The high specificity of antibodies for their antigen allows a fine discrimination of target conformations and post-translational modifications, making antibodies the first choice tool to interrogate ...the proteome. We describe here an approach based on a large-scale intracellular expression and selection of antibody fragments in eukaryotic cells, so-called intrabodies, and the subsequent identification of their natural target within living cell. Starting from a phenotypic trait, this integrated system allows the identification of new therapeutic targets together with their companion inhibitory intrabody. We applied this system in a model of allergy and inflammation. We first cloned a large and highly diverse intrabody library both in a plasmid and a retroviral eukaryotic expression vector. After transfection in the RBL-2H3 rat basophilic leukemia cell line, we performed seven rounds of selection to isolate cells displaying a defect in Fc epsilon RI-induced degranulation. We used high throughput sequencing to identify intrabody sequences enriched during the course of selection. Only one intrabody was common to both plasmid and retroviral selections, and was used to capture and identify its target from cell extracts. Mass spectrometry analysis identified protein RGD1311164 (C12orf4), with no previously described function. Our data demonstrate that RGD1311164 is a cytoplasmic protein implicated in the early signaling events following Fc epsilon RI-induced cell activation. This work illustrates the strength of the intrabody-based in-cell selection, which allowed the identification of a new player in mast cell activation together with its specific inhibitor intrabody.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We search for new charmless decays of neutral b hadrons to pairs of charged hadrons, using 1 fb(-1) of data collected by the CDF II detector at the Fermilab Tevatron. We report the first observation ...of the Bs0-->K-pi+ decay and measure B(Bs0-->K-pi+)=(5.0+/-0.7(stat)+/-0.8(syst))x10(-6). We also report the first observation of charmless b-baryon decays, and measure B(Lambdab0-->ppi-)=(3.5+/-0.6(stat)+/-0.9(syst))x10(-6) and B(Lambdab0-->pK-)=(5.6+/-0.8(stat)+/-1.5(syst))x10(-6). No evidence is found for other modes, and we set the limit B(Bs0-->pi+pi;-)<1.2x10(-6) at 90% C.L.
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CMK, CTK, FMFMET, IJS, NUK, PNG, UM
We report a measurement of the single-top-quark production cross section in 2.2 fb;{-1} of pp collision data collected by the Collider Detector at Fermilab at sqrts=1.96 TeV. Candidate events are ...classified as signal-like by three parallel analyses which use likelihood, matrix element, and neural network discriminants. These results are combined in order to improve the sensitivity. We observe a signal consistent with the standard model prediction, but inconsistent with the background-only model by 3.7 standard deviations with a median expected sensitivity of 4.9 standard deviations. We measure a cross section of 2.2(-0.6)(+0.7)(stat+syst) pb, extract the Cabibbo-Kobayashi-Maskawa matrix-element value |V(tb)|=0.88(-0.12)(+0.13)(stat+syst)+/-0.07(theory), and set the limit |V(tb)|>0.66 at the 95% C.L.
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CMK, CTK, FMFMET, IJS, NUK, PNG, UM
The advent of AC superconducting wires with ultra-fine filaments promoted the development of superconducting transformers, but most superconducting transformers with large capacity had been single ...phase ones. This paper describes the development of a 100 kVA three phase superconducting transformer (440/220 V). Fundamental characteristics of the transformer are obtained through no-load tests, short circuit tests and load tests under various conditions. The transformer cores are made of amorphous films and immersed in liquid helium with transformer windings for simplifying the structure of it. The losses and efficiency are analyzed.
Somatic cell genetic alterations are a hallmark of tumor development and progression. Although various technologies have been developed and utilized to identify genetic aberrations, identifying ...genetic translocations at the chromosomal level is still a challenging task. High density SNP microarrays are useful to measure DNA copy number variation (CNV) across the genome. Utilizing SNP array data of cancer cell lines and patient samples, we evaluated the CNV and copy number breakpoints for several known fusion genes implicated in tumorigenesis. This analysis demonstrated the potential utility of SNP array data for the prediction of genetic aberrations via translocations based on identifying copy number breakpoints within the target genes. Genome-wide analysis was also performed to identify genes harboring copy number breakpoints across 820 cancer cell lines. Candidate oncogenes were identified that are linked to potential translocations in specific cancer cell lines.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Unicameral bone cyst (UBC) is a benign cystic lesion in children which is prone to fracture. Various treatments are available, but recurrence after different types of percutaneous injection therapy ...can cause bone destruction and pathologic fracture. The potential therapeutic effects of anti-resorptive agents, such as bisphosphonates, have not been investigated for UBC. The objective of this study was to characterize the cells from the fibro-cellular membrane of unicameral bone cyst (UBC cells) and to determine whether zoledronate, a nitrogen-containing bisphosphonate, could induce apoptosis in UBC cells. Flow cytometry and immunoblotting were performed in order to determine whether zoledronate induced apoptosis. Cells derived from normal human trabecular bones were used as controls against UBC cells to compare the effect of zoledronate in inducing apoptosis. Immunohisto/cytochemistry (IHC/ICC) and mini-array analyses were performed on tissues and cultured cells. Isolated peripheral blood mononuclear cells were incubated with conditioned media from the UBC cells to determine whether they are capable of inducing osteoclastogenesis. UBC membrane is composed of cells staining positively with CD68, SDF-1, STRO-1 and RANKL, but in vitro cells showed no staining with antibodies to CD68 and STRO-1, suggesting that there was a clonal selection of stromal cells during cell culture. UBC cells also express RUNX2 (runt-related transcription factor-2, core binding factor-1), a key transcription factor for osteoblastic differentiation. In addition, media collected from UBC cells induced a generation of multi-nucleated osteoclast-like cells of peripheral blood mononuclear cells. Zoledronate induced apoptosis of UBC cells in a dose-dependent manner. Apoptosis was evidenced by induction of the active cleaved form of caspase-3. The baseline apoptotic fractions were similar in UBC cells and trabecular bone cells. However, in the overall apoptotic fractions in this study, trabecular bone cells showed 17.2% of apoptosis, significantly lower than 24.2% of UBC cells (
p-value
=
0.007). With the various zoledronate concentrations, mean apoptotic fractions of trabecular bone cells was 19.2%, significantly lower than 27.8% of UBC cells (
p-value
=
0.040). With GGOH co-treatment in various zoledronate concentrations, 15.1% apoptosis was shown in trabecular bone cells, which was not significantly lower than 20.6% of UBC cells (
p-value
=
0.076). This data suggests that zoledronate causes apoptosis in both UBC and trabecular bone cells by inhibition of the mevalonate pathway. In addition to the known anti-osteoclastogenic effect of bisphosphonates, the GGOH inhibitory effects of zoledronate were more prominent in UBC cells than trabecular bone cells, indicating their potential therapeutic role in UBC.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The alphaviruses are a group of 26 mosquitoborne viruses that cause a variety of human diseases. Many of the New World alphaviruses cause encephalitis, whereas the Old World viruses more typically ...cause fever, rash, and arthralgia. The genome is a single-stranded nonsegmented RNA molecule of + polarity; it is about 11,700 nucleotides in length. Several alphavirus genomes have been sequenced in whole or in part, and these sequences demonstrate that alphaviruses have descended from a common ancestor by divergent evolution. We have now obtained the sequence of the 3′-terminal 4288 nucleotides of the RNA of the New World Alphavirus western equine encephalitis virus (WEEV). Comparisons of the nucleotide and amino acid sequences of WEEV with those of other alphaviruses clearly show that WEEV is recombinant. The sequences of the capsid protein and of the (untranslated) 3′-terminal 80 nucleotides of WEEV are closely related to the corresponding sequences of the New World Alphavirus eastern equine encephalitis virus (EEEV), whereas the sequences of glycoproteins E2 and E1 of WEEV are more closely related to those of an Old World virus, Sindbis virus. Thus, WEEV appears to have arisen by recombination between an EEEV-like virus and a Sindbis-like virus to give rise to a new virus with the encephalogenic properties of EEEV but the antigenic specificity of Sindbis virus. There has been speculation that recombination might play an important role in the evolution of RNA viruses. The current finding that a widespread and successful RNA virus is recombinant provides support for such an hypothesis.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
We have isolated a cDNA clone after reverse transcription of the genomic RNA of Asibi yellow fever virus whose structure suggests it was formed by self-priming from a 3'-terminal hairpin of 87 ...nucleotides in the genomic RNA. We have also isolated a clone from cDNA made to Murray Valley encephalitis virus RNA that also appears to have arisen by self-priming from a 3'-terminal structure very similar or identical to that of yellow fever. In addition, 3'-terminal sequencing of the S1 strain of dengue 2 RNA shows that this RNA is also capable of forming a 3'-terminal hairpin of 79 nucleotides. Furthermore, we have identified two 20-nucleotide sequence elements which are present in the 3' untranslated region of all three viruses; one of these sequence elements is repeated in Murray Valley encephalitis and dengue 2 RNA but not in yellow fever RNA. In all three viruses, which represent the three major serological subgroups of the mosquito-borne flaviviruses, the 3'-proximal conserved sequence element, which is found immediately adjacent to the potential 3'-terminal hairpin, is complementary to another conserved domain near the 5' end of the viral RNAs, suggesting that flavivirus RNAs can cyclize (calculated delta G less than -11 kcal; 1 kcal = 4.184 kJ).
Authors developed a dual polymetal (W/WNx/poly-Si) gate complementary MOS down to a 0.15 mu m gate length. The short-channel effects are effectively suppressed and a saturation current of 300 mu A/mu ...m is obtained for nMOS and 110 mu A/mu m is observed for pMOS at a 0.15 mu m gate length. The lower saturation current of pMOS is attributed both to the p+-doped poly gate depletion and to the hole mobility degradation due to the increased vertical electric field in the surface-channel pMOS. B penetration is not observed with pure SiO2 gate dielectrics. The gate induced drain leakage current could be markedly reduced by optimizing the well doping below the gate edge. 14 refs.
Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides associated with cell surface class I major histocompatibility complex (MHC) molecules. This association presumably occurs between ...newly synthesized class I MHC molecules and peptide fragments in a pre-Golgi compartment. Little is known about the factors that regulate the formation of these antigenic peptide fragments within the cell. To examine the role of residues within a core epitope and in the flanking sequences for the generation and presentation of the newly synthesized peptide fragment recognized by CD8+ CTL, we have mutagenized the coding sequence for the CTL epitope spanning residues 202-221 in the influenza A/Japan/57 hemagglutinin (HA). In this study over 60 substitution mutations in the epitope were tested for their effects on target cell sensitization using a cytoplasmic viral expression system. The HA202-221 site contains two overlapping subsites defined by CTL clones 11-1 and 40-2. Mutations in HA residues 204-213 or residues 210-219 often abolished target cell lysis by CTL clones 11-1 and 40-2, respectively. Although residues outside the core epitope did not usually affect the ability to be lysed by CTL clones, substitution of a Gly residue for Val-214 abolished lysis by clone 11-1. These data suggest that residues within a site that affect MHC binding and T cell receptor recognition appear to play the predominant role in dictating the formation of the antigenic complex recognized by CD8+ CTL, and therefore the antigenicity of the protein antigen presented to CD8+ T cells. Most alterations in residues flanking the endogenously expressed epitope do not appreciably affect the generation and recognition of the site.
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