage.
Authors: Friedman A , Weiner H L
Published: May 2006

Oral tolerance was generated to hen egg white lysozyme in the mouse or to guinea pig myelin basic protein in the rat by a low-dose (1 mg) or a high-dose (5-20 mg) feeding regimen. High doses of antigen induced tolerance characterized by anergy with little or no active suppression and increased secretion of interleukin 4 (IL-4). Anergy was shown by an increase in frequency of IL-2-secreting cells following culture in recombinant IL-2. Low doses of antigen induced tolerance characterized by antigen-driven active suppression with increased secretion of transforming growth factor beta (TGF-beta) and IL-4 and minimal anergy. Without further immunization, spleen cells from animals orally tolerized by both regimens secreted increased levels of IL-4 and TGF-beta in an antigen-specific manner. Animals fed high doses secreted more IL-4 and less TGF-beta, whereas those fed low doses secreted more TGF-beta and less IL-4. These results demonstrate that the two feeding regimens induced cell populations that differed in their cytokine secretion profile and their capacity to actively suppress in vitro and to induce anergy. Our results provide a basis for distinguishing different forms of antigen-driven peripheral tolerance and have important implications for orally induced antigen-specific modulation of human autoimmune diseases.
Frog diazepam-binding inhibitor: peptide sequence, cDNA cloning, and expression in the brain.
Authors: Lihrmann I , Plaquevent J C , Tostivint H , Raijmakers R , Tonon M C , Conlon J M , Vaudry H
Published: May 2006

Three peptides derived from diazepam-binding inhibitor (DBI) were isolated in pure form from the brain of the frog Rana ridibunda. The primary structures of these peptides showed that they correspond to mammalian DBI-(1-39), DBI-(58-87), and DBI-(70-87). A set of degenerate primers, whose design was based on the amino acid sequence data, was used to screen a frog brain cDNA library. The cloned cDNA encodes an 87-amino acid polypeptide, which exhibits 68% similarity with porcine and bovine DBI. Frog DBI contains two paired basic amino acids (Lys-Lys) at positions 14-15 and 62-63 and a single cysteine within the biologically active region of the molecule. Northern blot analysis showed that DBI mRNA is expressed at a high level in the brain but is virtually absent in peripheral tissues. The distribution of DBI mRNA and DBI-like immunoreactivity in the frog brain was studied by in situ hybridization and immunocytochemistry. Both approaches revealed that the DBI gene is expressed in ependymal cells and circumventricular organs lining the ventricular cavity. Since amphibia diverged from mammals at least 250 million years ago, the data show that evolutionary pressure has acted to conserve the structure of DBI in the vertebrate phylum. The distribution of both DBI mRNA and DBI-like immunoreactivity indicates that DBI is selectively expressed in glial cells.
Kinetic characterization of intramolecular and intermolecular hammerhead RNAs with stem II deletions.
Authors: Long D M , Uhlenbeck O C
Published: May 2006

A method is described to obtain intramolecular cleavage rates for the hammerhead ribozyme during in vitro transcription. By avoiding RNA purification and renaturation, the potential for formation of inactive conformations of the RNA is minimized. By showing that an intramolecular hammerhead and a closely related intermolecular hammerhead cleave at the same rate under a given set of conditions, we confirm that both reactions probably have the same rate-limiting step. An in vitro selection strategy was used to isolate active hammerheads from a library of molecules where six randomized nucleotides replaced stem-loop II. The sequence and number of nucleotides which replace stem-loop II have large effects on hammerhead cleavage activity. The relative activities of three sequences selected from the intramolecular library are the same when the sequences are transferred into an intermolecular hammerhead background.
Analytic inversion in synthetic aperture radar.
Authors: Rothaus O S
Published: May 2006

A method of processing synthetic aperture radar signals that avoids some of the approximations currently in use that appear to be responsible for severe phase distortions is described. As a practical matter, this method requires N3 numerical operations, as opposed to the N2 ln N currently the case, but N3 is now easily managed, for N in the range of interest.
Calorie restriction delays spontaneous tumorigenesis in p53-knockout transgenic mice.
Authors: Hursting S D , Perkins S N , Phang J M
Published: May 2006

Transgenic mice with both alleles of the p53 tumor suppressor gene (frequently mutated in human tumors) knocked out by gene targeting provide a potentially useful tumorigenesis model because these mice rapidly develop spontaneous tumors. To determine whether tumorigenesis in p53-knockout mice is sensitive to experimental manipulation, tumor development in response to calorie restriction (CR; a potent inhibitor of rodent tumors) was evaluated. Tumor development was monitored for 48 weeks in male nullizygous p53-knockout and wild-type littermate mice (28-30 per treatment group) fed ad libitum (AL) or restricted to 60% of AL carbohydrate calorie intake. CR:p53-knockout mice (median survival = 25 weeks) experienced a delay in tumor onset and subsequent mortality (P = 0.0002) relative to AL:p53-knockout mice (median survival = 16 weeks). Tumor development and mortality in wild-type littermates on either diet treatment were < 4% through 48 weeks. Cell cycle analyses were performed on splenocytes from p53-knockout mice and wild-type littermates after 4 weeks of AL feeding or CR (5 per group). The percentage of splenocytes in S phase of the cell cycle was 3-fold higher for p53-knockout mice than wild-type mice (P < 0.001), and CR reduced the percentage of S-phase splenocytes in both p53-knockout and wild-type mice (P = 0.012). These data demonstrate that tumor development in p53-knockout mice genetically predisposed to tumors can be delayed by CR (possibly via cell cycle modulation) and suggest that these mice provide a very useful model of spontaneous tumorigenesis.
Cloning and characterization of a basic helix-loop-helix protein expressed in early mesoderm and the developing somites.
Authors: Quertermous E E , Hidai H , Blanar M A , Quertermous T
Published: May 2006

Basic helix-loop-helix (bHLH) heterodimer protein complexes regulate transcription of genes during the processes of differentiation and development. To study the molecular basis of early mesodermal differentiation, we sought to identify bHLH proteins from cells of mesodermal origin. By using an interaction cloning strategy with a radiolabled recombinant bHLH protein, E12, a clone encoding a potential heterodimer partner was isolated from an endothelial cell library. This gene (bHLH-EC2) is most homologous to Twist but shares similarity within the bHLH domain with TAL1 and other members of this family. bHLH-EC2 is expressed in cultured endothelial cells and in embryonic stem cell, erythroleukemia, and muscle cell lines in a differentiation-dependent manner. In situ hybridization studies of mouse embryos reveal that bHLH-EC2 is expressed throughout the primitive mesoderm as early as 7.5 days postcoitum. Expression then becomes restricted to the paraxial mesoderm and to the dermamyotome of the developing somite. Expression of bHLH-EC2 in cells destined to become myoblasts thus predates expression of myogenic bHLH factors. bHLH-EC2 is expressed in early endothelial and hematopoietic cells in vivo, as shown by RNA studies of embryonic yolk sac and cultured cells derived from yolk sac explants. These findings suggest that bHLH-EC2 plays a role in the development of multiple cell types derived from the primitive mesoderm.
Transient transfection of the enteric parasite Entamoeba histolytica and expression of firefly luciferase.
Authors: Purdy J E , Mann B J , Pho L T , Petri W A
Published: May 2006

Development of DNA-mediated transfection in Entamoeba histolytica will facilitate basic research toward the control of this protozoan parasite. A transient transfection system was established by using the firefly luciferase gene ligated to the 5' and 3' flanking regions of the amebic hgl1 gene. The optimal construct tested encoded an hgl1-luciferase fusion protein and contained 1 kb of 5' flanking sequence with 16 bases of coding sequence from the hgl1 gene ligated in-frame to the luciferase start codon and 2.3 kb of 3' flanking sequence from hgl1 ligated 3' to the luciferase stop codon. Optimal electroporation conditions in strain HM-1:IMSS trophozoites when using this construct were 500 microF and 500 V/cm, which resulted in luciferase activity up to 5000-fold above background 9-12 hr after electroporation. Constructs that contained the luciferase gene without amebic flanking sequences or that contained a simian virus 40 promoter, enhancer, and polyadenylylation signal produced only background levels of luciferase activity. The ability to introduce and express genes in amebae will now permit a genetic analysis of the virulence of this organism, which remains a serious threat to world health.
The Epstein-Barr virus nuclear antigen 2 transactivator is directed to response elements by the J kappa recombination signal binding protein.
Authors: Grossman S R , Johannsen E , Tong X , Yalamanchili R , Kieff E
Published: May 2006

Epstein-Barr virus nuclear antigen 2 (EBNA-2) plays an essential role in primary B-lymphocyte growth transformation. EBNA-2 is an acidic transcriptional transactivator that is brought to virus and cell EBNA-2 response elements by interaction with a factor that recognizes the double-stranded sequence MNYYGTGGGAA, where M is A or C, N is any nucleotide, and Y is a pyrimidine. A 63-kDa protein that recognizes this DNA sequence has now been purified by S-Sepharose and oligonucleotide affinity chromatography. p63 peptide sequence is identical to the predicted amino acid sequence for the human J kappa immunoglobulin recombination signal binding protein. Purified or recombinant in vitro-translated J kappa binds to the MNYYGTGGGAA EBNA-2 response element sequence and interacts with EBNA-2. Surprisingly, J kappa does not bind to the J kappa 1 heptamer recombination signal sequence (CACTGTG), and its prior identification as a heptamer binding protein was most likely due to the addition of a BamHI restriction site to the native heptamer creating a near EBNA-2 response element consensus (CACTGTGGGAT).
Promotion of parallel DNA quadruplexes by a yeast telomere binding protein: a circular dichroism study.
Authors: Giraldo R , Suzuki M , Chapman L , Rhodes D
Published: May 2006

Repressor-activator protein 1 (RAP1) has an essential role in the maintenance of yeast telomeres. Yeast telomeric DNA consists of simple repeated G-rich sequences that are bound by RAP1. We have found that RAP1, in addition to its known binding activity for double-stranded DNA, interacts with the G-rich strand containing guanine base (G)-tetrads. We show here using circular dichroism spectroscopy that RAP1 promotes the formation of one particular type of DNA quadruplex, parallel G4-DNA. Furthermore, RAP1 is able to bind to both preformed parallel and antiparallel DNA quadruplexes. These results have implications for the possible use of DNA quadruplexes in telomere-telomere association in vivo.
Structure and expression of the hairless gene of mice.
Authors: Cachon-Gonzalez M B , Fenner S , Coffin J M , Moran C , Best S , Stoye J P
Published: May 2006

The hairless mutation of mice was caused by insertion of a murine leukemia virus. Starting with sequences flanking the provirus, a series of overlapping clones surrounding the viral integration site were obtained. By using a combination of sequencing, PCR, and exon-trapping techniques, the hairless gene was identified. It encodes a predicted protein of 1182 amino acids, including a potential zinc-finger domain. The expression patterns of the gene closely reflect the phenotype of animals carrying the hairless mutation.
Mode of action of anti-lipopolysaccharide-binding protein antibodies for prevention of endotoxemic shock in mice.
Authors: Gallay P , Heumann D , Le Roy D , Barras C , Glauser M P
Published: May 2006

Lipopolysaccharide (LPS)-binding protein (LBP) has been shown to regulate the response of monocytes to LPS in vitro. In a previous study, polyclonal anti-LBP IgGs were found to protect D-galactosamine-sensitized mice against a lethal endotoxemic shock induced by a low challenge of LPS or lipid A when administered simultaneously with endotoxin. In the present study, we investigated the mode of action of these anti-LBP IgGs. In vitro, we demonstrated that they interfere with LPS binding to monocytes or polymorphonuclear cells in different ways: by the mere prevention of binding of LPS to LBP thus preventing the binding of LPS to CD14, or by reacting with LPS-LBP complexes thus mediating their binding to complement or Fc receptors on monocytes and on polymorphonuclear cells. In vivo, we demonstrated that anti-LBP IgGs afforded protection against lethal endotoxemic shock by one of two mechanisms. First, LBP blockade by pretreatment with anti-LBP IgG allowed protection against a low dose of LPS (100 ng). This protection occurred despite LPS levels in blood similar to those in control mice but in the absence of detectable tumor necrosis factor (TNF). This demonstrated that anti-LBP IgG could block the LBP-mediated TNF release upon LPS challenge. In contrast, anti-LBP IgG did not afford protection in mice not sensitized with D-galactosamine and challenged with high-dose LPS (1 mg), confirming that LPS at high concentrations could stimulate cells independently of the LBP pathway. Second, anti-LBP treatment administered simultaneously with LPS challenge protected mice against both low and high doses of LPS. Unlike after pretreatment with anti-LBP IgG, this protection was accompanied by a decrease of circulating LPS, suggesting that anti-LBP IgG in these conditions facilitated clearance of LPS probably by clearing LPS-LBP complexes. These data and the fact that LBP binds to all LPS through lipid A suggest that antibody directed to LBP could be a candidate for therapeutic strategies in endotoxemic shock.
Strand displacement amplification as an in vitro model for rolling-circle replication: deletion formation and evolution during serial transfer.
Authors: Walter N G , Strunk G
Published: May 2006

Strand displacement amplification is an isothermal DNA amplification reaction based on a restriction endonuclease nicking its recognition site and a polymerase extending the nick at its 3' end, displacing the downstream strand. The reaction resembles rolling-circle replication of single-stranded phages and small plasmids. The displaced sense strand serves as target for an antisense reaction and vice versa, resulting in exponential growth and the autocatalytic nature of this in vitro reaction as long as the template is the limiting agent. We describe the optimization of strand displacement amplification for in vitro evolution experiments under serial transfer conditions. The reaction was followed and controlled by use of the fluorescent dye thiazole orange binding to the amplified DNA. We were able to maintain exponential growth conditions with a doubling time of 3.0 min throughout 100 transfers or approximately 350 molecular generations by using an automatic handling device. Homology of in vitro amplification with rolling-circle replication was mirrored by the occurring evolutionary processes. Deletion events most likely caused by a slipped mispairing mechanism as postulated for in vivo replication took place. Under our conditions, the mutation rate was high and a molecular quasi-species formed with a mutant lacking internal hairpin formation ability and thus outgrowing all other species under dGTP/dCTP deficiency.
A point mutation in Euglena gracilis chloroplast tRNA(Glu) uncouples protein and chlorophyll biosynthesis.
Authors: Stange-Thomann N , Thomann H U , Lloyd A J , Lyman H , Söll D
Published: May 2006

The universal precursor of tetrapyrrole pigments (e.g., chlorophylls and hemes) is 5-aminolevulinic acid (ALA), which in Euglena gracilis chloroplasts is derived via the two-step C5 pathway from glutamate charged to tRNA(Glu). The first enzyme in this pathway, Glu-tRNA reductase (GluTR) catalyzes the reduction of glutamyl-tRNA(Glu) (Glu-tRNA) to glutamate 1-semialdehyde (GSA) with the release of the uncharged tRNA(Glu). The second enzyme, GSA-2,1-aminomutase, converts GSA to ALA. tRNA(Glu) is a specific cofactor for the NADPH-dependent reduction by GluTR, an enzyme that recognizes the tRNA in a sequence-specific manner. This RNA is the normal tRNA(Glu), a dual-function molecule participating both in protein and in ALA and, hence, chlorophyll biosynthesis. A chlorophyll-deficient mutant of E. gracilis (Y9ZNalL) does not synthesize ALA from glutamate, although it contains GluTR and GSA-2,1-aminomutase activity. The tRNA(Glu) isolated from the mutant can still be acylated with glutamate in vitro and in vivo. Furthermore, it supports chloroplast protein synthesis; however, it is a poor substrate for GluTR. Sequence analysis of the tRNA and of its gene revealed a C56-->U mutation in the resulting gene product. C56 is therefore an important identity element for GluTR. Thus, a point mutation in the T loop of tRNA uncouples protein from chlorophyll biosynthesis.