bluuebunny/crossref_metadata_2025 huggingface dataset:
Model observers for assessment of image quality.
Authors: Barrett H H , Yao J , Rolland J P , Myers K J
Published: May 2006

Image quality can be defined objectively in terms of the performance of some "observer" (either a human or a mathematical model) for some task of practical interest. If the end user of the image will be a human, model observers are used to predict the task performance of the human, as measured by psychophysical studies, and hence to serve as the basis for optimization of image quality. In this paper, we consider the task of detection of a weak signal in a noisy image. The mathematical observers considered include the ideal Bayesian, the nonprewhitening matched filter, a model based on linear-discriminant analysis and referred to as the Hotelling observer, and the Hotelling and Bayesian observers modified to account for the spatial-frequency-selective channels in the human visual system. The theory behind these observer models is briefly reviewed, and several psychophysical studies relating to the choice among them are summarized. Only the Hotelling model with channels is mathematically tractable in all cases considered here and capable of accounting for all of these data. This model requires no adjustment of parameters to fit the data and is relatively insensitive to the details of the channel mechanism. We therefore suggest it as a useful model observer for the purpose of assessing and optimizing image quality with respect to simple detection tasks.
Use of a gene encoding a suppressor tRNA as a reporter of transcription: analyzing the action of the Nun protein of bacteriophage HK022.
Authors: Sloan S B , Weisberg R A
Published: May 2006

The Nun protein of phage HK022 blocks the expression of genes that lie downstream of the nut sites of phage lambda. Nun is believed to act by promoting premature termination of transcription at or near these sites. To test this hypothesis and to facilitate mapping the sites of termination, we inserted a gene encoding a suppressor tRNA immediately downstream of the lambda nutL site and determined the effect of Nun on tRNA level. We found that Nun severely reduced the accumulation of mature, biologically active tRNA and promoted the accumulation of short, promoter-proximal transcripts whose 3' ends were dispersed over a 100-nucleotide region downstream of nutL. These results are consistent with the hypothesis that Nun terminates transcription within the region immediately downstream of nutL and are inconsistent with the hypothesis that the only action of Nun is to prevent translation of genes located downstream of the nut site. The stability, small size, and easily assayable biological function of suppressor tRNA recommend it as a reporter of transcription in other systems.
Gene sequence tags from Plasmodium falciparum genomic DNA fragments prepared by the "genease" activity of mung bean nuclease.
Authors: Reddy G R , Chakrabarti D , Schuster S M , Ferl R J , Almira E C , Dame J B
Published: May 2006

A genes-first approach to genome sequencing is described which efficiently generates gene sequence tags from genomic DNA. Mung bean nuclease (EC 3.1.30.1) cleaves the genomic DNA of many organisms before and after genes and within some introns. Analysis of gene sequence tags prepared from mung bean nuclease-digested Plasmodium falciparum DNA demonstrates that this method has several advantages over the popular cDNA expressed sequence tag approach. To date, 673 sequence tags containing over 215 kb of sequence have been generated from 400 clones. Sixty clones (15%) have significant similarity to sequences in the protein and translated nucleic acid data bases. These represent 51 unique genes, of which only 5 encode previously known P. falciparum proteins. The identified proteins include those expressed in erythrocytic, exoerythrocytic, and gametocytic stages of the parasite. Thirty percent of clones identified appear to carry complete coding regions. The spacer DNA separating genes is rarely cloned. These gene sequence tags will form a useful data base from which to initiate projects to develop new therapeutics, vaccines, and strategies to control human malaria.
Genomic clone for sandbar shark lambda light chain: generation of diversity in the absence of gene rearrangement.
Authors: Hohman V S , Schuchman D B , Schluter S F , Marchalonis J J
Published: May 2006

While the general structure of immunoglobulin chains has remained relatively unchanged throughout evolution, the organization of the genes encoding these molecules differs substantially. To understand how the rearranging immunoglobulin system arose, it is necessary to examine living representatives of the most early vertebrate phyla. Elasmo-branches, which include the sharks, skates, and rays, are the most primitive phylogenetic class of vertebrates from which immunoglobulin DNA sequences have been obtained. In the sandbar shark (Carcharhinus plumbeus), the genes are arranged in individual clusters in which a single variable (V), joining (J), and constant (C) region gene, along with upstream regulatory elements, span a distance of approximately 4.4 kb or approximately 5.8 kb. We report the complete sequence of a genomic clone encoding sandbar shark lambda light chain. A unique finding of our study is that the V and J genes are fused in the germ line. Three additional clones have been shown by DNA sequencing to also have fused V and J genes. The four clones have complementarity-determining regions 3 of various lengths and amino acid sequence variability similar to the products of rearranged genes. Furthermore, analysis by polymerase chain reaction technology revealed an additional 26 genomic clones demonstrating fusion of the V and J segments. Therefore, VJ fusion is the prominent organizational feature of sandbar shark immunoglobulin light chain genes. This finding raises questions concerning the necessity of recombination to produce an antibody repertoire capable of reacting against a diverse array of antigens.
Rapid interchange between two distinct forms of cyanobacterial photosystem II reaction-center protein D1 in response to photoinhibition.
Authors: Clarke A K , Soitamo A , Gustafsson P , Oquist G
Published: May 2006

We have studied photoinhibition of photosynthesis in the cyanobacterium Synechococcus sp. PCC 7942, which possesses two distinct forms of the photosystem II reaction-center protein D1 (D1:1 and D1:2). We report here that when cells adapted to a growth irradiance of 50 mumol.m-2.s-1 are exposed to an irradiance of 500 mumol.m-2.s-1, the normally predominant D1 form (D1:1) is rapidly replaced with the alternative D1:2. This interchange is not only complete within the first hour of photoinhibition but is also fully reversible once cells are returned to 50 mumol.m-2 x s-1. By using a mutant that synthesizes only D1:1, we show that the failure to replace D1:1 with D1:2 during photoinhibition results in severe loss of photosynthetic activity as well as a diminished capacity to recover after the stress period. We believe that this interchange between D1 forms may constitute an active component in a protection mechanism unique among photosynthetic organisms that enables cyanobacteria to effectively cope with and recover from photoinhibition.
RNA sequence of astrovirus: distinctive genomic organization and a putative retrovirus-like ribosomal frameshifting signal that directs the viral replicase synthesis.
Authors: Jiang B , Monroe S S , Koonin E V , Stine S E , Glass R I
Published: May 2006

The genomic RNA of human astrovirus was sequenced and found to contain 6797 nt organized into three open reading frames (1a, 1b, and 2). A potential ribosomal frameshift site identified in the overlap region of open reading frames 1a and 1b consists of a "shifty" heptanucleotide and an RNA stem-loop structure that closely resemble those at the gag-pro junction of some retroviruses. This translation frame-shift may result in the suppression of in-frame amber termination at the end of open reading frame 1a and the synthesis of a nonstructural, fusion polyprotein that contains the putative protease and RNA-dependent RNA polymerase. Comparative sequence analysis indicated that the protease and polymerase of astrovirus are only distantly related to the respective enzymes of other positive-strand RNA viruses. The astrovirus polyprotein lacks the RNA helicase domain typical of other positive-strand RNA viruses of similar genome size. The genomic organization and expression strategy of astrovirus, with the protease and the polymerase brought together by predicted frameshift, most closely resembled those of plant leuteoviruses. Specific features of the sequence and genomic organization support the classification of astroviruses as an additional family of positive-strand RNA viruses, designated Astroviridae.
Salmonella induces the formation of filamentous structures containing lysosomal membrane glycoproteins in epithelial cells.
Authors: Garcia-del Portillo F , Zwick M B , Leung K Y , Finlay B B
Published: May 2006

Salmonella species invade and replicate within epithelial cells in membrane-bound vacuoles. In this report we show that upon infection of HeLa epithelial cells, Salmonella typhimurium residues in vacuoles that contain lysosomal membrane glycoproteins (lgps). Four to six hours after invasion, intracellular bacteria induce the formation of stable filamentous structures containing lgps that are connected to the bacteria-containing vacuoles. Formation of these lgp-rich structures requires viable intracellular bacteria and is blocked by inhibitors of vacuolar acidification. These structures are not present in uninfected cells or in cells infected with another invasive bacteria, Yersinia enterocolitica. Tracers added to the extracellular medium are not delivered to the Salmonella-induced filaments, suggesting that these structures are different from previously described tubular lysosomes. Initiation of intracellular bacterial replication correlates with formation of these lgp-containing filaments. Certain avirulent Salmonella mutants that are defective for intracellular replication fail to induce formation of these structures. These observations suggest that Salmonella-induced filaments containing lgps are linked to intracellular bacterial replication.
Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector.
Authors: Flotte T R , Afione S A , Conrad C , McGrath S A , Solow R , Oka H , Zeitlin P L , Guggino W B , Carter B J
Published: May 2006

Adeno-associated virus (AAV) vectors expressing the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA complement the cystic fibrosis (CF) defect in vitro. Unlike other DNA virus vectors, AAV is a stably integrating virus, which could make possible long-term in vivo complementation of the CF defect in the airway epithelium. We report AAV-CFTR gene transfer and expression after infection of primary CF nasal polyp cells and after in vivo delivery of AAV-CFTR vector to one lobe of the rabbit lung through a fiberoptic bronchoscope. In the rabbit, vector DNA could be detected in the infected lobe up to 6 months after administration. A 26-amino acid polypeptide sequence unique to the recombinant AAV-CFTR protein was used to generate both oligonucleotide probes and a polyclonal antibody which allowed the unambiguous identification of vector RNA and CFTR protein expression. With these reagents, CFTR RNA and protein were detected in the airway epithelium of the infected lobe for up to 6 months after vector administration. AAV vectors do, therefore, efficiently promote in vivo gene transfer to the airway epithelium which is stable over several months. These findings indicate that AAV-CFTR vectors could potentially be very useful for gene therapy.
Permeability properties of a large gated channel within the ferric enterobactin receptor, FepA.
Authors: Liu J , Rutz J M , Feix J B , Klebba P E
Published: May 2006

FepA is an Escherichia coli outer membrane receptor protein for the siderophore ferric enterobactin. Prior studies conducted in vivo suggested that FepA and other TonB-dependent outer membrane proteins transport ligands by a gated-channel mechanism. To corroborate and extend these findings we have determined the permeability properties of the FepA channel in vitro, by measuring the diffusion rates of hydrophilic nonelectrolytes through the FepA channel in liposome swelling experiments. Like porins, the FepA deletion mutant delta RV showed a size-dependent permeability to oligosaccharides, indicating that it forms a nonspecific, hydrophilic pore. Unlike OmpF and other E. coli porins, however, delta RV proteoliposomes transported stachyose (666 Da) and ferrichrome (740 Da). These data, and other uptake results with a series of maltodextrins of increasing size, confirm the existence of a channel domain within FepA that is considerably larger than OmpF-type pores. These results represent a reconstitution of the channel function of a TonB-dependent receptor protein and establish that FepA contains the largest channel that has been characterized in the E. coli outer membrane.
Biliary glycoprotein, a potential human cell adhesion molecule, is down-regulated in colorectal carcinomas.
Authors: Neumaier M , Paululat S , Chan A , Matthaes P , Wagener C
Published: May 2006

Biliary glycoprotein (BGP) is the human homologue of a cell adhesion molecule (CAM) of the rat designated Cell-CAM. The BGP gene is a member of the carcinoembryonic antigen gene family, which belongs to the immunoglobulin superfamily. BGP is expressed in cells of epithelial and myeloid origin. In granulocytes, BGP is a main antigen of the CD66 cluster of differentiation antigens that mediate the binding to endothelial E-selectin. Since BGP is a major human CAM, the expression of BGP was studied in 21 colorectal carcinoma tissue specimens and in the respective adjacent normal mucosae. As an internal control for epithelial mRNA, the expression of cytokeratin 18 was evaluated in parallel. In addition, the expression of carcinoembryonic antigen and nonspecific crossreacting antigen, which are highly homologous to BGP, was investigated. Two BGP mRNAs of 3.9 and 1.5 kilobases were detected in the normal colonic mucosa samples. The median of the tumor-to-normal ratios of mRNA expression was 0.2 for both BGP mRNAs. In contrast, the median was 1.2 for cytokeratin, 1.0 for carcinoembryonic antigen, and 1.4 for nonspecific crossreacting antigen. Relative to cytokeratin 18 expression, the expression of BGP was reduced to < or = 0.1 in half of the tumors and to < or = 0.4 in > 80% of the tumors. These findings indicate that the loss or reduced expression of the adhesion molecule BGP is a major event in colorectal carcinogenesis.
The precursor region of a protein active in sperm-egg fusion contains a metalloprotease and a disintegrin domain: structural, functional, and evolutionary implications.
Authors: Wolfsberg T G , Bazan J F , Blobel C P , Myles D G , Primakoff P , White J M
Published: May 2006

PH-30, a sperm surface protein involved in sperm-egg fusion, is composed of two subunits, alpha and beta, which are synthesized as precursors and processed, during sperm development, to yield the mature forms. The mature PH-30 alpha/beta complex resembles certain viral fusion proteins in membrane topology and predicted binding and fusion functions. Furthermore, the mature subunits are similar in sequence to each other and to a family of disintegrin domain-containing snake venom proteins. We report here the sequences of the PH-30 alpha and beta precursor regions. Their domain organizations are similar to each other and to precursors of snake venom metalloproteases and disintegrins. The alpha precursor region contains, from amino to carboxyl terminus, pro, metalloprotease, and disintegrin domains. The beta precursor region contains pro and metalloprotease domains. Residues diagnostic of a catalytically active metalloprotease are present in the alpha, but not the beta, precursor region. We propose that the active sites of the PH-30 alpha and snake venom metalloproteases are structurally similar to that of astacin. PH-30, acting through its metalloprotease and/or disintegrin domains, could be involved in sperm development as well as sperm-egg binding and fusion. Phylogenetic analysis indicates that PH-30 stems from a multidomain ancestral protein.
Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins.
Authors: Pan K M , Baldwin M , Nguyen J , Gasset M , Serban A , Groth D , Mehlhorn I , Huang Z , Fletterick R J , Cohen F E
Published: May 2006

Prions are composed largely, if not entirely, of prion protein (PrPSc in the case of scrapie). Although the formation of PrPSc from the cellular prion protein (PrPC) is a post-translational process, no candidate chemical modification was identified, suggesting that a conformational change features in PrPSc synthesis. To assess this possibility, we purified both PrPC and PrPSc by using nondenaturing procedures and determined the secondary structure of each. Fourier-transform infrared (FTIR) spectroscopy demonstrated that PrPC has a high alpha-helix content (42%) and no beta-sheet (3%), findings that were confirmed by circular dichroism measurements. In contrast, the beta-sheet content of PrPSc was 43% and the alpha-helix 30% as measured by FTIR. As determined in earlier studies, N-terminally truncated PrPSc derived by limited proteolysis, designated PrP 27-30, has an even higher beta-sheet content (54%) and a lower alpha-helix content (21%). Neither PrPC nor PrPSc formed aggregates detectable by electron microscopy, while PrP 27-30 polymerized into rod-shaped amyloids. While the foregoing findings argue that the conversion of alpha-helices into beta-sheets underlies the formation of PrPSc, we cannot eliminate the possibility that an undetected chemical modification of a small fraction of PrPSc initiates this process. Since PrPSc seems to be the only component of the "infectious" prion particle, it is likely that this conformational transition is a fundamental event in the propagation of prions.
Expression of nerve growth factor and nerve growth factor receptor tyrosine kinase Trk in activated CD4-positive T-cell clones.
Authors: Ehrhard P B , Erb P , Graumann U , Otten U
Published: May 2006

Recent evidence suggests that nerve growth factor (NGF), in addition to its neurotrophic functions, acts as an immunomodulator mediating "cross-talk" between neuronal and immune cells, including T lymphocytes. We have analyzed murine CD4+ T-cell clones for their ability to express transcripts encoding NGF, low-affinity NGF receptor, and trk protooncogene, the signal-transducing receptor subunit for NGF. We show that two CD4+ T-helper (Th) clones, Th0-type clone 8/37 and Th2-type clone D10.G4.1, express NGF and Trk mRNA after appropriate activation with mitogen or with antigen and antigen-presenting cells. NGF and trk induction occurred to a similar extent and over a similar time course in activated 8/37 T cells, raising the possibility that NGF and trk genes are under coordinate control. NGF and NGF receptor expression does not seem to be a universal property of all activated CD4+ T cells, since Th1-type clone 9/9 did not express any of the transcripts after either stimulation. The absence of low-affinity NGF receptor mRNA in resting and activated T cells implies that the low-affinity NGF receptor is not involved in NGF signal transduction in CD4+ T cells. Our finding that activated CD4+ T-cell clones not only express Trk but also synthesize and release biologically active NGF implicates NGF as an autocrine and/or paracrine factor in the development and regulation of immune responses.
Human bradykinin B2 receptors isolated by receptor-specific monoclonal antibodies are tyrosine phosphorylated.
Authors: Jong Y J , Dalemar L R , Wilhelm B , Baenziger N L
Published: May 2006

We report the immunoaffinity isolation of bradykinin B2 receptors in a tyrosine-phosphorylated state from WI-38 human lung fibroblasts. We generated six monoclonal antibodies directed against B2 bradykinin receptor biologic activity mediating prostaglandin E2 production in WI-38. These cells express a repertoire of bradykinin receptor affinity forms with closely correlated biologic activity and [3H]bradykinin binding. Some of the monoclonal antibodies selectively recognize intermediate-affinity (Kd = 5.6 nM) or low-affinity (Kd = 42 nM) receptor forms, whereas others recognize epitopes common to both. The monoclonal antibodies block bradykinin binding and biologic activity. Immunoaffinity chromatography on an immobilized monoclonal antibody of intermediate- plus low-affinity specificity yields WI-38 B2 receptors with intact [3H]bradykinin binding activity and a molecular mass of 78 kDa. The same band is immunoblotted by all the monoclonal antibodies, indicating a similar molecular mass for receptor forms of different affinity. Anti-phosphotyrosine antibodies demonstrate that the receptors are tyrosine phosphorylated, with implications for receptor function and regulation. Genistein completely inhibits bradykinin-mediated prostaglandin E2 production with an IC50 of 8 microM, indicating that tyrosine kinase activity is critical for the signal transduction leading to arachidonic acid release.
Cooperative inhibition of NF-kappa B and Tat-induced superactivation of human immunodeficiency virus type 1 long terminal repeat.
Authors: Biswas D K , Ahlers C M , Dezube B J , Pardee A B
Published: May 2006

Human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-regulated gene expression is stimulated independently by the cellular trans-activator NF-kappa B and the viral protein Tat. Noncytotoxic concentrations of the drug pentoxifylline (PTX) inhibited interaction of NF-kappa B with its motif and the stimulation of HIV-1 LTR-driven gene expression in Jurkat cells. Tat protein (from a cotransfected Tat-expression vector) also induced activation of HIV-1 LTR-driven gene expression. This activation was unaffected by PTX when NF-kappa B sites in the HIV-1 LTR were mutated, suggesting that this drug does not directly influence Tat function, which, however, was inhibited by the Tat-inhibitor Ro 24-7429. Transient reporter gene expression regulated by HIV-1 LTR with wild-type NF-kappa B motifs in the presence of Tat protein was 10- to 60-fold higher than in the presence of either of the trans-activators alone, demonstrating superactivation of HIV-1 LTR by the concerted action of both the trans-activators. Treatment of cells with either PTX or Ro 24-7429 inhibited this superactivation of the HIV-1 LTR. The inhibitory effect of these two drugs in combination, at concentrations that alone did not significantly influence viral promoter activity, was far more than additive. A cooperative action of PTX (NF-kappa B inhibitor) and Ro 24-7429 (Tat inhibitor) on HIV-1 LTR-regulated gene expression is suggested. Concentrations of the drugs that induced maximum inhibition of HIV-1 LTR through their cooperative action are far below cytotoxic levels. Thus, the combination of these two inhibitors could be very effective for anti-HIV therapy.
Transformation of human T-cell clones by Herpesvirus saimiri: intact antigen recognition by autonomously growing myelin basic protein-specific T cells.
Authors: Weber F , Meinl E , Drexler K , Czlonkowska A , Huber S , Fickenscher H , Müller-Fleckenstein I , Fleckenstein B , Wekerle H , Hohlfeld R
Published: May 2006

Herpesvirus saimiri has recently been shown to immortalize human T cells. It was unknown, however, whether Herpesvirus saimiri transformation affects T-cell receptor (TCR) expression and signal transduction. In the present study, we have transformed CD4+ human T-cell clones specific for human myelin basic protein. The transformed T cells were grown in interleukin 2 and divided in the absence of antigen and antigen-presenting cells. They retained the membrane phenotype of activated T cells and secreted the cytokines interferon gamma and lymphotoxin, but interleukin 4 was not detected. Further, the transformed T cells continued to express the original TCR as demonstrated by TCR variable-region-V beta-specific monoclonal antibodies and TCR sequencing. Antigen-specific recognition and signal transduction by the TCR were demonstrated by myelin-basic-protein-induced HLA-DR-restricted secretion of interferon gamma and lymphotoxin and by myelin-basic-protein-specific proliferation. Antigen specificity and reactivity have been maintained for > 1 year after transformation. Transformation with Herpesvirus saimiri now allows the production of virtually unlimited numbers of (auto)antigen-specific T cells expressing functional TCR and a stable membrane phenotype. This technology will facilitate studies of the pathogenesis of putative autoimmune diseases, such as multiple sclerosis, and may be of help in TCR-targeted immunotherapy.
Sperm 1: a POU-domain gene transiently expressed immediately before meiosis I in the male germ cell.
Authors: Andersen B , Pearse R V , Schlegel P N , Cichon Z , Schonemann M D , Bardin C W , Rosenfeld M G
Published: May 2006

Members of the POU-domain gene family encode for transcriptional regulatory molecules that are important for terminal differentiation of several organ systems, including anterior pituitary, sensory neurons, and B lymphocytes. We have identified a POU-domain factor, referred to as sperm 1 (Sprm-1). This factor is most related to the transactivator Oct-3/4, which is expressed in the early embryo, primordial germ cells, and the egg. However, in contrast with Oct-3/4, rat Sprm-1 is selectively expressed during a 36- to 48-hr period immediately preceding meiosis I in male germ cells. Although the POU-domain of Sprm-1 is divergent from the POU-domains of Oct-1 and Oct-2, random-site-selection assay reveals that Sprm-1 preferentially binds to a specific variant of the classic octamer DNA-response element in which the optimal sequence differs from that preferred by Oct-1 and Pit-1. These data suggest that the Sprm-1 gene encodes a DNA-binding protein that may exert a regulatory function in meiotic events that are required for terminal differentiation of the male germ cell.
Opiate-like substances in an invertebrate, an opiate receptor on invertebrate and human immunocytes, and a role in immunosuppression.
Authors: Stefano G B , Digenis A , Spector S , Leung M K , Bilfinger T V , Makman M H , Scharrer B , Abumrad N N
Published: May 2006

The presence of morphine-like and codeine-like substances was demonstrated in the pedal ganglia, hemolymph, and mantle tissues of the mollusc Mytilus edulis. The pharmacological activities of the endogenous morphine-like material resemble those of authentic morphine. Both substances were found to counteract, in a dose-dependent manner, the stimulatory effect of tumor necrosis factor alpha or interleukin 1 alpha on human monocytes and Mytilus immunocytes, when added simultaneously to the incubation medium. The immunosuppressive effect of this opiate material expresses itself in a lowering of chemotactic activity, cellular velocity, and adherence. Codeine mimics the activity of authentic morphine, but only at much higher concentrations. Specific high-affinity receptor sites (mu 3) for morphine have been identified on human monocytes and Mytilus immunocytes. In Mytilus recovering from experimentally induced stress, the return of "altered" immunocytes to a more inactive state appears to be due to a significant rise in the content of morphine-like material in the pedal ganglia and hemolymph at this time. Thus, morphine may have a role in calming or terminating the state of immune alertness.
Dominant T-cell-receptor beta chain variable region V beta 14+ clones in juvenile rheumatoid arthritis.
Authors: Grom A A , Thompson S D , Luyrink L , Passo M , Choi E , Glass D N
Published: May 2006

The characteristic histopathology and major histocompatibility complex associations in juvenile rheumatoid arthritis suggest an oligoclonal antigen-specific T-cell population may be critical to pathogenesis. To test this, we analyzed the T-cell repertoire of a polyarticular HLA-DR4+ juvenile rheumatoid arthritis patient with an aggressive form of disease that required arthrocentesis of the knee joints and early replacement of both hip joints. A comparison of T-cell-receptor beta chain variable region (V beta) gene expression in peripheral blood and synovial fluid performed by semiquantitation of cDNA samples amplified by the PCR revealed overexpression of the T-cell-receptor V beta 14 gene family. To determine the nature of V beta 14 overexpression, we sequenced randomly cloned amplification products derived from two synovial fluid, two synovial tissue, and three peripheral blood samples by using a V beta 14/beta chain constant region primer pair. Sequence data showed that the T-cell response in the synovia was oligoclonal. Of four clones found, one was present in all joints examined and persisted over time. This clone accounted for 67% and 74% of all V beta 14+ clones sequenced in two synovial fluid samples and 75% and 40% in two synovial tissue samples. This clone was also found at a lesser frequency in peripheral blood samples. Further studies provided evidence for the presence of oligoclonally expanded populations of T cells utilizing the V beta 14 T-cell receptor in 6 of 27 patients examined. In contrast to the remaining patients studied, 3 with a late onset polyarticular course who exhibited especially marked clonality were characterized by features typical of adult rheumatoid arthritis (IgM rheumatoid factor-positive and HLA-DR4+). These data suggest a role for V beta 14+ T cells in a group of juvenile rheumatoid arthritis patients.
Glyceraldehyde-3-phosphate dehydrogenase is required for the transport of nitric oxide in platelets.
Authors: McDonald B , Reep B , Lapetina E G , Molina y Vedia L
Published: May 2006

Nitric oxide (NO) or NO-generating compounds like sodium nitroprusside (SNP) increase cellular levels of cGMP and produce S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase [GAPDH; D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12]. In search of a reagent that could discriminate between these two effects, we used the sesquiterpene antibiotic koningic acid, which binds to GAPDH at the Cys-149 of the active site. Koningic acid inhibited basal and sodium nitroprusside-stimulated NAD-dependent covalent modification of purified rabbit muscle GAPDH in a dose-dependent manner. Furthermore, we tested the effect of koningic acid on human platelets. Approximately 90% of GAPDH is present in the cytosol of human platelets, and the exposure of platelet cytosol to koningic acid inhibited GAPDH activity, while the soluble guanylyl cyclase (basal and sodium nitroprusside-stimulated) activity remained unaltered. Pretreatment of intact platelets with koningic acid slowed the rate of aggregation induced by a submaximal concentration of thrombin. In addition, the antibiotic also inhibited the cGMP increases triggered by SNP, S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosyndomidine (SIN-1) but failed to prevent an increase in cGMP caused by nitrosylated albumin. Under the same conditions, koningic acid also inhibited basal and SNP- SNAP-, and SIN-1-stimulated NAD-dependent modification of GAPDH and its enzymatic activity. These results suggest that the mechanism of delivery of NO from SNP, SNAP, and SIN-1 to platelets may require the active form of GAPDH. When NO is delivered by nitrosylated albumin, active GAPDH was not necessary.
The receptor for interleukin 3 is selectively induced in human endothelial cells by tumor necrosis factor alpha and potentiates interleukin 8 secretion and neutrophil transmigration.
Authors: Korpelainen E I , Gamble J R , Smith W B , Goodall G J , Qiyu S , Woodcock J M , Dottore M , Vadas M A , Lopez A F
Published: May 2006

Interleukin (IL)-3 stimulates hemopoiesis in vitro. However, IL-3 is not normally found in bone marrow, raising doubts as to the in vivo role of IL-3. We have found that human umbilical vein endothelial cells (HUVEC) express functional high-affinity receptors for IL-3 after stimulation with tumor necrosis factor alpha (TNF-alpha), IL-1 beta, or lipopolysaccharide, and that this receptor is involved in inflammatory phenomena. TNF-alpha caused time- and dose-dependent up-regulation of mRNA for the IL-3 receptor alpha and beta chains, with maximal effects occurring 16-36 h after stimulation with TNF-alpha at 100 units/ml. Induction of mRNA correlated with protein expression on the cell surface as judged by monoclonal antibody staining and by the ability of HUVEC to specifically bind 125I-labeled IL-3. Scatchard analysis under optimal conditions of TNF-alpha stimulation revealed approximately 1500 IL-3 receptors per cell, which were of a high-affinity class (Kd = 500 pM) only. In contrast to a previous report, receptors for granulocyte-macrophage colony-stimulating factor could not be detected. IL-3 binding to TNF-alpha-activated HUVEC enhanced IL-8 production, E-selection expression, and neutrophil transmigration. The selective induction of a functional IL-3 receptor on endothelial cells suggests that, beyond hemopoiesis, IL-3 may have an important role in chronic inflammation and in allergic diseases.
Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene.
Authors: Lubahn D B , Moyer J S , Golding T S , Couse J F , Korach K S , Smithies O
Published: May 2006

Estrogen receptor and its ligand, estradiol, have long been thought to be essential for survival, fertility, and female sexual differentiation and development. Consistent with this proposed crucial role, no human estrogen receptor gene mutations are known, unlike the androgen receptor, where many loss of function mutations have been found. We have generated mutant mice lacking responsiveness to estradiol by disrupting the estrogen receptor gene by gene targeting. Both male and female animals survive to adulthood with normal gross external phenotypes. Females are infertile; males have a decreased fertility. Females have hypoplastic uteri and hyperemic ovaries with no detectable corpora lutea. In adult wild-type and heterozygous females, 3-day estradiol treatment at 40 micrograms/kg stimulates a 3- to 4-fold increase in uterine wet weight and alters vaginal cornification, but the uteri and vagina do not respond in the animals with the estrogen receptor gene disruption. Prenatal male and female reproductive tract development can therefore occur in the absence of estradiol receptor-mediated responsiveness.
Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease.
Authors: Uéda K , Fukushima H , Masliah E , Xia Y , Iwai A , Yoshimoto M , Otero D A , Kondo J , Ihara Y , Saitoh T
Published: May 2006

A neuropathological hallmark of Alzheimer disease (AD) is a widespread amyloid deposition. We analyzed the entire amino acid sequences in an amyloid preparation and found, in addition to the major beta/A4-protein (A beta) fragment, two unknown peptides. We raised antibodies against synthetic peptides using subsequences of these peptides. These antibodies immunostained amyloid in neuritic and diffuse plaques as well as vascular amyloid. Electron microscopic analysis demonstrated that the immunostaining was localized on amyloid fibrils. We have isolated an apparently full-length cDNA encoding a 140-amino-acid protein within which two previously unreported amyloid sequences are encoded in tandem in the most hydrophobic domain. We tentatively named this 35-amino acid peptide NAC (non-A beta component of AD amyloid) and its precursor NACP. NAC is the second component, after A beta, identified chemically in the purified AD amyloid preparation. Secondary structure predictions indicate that the NAC peptide sequence has a strong tendency to form beta-structures consistent with its association with amyloid. NACP is detected as a M(r) 19,000 protein in the cytosolic fraction of brain homogenates and comigrates on immunoblots with NACP synthesized in Escherichia coli from NACP cDNA. NACP mRNA is expressed principally in brain but is also expressed in low concentrations in all tissues examined except in liver, suggesting its ubiquitous and brain-specific functions. The availability of the cDNA encoding full-length NACP should help to elucidate the mechanisms of amyloidosis in AD.
Isolation of cDNA encoding the human NF-E2 protein.
Authors: Chan J Y , Han X L , Kan Y W
Published: May 2006

The human homolog of mouse NF-E2 was isolated from the K562 cell line and found to encode a member of the basic leucine-zipper family of DNA-binding regulatory proteins. The deduced amino acid sequence of the mouse and human proteins exhibited near identity. Comparison to the related protein, Nrf1, revealed significant homologies at isolated regions, particularly within the basic domain, suggesting that NF-E2 and Nrf1 are members of a distinct subfamily of basic leucine-zipper proteins that share similar DNA-binding properties. High levels of human NF-E2 mRNA were observed in human erythroleukemic cell lines examined. Extensive survey of human tissue samples found NF-E2 expression not limited to erythropoeitic organs. Expression in the colon and testis suggests that NF-E2 may participate in the regulation of genes other than globin.
Human cytomegalovirus induces JC virus DNA replication in human fibroblasts.
Authors: Heilbronn R , Albrecht I , Stephan S , Bürkle A , zur Hausen H
Published: May 2006

JC virus, a human papovavirus, is the causative agent of the demyelinating brain disease progressive multifocal leucoencephalopathy (PML). PML is a rare but fatal disease which develops as a complication of severe immunosuppression. Latent JC virus is harbored by many asymptomatic carriers and is transiently reactivated from the latent state upon immunosuppression. JC virus has a very restricted host range, with human glial cells being the only tissue in which it can replicate at reasonable efficiency. Evidence that latent human cytomegalovirus is harbored in the kidney similar to latent JC virus led to the speculation that during episodes of impaired immunocompetence, cytomegalovirus might serve as helper virus for JC virus replication in otherwise nonpermissive cells. We show here that cytomegalovirus infection indeed leads to considerable JC virus DNA replication in cultured human fibroblasts that are nonpermissive for the replication of JC virus alone. Cytomegalovirus-mediated JC virus replication is dependent on the JC virus origin of replication and T antigen. Ganciclovir-induced inhibition of cytomegalovirus replication is associated with a concomitant inhibition of JC virus replication. These results suggest that reactivation of cytomegalovirus during episodes of immunosuppression might lead to activation of latent JC virus, which would enhance the probability of subsequent PML development. Ganciclovir-induced repression of both cytomegalovirus and JC virus replication may form the rational basis for the development of an approach toward treatment or prevention of PML.
Isolation of Hsp90 mutants by screening for decreased steroid receptor function.
Authors: Bohen S P , Yamamoto K R
Published: May 2006

The 90-kDa heat shock protein Hsp90 represents a highly conserved strongly expressed gene family; in Saccharomyces cerevisiae, Hsp90 proteins are essential for cell viability. Hsp90 interacts with certain cellular proteins, including steroid hormone receptors, tyrosine and serine/threonine kinases, and other heat shock proteins, but its biological functions are not understood. The unliganded glucocorticoid receptor must interact with Hsp90 to acquire competence for high-affinity hormone binding and subsequent transcriptional regulation. By screening in yeast for defects in glucocorticoid receptor function, Hsp90 mutants were isolated. Four such mutants are described, all of which interact with the glucocorticoid receptor but display distinct defects in ligand responsiveness and differences in growth and resistance to high temperature.
Identification of a cytochrome P450 gene by reverse transcription--PCR using degenerate primers containing inosine.
Authors: Shen Z , Wells R L , Liu J , Elkind M M
Published: May 2006

A cytochrome P450-like gene, tentatively named P450CMEF, was amplified by a mixed oligonucleotide-primed amplification of cDNA from C3H mouse embryo fibroblast cells, designated 10T1/2, that had been treated with 7,12-dimethylbenz[a]anthracene (DMBA) or benz[a]anthracene (BA). A set of inosine-containing degenerate primers that were targeted to two conserved regions of known cytochrome P450 cDNAs were used. One primer was coded for the well-described and conserved heme-binding region of P450 enzymes, and the second was designed based upon other considerations of homology among P450 molecules. One of the four PCR-amplified cDNA products hybridized to two major RNA bands, 4.2 and 5.3 kb, that were induced by DMBA or BA. The amino acid sequence of the fragment deduced from the base-sequence data indicate that the amplified cDNA has a 50-55% identity with the cytochrome P450 subfamily 1A. The induction of P450CMEF mRNA preceded the induction of aryl hydrocarbon hydroxylase activity after DMBA or BA treatment, suggesting that the product of P450CMEF is involved in the metabolism of these polycyclic aromatic hydrocarbons in 10T1/2 cells. From the partial sequence of the cDNA identified by this procedure, we propose that P450CMEF is a member of the P450 superfamily, possibly in a subfamily of family 1, that is induced in 10T1/2 cells by DMBA and BA. This method should be useful in identifying additional P450 genes and genes in other gene families.
Cys4057 of apolipoprotein(a) is essential for lipoprotein(a) assembly.
Authors: Brunner C , Kraft H G , Utermann G , Müller H J
Published: May 2006

Lipoprotein(a) contains one copy each of apolipoprotein B-100 and apolipoprotein(a). It has been hypothesized that a disulfide bond might exist between Cys4057 of apolipoprotein(a) and Cys3734 in apolipoprotein B-100. To investigate the role of Cys4057 for lipoprotein(a) assembly, wild-type and in vitro mutagenized apolipoprotein(a) cDNA plasmids were expressed in the human hepatocarcinoma line HepG2. The mutant plasmids encoded apolipoprotein(a) species with Cys4057 exchanged to either serine or glycine. Untransfected HepG2 cells, although able to secrete apolipoprotein B-100-containing lipoproteins, do not synthesize detectable amounts of apolipoprotein(a). After transfection of wild-type plasmid, almost all apolipoprotein(a) in the culture supernatant was present in lipoprotein(a)-like particles as demonstrated by immunoblotting, density-gradient centrifugation, and ELISA. The same analysis performed with supernatants of cells transfected with plasmids mutated in codon 4057 revealed free apolipoprotein(a) glycoprotein without detectable amounts of lipoprotein-associated apolipoprotein(a). Our results strongly suggest the existence of a disulfide bridge between Cys4057 of apolipoprotein(a) and apolipoprotein B-100 within recombinant lipoprotein(a) particles. Furthermore, they indicate that disulfide bridge formation is essential for assembly of the lipoprotein(a)-like complex produced by HepG2 cells and suggest a similar role of Cys4057 during lipoprotein(a) assembly in vivo.
Discovery of biologically active peptides in random libraries: solution-phase testing after staged orthogonal release from resin beads.
Authors: Salmon S E , Lam K S , Lebl M , Kandola A , Khattri P S , Wade S , Pátek M , Kocis P , Krchnák V , Thorpe D
Published: May 2006

To speed drug discovery, we developed an approach for identification of individual peptides with a desired biological activity from a library containing millions of peptides. The approach uses sequential orthogonal release of chemically synthesized peptides from insoluble beads, followed by testing in solution. In this system, each bead within a library of beads has one peptide sequence, but peptide molecules are attached to the bead with three types of chemical linkers, including two linkers cleavable at different pH optima. An uncleavable linker keeps some peptide attached to the bead for sequencing positives from the solution assay. Applicability of this discovery technique was documented by identifying ligands for a monoclonal antibody and for the human platelet fibrinogen receptor, glycoprotein IIb/IIIa.
IgG from amyotrophic lateral sclerosis patients increases current through P-type calcium channels in mammalian cerebellar Purkinje cells and in isolated channel protein in lipid bilayer.
Authors: Llinás R , Sugimori M , Cherksey B D , Smith R G , Delbono O , Stefani E , Appel S
Published: May 2006

The effect of the IgG from amyotrophic lateral sclerosis (ALS) patients was tested on the voltage-dependent barium currents (IBa) in mammalian dissociated Purkinje cells and in isolated P-type calcium channels in lipid bilayers. Whole cell clamp of Purkinje cells demonstrates that ALS IgG increases the amplitude of IBa without modifying their voltage kinetics. This increased IBa could be blocked by a purified nonpeptide toxin from Agelenopsis aperta venom (purified funnel-web spider toxin) or by a synthetic polyamine analog (synthetic funnel-web spider toxin) and by a peptide toxin from the same spider venom, omega-Aga-IVA. Similar results were obtained on single-channel recordings from purified P channel protein. The addition of ALS IgG increased single-channel IBa open time without affecting slope conductance. The results described above were not seen with normal human IgG nor with boiled ALS IgG. It is concluded that ALS IgG enhances inward current through P-type calcium channels. Since P-type Ca2+ channels are present in motoneuron axon terminals, we propose that the enhanced calcium current triggered by ALS IgG may contribute to neuronal damage in ALS.
Methotrexate resistance in an in vivo mouse tumor due to a non-active-site dihydrofolate reductase mutation.
Authors: Dicker A P , Waltham M C , Volkenandt M , Schweitzer B I , Otter G M , Schmid F A , Sirotnak F M , Bertino J R
Published: May 2006

A series of methotrexate (MTX)-resistant L1210 leukemia murine ascites tumors were developed in vivo and analyzed for drug resistance. Three of 20 tumors studied expressed an altered dihydrofolate reductase (DHFR) and each was identical, having a C to T base transition at nucleotide 46 in the DHFR gene as demonstrated by PCR and direct sequencing. This transition results in a Gly to Trp substitution at amino acid 15 of the enzyme. Purified altered enzyme displays significantly lower binding affinity for the antifolates MTX, trimetrexate, edatrexate, and trimethoprim with respective Ki values 165-, 76-, 30-, and 28-fold higher than values obtained for enzyme isolated from parental tumor (wild-type enzyme). Substrate (dihydrofolate) and cofactor (NADPH) binding is also diminished for the mutant enzyme, although to a lesser extent (17.3- and 3.6-fold higher Km, respectively). Gly-15 is highly conserved for all vertebrate species of DHFR but has no known interaction(s), either directly or indirectly, with bound cofactor, substrate, or inhibitor. Protein molecular modeling reveals that the affected residue is 9-12 A away from the enzyme active site and located in a region analogous to the mobile Met-20 loop domain characterized for Escherichia coli DHFR.