gebhart/patrepeval huggingface dataset:
The Serpin Inhibitory Mechanism Is Critically Dependent on the Length of the Reactive Center Loop (2001)

The recent crystallographic structure of a serpin-protease complex revealed that protease inactivation results from a disruption of the catalytic site architecture caused by the displacement of the catalytic serine. We hypothesize that inhibition depends on the length of the N-terminal portion of the reactive center loop, to which the active serine is covalently attached. To test this, alpha(1)-antitrypsin Pittsburgh variants were prepared with lengthened and shortened reactive center loops. The rates of inhibition of factor Xa and of complex dissociation were measured. The addition of one residue reduced the stability of the complex more than 200,000-fold, and the addition of two residues reduced it by more than 1,000,000-fold, whereas the deletion of one or two residues lowered the efficiency of inhibition and increased the stability of the complex (2-fold). The deletion of more than two residues completely converted the serpin into a substrate. Similar results were obtained for the alpha(1)-antitrypsin variants with thrombin and for PAI-1 and PAI-2 with their common target tissue plasminogen activator. We conclude that the length of the serpin reactive center loop is critical for its mechanism of inhibition and is precisely regulated to balance the efficiency of inhibition and stability of the final complex.
Alpha-methylacyl-CoA racemase: a new molecular marker for prostate cancer. (2002)

Identification of genes that are dysregulated in association with prostate carcinogenesis can provide disease markers and clues relevant to disease etiology. Of particular interest as candidate markers of disease are those genes that are frequently overexpressed. In this study, we describe a gene, alpha-methylacyl-CoA racemase (AMACR), whose expression is consistently up-regulated in prostate cancer. Analysis of mRNA levels of AMACR revealed an average up-regulation of approximately 9 fold in clinical prostate cancer specimens compared with normal. Western blot and immunohistochemical analysis confirms the up-regulation at the protein level and localizes the enzyme predominantly to the peroxisomal compartment of prostate cancer cells. A detailed immunohistochemical analysis of samples from 168 primary prostate cancer cases using both standard slides and tissue microarrays demonstrates that both prostate carcinomas and the presumed precursor lesion (high-grade prostatic intraepithelial neoplasia) consistently scored significantly higher than matched normal prostate epithelium; 88% of the carcinomas had a staining score higher than the highest score observed for any sample of normal prostate epithelium. Both untreated metastases (n = 32 patients) and hormone refractory prostate cancers (n = 14 patients) were generally strongly positive for AMACR. To extend the utility of this marker for prostate cancer diagnosis, we combined staining for cytoplasmic AMACR with staining for the nuclear protein, p63, a basal cell marker in the prostate that is absent in prostate cancer. In a simple assay that can be useful for the diagnosis of prostate cancer on both biopsy and surgical specimens, combined staining for p63 and AMACR resulted in a staining pattern that greatly facilitated the identification of malignant prostate cells. The enzyme encoded by the AMACR gene plays a critical role in peroxisomal beta oxidation of branched chain fatty acid molecules. These observations could have important epidemiological and preventive implications for prostate cancer, as the main sources of branched chain fatty acids are dairy products and beef, the consumption of which has been associated with an increased risk for prostate cancer in multiple studies. On the basis of its consistency and magnitude of cancer cell-specific expression, we propose AMACR as an important new marker of prostate cancer and that its use in combination with p63 staining will form the basis for an improved staining method for the identification of prostate carcinomas. Furthermore, the absence of AMACR staining in the vast majority of normal tissues coupled with its enzymatic activity makes AMACR the ideal candidate for development of molecular probes for the noninvasive identification of prostate cancer by imaging modalities.
Flex on cap-solder paste bumping (2002)

Soldered flip chips require the formation of a solder bump on the semiconductor device. Traditional solder deposition methods of evaporation and plating have limitations both from a cost structure as well as in capability to meet product requirements. Presented in this paper are the capabilities and reliability results of the FOC (Flex on Cap) solder paste bumping process developed by Delco Electronics and practised by flip chip technologies. This paper describes the fine pitch capabilities, solder alloys, bump height uniformity, alloy control and reliability data for the FOC process. The solder paste process permits cost effective bumping of low alpha solders either W/Pb or Pb free alloys. The importance of the UBM (Under Bump Metalization) for reliable eutectic Pb/Sn and other high Sn content solders that are important for the DCA (Direct Chip Attach) market is shown. The system level cost savings of having all the necessary solder present on the IC, eliminating the deposition of solder on the board, are given.
Expression of colony-stimulating factor 1 receptor during prostate development and prostate cancer progression (2002)

Colony-stimulating factor-1 receptor (CSF-1R) is the major regulator of macrophage development and is associated with epithelial cancers of the breast and ovary. Immunohistochemistry analysis of murine prostate development demonstrated epithelial expression of CSF-1R during the protrusion of prostatic buds from the urogenital sinus, during the prepubertal and androgen-driven proliferative expansion and branching of the gland, with a decline in older animals. Models of murine prostate cancer showed CSF-1R expression in areas of carcinoma- and tumor-associated macrophages. Several human prostate cancer cell lines and primary cultures of human prostate epithelial cells had low but detectable levels of CSF-1R. Human prostatectomy samples showed low or undetectable levels of receptor in normal glands or benign prostatic hypertrophy specimens. Staining was strongest in areas of prostatic intraepithelial neoplasia or carcinoma of Gleason histological grade 3 or 4. The activated form of the receptor reactive with antibodies specific for phosphotyrosine modified peptide sequences was observed in samples of metastatic prostate cancer. Immunohistochemistry showed strong expression of CSF-1R by macrophage lineage cells, including villous macrophages and the syncytiotrophoblast layer of placenta, Kupper cells in the liver, and histiocytes infiltrating near prostate cancers. These observations correlate CSF-1R expression with changes in the growth and development of the normal and neoplastic prostate.
BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies (2006)

The tricarboxylate reagent benzene-1,3,5-triacetic acid (BTA) was used to attach 5'-aminated DNA primers and templates on an aminosilanized glass surface for subsequent generation of DNA colonies by in situ solid-phase amplification. We have characterized the derivatized surfaces for the chemical attachment of oligonucleotides and evaluate the properties relevant for the amplification process: surface density, thermal stability towards thermocycling, functionalization reproducibility and storage stability. The derivatization process, first developed for glass slides, was then adapted to microfabricated glass channels containing integrated fluidic connections. This implementation resulted in an important reduction of reaction times, consumption of reagents and process automation. Innovative analytical methods for the characterization of attached DNA were developed for assessing the surface immobilized DNA content after amplification. The results obtained showed that the BTA chemistry is compatible and suitable for forming highly dense arrays of DNA colonies with optimal surface coverage of about 10 million colonies/cm(2) from the amplification of initial single-template DNA molecules immobilized. We also demonstrate that the dsDNA colonies generated can be quantitatively processed in situ by restriction enzymes digestion. DNA colonies generated using the BTA reagent can be used for further sequence analysis in an unprecedented parallel fashion for low-cost genomic studies.
Evaluation of Live Attenuated Influenza A Virus H6 Vaccines in Mice and Ferrets (2008)

Avian influenza A virus A/teal/HK/W312/97 (H6N1) possesses seven gene segments that are highly homologous to those of highly pathogenic human influenza H5N1 viruses, suggesting that a W312-like H6N1 virus might have been involved in the generation of the A/HK/97 H5N1 viruses. The continuous circulation and reassortment of influenza H6 subtype viruses in birds highlight the need to develop an H6 vaccine to prevent potential influenza pandemics caused by the H6 viruses. Based on the serum antibody cross-reactivity data obtained from 14 different H6 viruses from Eurasian and North American lineages, A/duck/HK/182/77, A/teal/HK/W312/97, and A/mallard/Alberta/89/85 were selected to produce live attenuated H6 candidate vaccines. Each of the H6 vaccine strains is a 6:2 reassortant ca virus containing HA and NA gene segments from an H6 virus and the six internal gene segments from cold-adapted A/Ann Arbor/6/60 (AA ca), the master donor virus that is used to make live attenuated influenza virus FluMist (intranasal) vaccine. All three H6 vaccine candidates exhibited phenotypic properties of temperature sensitivity (ts), ca, and attenuation (att) conferred by the internal gene segments from AA ca. Intranasal administration of a single dose of the three H6 ca vaccine viruses induced neutralizing antibodies in mice and ferrets and fully protected mice and ferrets from homologous wild-type (wt) virus challenge. Among the three H6 vaccine candidates, the A/teal/HK/W312/97 ca virus provided the broadest cross-protection against challenge with three antigenically distinct H6 wt viruses. These data support the rationale for further evaluating the A/teal/HK/W312/97 ca vaccine in humans.
THE STRUCTURE AND FUNCTION OF PROTEINS INVOLVED IN MAMMALIAN PRE-mRNA SPLICING (1996)

Intervening sequences are removed from nuclear pre-mRNAs in a well-defined multi-step pathway. Small nuclear ribonucleoprotein particles (snRNPs) and numerous protein factors are essential for the formation of the active spliceosome in which intron excision proceeds in two successive transesterification reactions. Important elements for catalysis are the RNA moieties of the snRNPs that align the pre-mRNA splice sites in the active center of the spliceosome. Although pre-mRNA splicing is almost certainly RNA-mediated, both snRNA-associated proteins and non-snRNP splicing factors participate in each step of the splicing reaction. Splicing proteins exert auxiliary functions in the recognition, selection, and juxtaposition of the splice sites and drive conformational changes during spliceosome assembly and catalysis. Many splicing factors have been isolated in recent years and corresponding cDNAs have been cloned. This review summarizes the structure and function of mammalian proteins which are essential components of the constitutive splicing machinery.
Characterizing acetogenic metabolism using a genome-scale metabolic reconstruction of Clostridium ljungdahlii (2013)

iHN637 represents a predictive model that recapitulates experimental data, and provides valuable insights into the metabolic response of C. ljungdahlii to genetic perturbations under various growth conditions. Thus, the model will be instrumental in guiding metabolic engineering of C. ljungdahlii for the industrial production of biocommodities and biofuels.
Wnt signals across the plasma membrane to activate the β-catenin pathway by forming oligomers containing its receptors, Frizzled and LRP (2004)

Wnt-induced signaling via beta-catenin plays crucial roles in animal development and tumorigenesis. Both a seven-transmembrane protein in the Frizzled family and a single transmembrane protein in the LRP family (LDL-receptor-related protein 5/6 or Arrow) are essential for efficiently transducing a signal from Wnt, an extracellular ligand, to an intracellular pathway that stabilizes beta-catenin by interfering with its rate of destruction. However, the molecular mechanism by which these two types of membrane receptors synergize to transmit the Wnt signal is not known. We have used mutant and chimeric forms of Frizzled, LRP and Wnt proteins, small inhibitory RNAs, and assays for beta-catenin-mediated signaling and protein localization in Drosophila S2 cells and mammalian 293 cells to study transmission of a Wnt signal across the plasma membrane. Our findings are consistent with a mechanism by which Wnt protein binds to the extracellular domains of both LRP and Frizzled receptors, forming membrane-associated hetero-oligomers that interact with both Disheveled (via the intracellular portions of Frizzled) and Axin (via the intracellular domain of LRP). This model takes into account several observations reported here: the identification of intracellular residues of Frizzled required for beta-catenin signaling and for recruitment of Dvl to the plasma membrane; evidence that Wnt3A binds to the ectodomains of LRP and Frizzled; and demonstrations that a requirement for Wnt ligand can be abrogated by chimeric receptors that allow formation of Frizzled-LRP hetero-oligomers. In addition, the beta-catenin signaling mediated by ectopic expression of LRP is not dependent on Disheveled or Wnt, but can also be augmented by oligomerization of LRP receptors.
p38 MAP kinase negatively regulates endothelial cell survival, proliferation, and differentiation in FGF-2–stimulated angiogenesis (2002)

The p38 mitogen-activated protein kinase (p38) is activated in response to environmental stress and inflammatory cytokines. Although several growth factors, including fibroblast growth factor (FGF)-2, mediate activation of p38, the consequences for growth factor-dependent cellular functions have not been well defined. We investigated the role of p38 activation in FGF-2-induced angiogenesis. In collagen gel cultures, bovine capillary endothelial cells formed tubular growth-arrested structures in response to FGF-2. In these collagen gel cultures, p38 activation was induced more potently by FGF-2 treatment compared with that in proliferating cultures. Treatment with the p38 inhibitor SB202190 enhanced FGF-2-induced tubular morphogenesis by decreasing apoptosis, increasing DNA synthesis and cell proliferation, and enhancing the kinetics of cell differentiation including increased expression of the Notch ligand Jagged1. Overexpression of dominant negative mutants of the p38-activating kinases MKK3 and MKK6 also supported FGF-2-induced tubular morphogenesis. Sustained activation of p38 by FGF-2 was identified in vascular endothelial cells in vivo in the chick chorioallantoic membrane (CAM). SB202190 treatment enhanced FGF-2-induced neovascularization in the CAM, but the vessels displayed abnormal features indicative of hyperplasia of endothelial cells. These results implicate p38 in organization of new vessels and suggest that p38 is an essential regulator of FGF-2-driven angiogenesis.
Ca 2+ /Calmodulin–Dependent Protein Kinase Modulates Cardiac Ryanodine Receptor Phosphorylation and Sarcoplasmic Reticulum Ca 2+ Leak in Heart Failure (2005)

Abnormal release of Ca from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction and arrhythmogenesis in heart failure (HF). We previously demonstrated decreased Ca transient amplitude and SR Ca load associated with increased Na/Ca exchanger expression and enhanced diastolic SR Ca leak in an arrhythmogenic rabbit model of nonischemic HF. Here we assessed expression and phosphorylation status of key Ca handling proteins and measured SR Ca leak in control and HF rabbit myocytes. With HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin-dependent protein kinase II (CaMKII) expression were increased 50% to 100%. The RyR2 complex included more CaMKII (which was more activated) but less calmodulin, FKBP12.6, and phosphatases 1 and 2A. The RyR2 was more highly phosphorylated by both protein kinase A (PKA) and CaMKII. Total phospholamban phosphorylation was unaltered, although it was reduced at the PKA site and increased at the CaMKII site. SR Ca leak in intact HF myocytes (which is higher than in control) was reduced by inhibition of CaMKII but was unaltered by PKA inhibition. CaMKII inhibition also increased SR Ca content in HF myocytes. Our results suggest that CaMKII-dependent phosphorylation of RyR2 is involved in enhanced SR diastolic Ca leak and reduced SR Ca load in HF, and may thus contribute to arrhythmias and contractile dysfunction in HF.
DNA immunization: Effects of vehicle and route of administration on the induction of protective antiviral immunity (1996)

The effectiveness of DNA immunization has been demonstrated in several model systems, usually following intramuscular injection of DNA in saline, or topical administration to the skin. In this study we have compared DNA delivered by three routes (intramuscular, intravenous, and intraperitoneal) and, for each route, in two vehicles (cationic liposome complex and pH sensitive liposome). These two lipid vehicles were evaluated because they are frequently used in gene therapy studies, but their immunogenicity has not been extensively studied. Each of these six combinations has been evaluated not only by assay of marker gene expression in a variety of tissues, but also by measurement of biologically-relevant parameters of immunity induction of antibodies, cytotoxic T lymphocytes, and protection against viral challenge. By both criteria (marker gene expression and induced immunity), the outcomes vary markedly among the six combinations. The combination leading to maximal marker gene expression (DNA with cationic lipid, administered i.v.) also induces detectable antibodies and CTL, and is the only one of the six combinations to induce immune responses comparable to those seen following i.m. injection of DNA in saline. However, marker gene expression can be detected in other combinations in the absence of induced immunity thus the value of marker gene expression in predicting the protection induced by a microbial antigen is questionable suggesting that, when evaluating various promoter constructs, marker gene expression may not adequately replace the direct measurement of biological outcomes.
Mammalian Target of Rapamycin Inhibition Promotes Response to Epidermal Growth Factor Receptor Kinase Inhibitors in PTEN-Deficient and PTEN-Intact Glioblastoma Cells (2006)

The epidermal growth factor receptor (EGFR) is commonly amplified, overexpressed, and mutated in glioblastoma, making it a compelling molecular target for therapy. We have recently shown that coexpression of EGFRvIII and PTEN protein by glioblastoma cells is strongly associated with clinical response to EGFR kinase inhibitor therapy. PTEN loss, by dissociating inhibition of the EGFR from downstream phosphatidylinositol 3-kinase (PI3K) pathway inhibition, seems to act as a resistance factor. Because 40% to 50% of glioblastomas are PTEN deficient, a critical challenge is to identify strategies that promote responsiveness to EGFR kinase inhibitors in patients whose tumors lack PTEN. Here, we show that the mammalian target of rapamycin (mTOR) inhibitor rapamycin enhances the sensitivity of PTEN-deficient tumor cells to the EGFR kinase inhibitor erlotinib. In two isogenic model systems (U87MG glioblastoma cells expressing EGFR, EGFRvIII, and PTEN in relevant combinations, and SF295 glioblastoma cells in which PTEN protein expression has been stably restored), we show that combined EGFR/mTOR kinase inhibition inhibits tumor cell growth and has an additive effect on inhibiting downstream PI3K pathway signaling. We also show that combination therapy provides added benefit in promoting cell death in PTEN-deficient tumor cells. These studies provide strong rationale for combined mTOR/EGFR kinase inhibitor therapy in glioblastoma patients, particularly those with PTEN-deficient tumors.
Polyribonucleotides Containing a Phosphorothioate Backbone (1970)

The enzymatic synthesis of two polyribonucleotides, poly r( s̄ A s̄ U) and poly r( s̄ U) containing a sugar‐phosphorothioate instead of a sugar‐phosphate backbone, is described. The formation and thermal stability of double‐stranded polymers is not impaired by this modification. They can also serve as templates for RNA‐polymerase. Single stranded polymers can serve as messengers. Both double and single‐standed polymer exhibit some degree of resistance to nucleases.
Attenuating mutations in glycoproteins E1 and E2 of Sindbis virus produce a highly attenuated strain when combined in vitro (1990)

Alterations in either the E1 or the E2 glycoprotein of Sindbis virus can affect pathogenesis in animals. Previously, we identified two distinct E1 glycoprotein gene sequences which differed in their effect on pathogenesis. One had an attenuation phenotype following subcutaneous inoculation of neonatal mice (E1 Ala-72, Gly-75, and Ser-237), while the other was virulent (E1 Val-72, Asp-75, and Ala-237). In this study, we examined the basis for this difference in pathogenesis by using a full-length cDNA clone of Sindbis virus from which infectious RNA could be transcribed in vitro. The relative contribution of each E1 residue to the pathogenesis phenotype was determined by using site-directed mutagenesis to alter each codon individually and in combination. Residues 75 and 237, in combination, appeared to be the major E1 determinants affecting pathogenesis. In addition, the effect of directly combining independently attenuating E1 and E2 mutations in the same virus was examined. The attenuating E1 sequences characterized in this study were coupled to a previously characterized attenuating mutation at E2 residue 114. The resulting recombinant virus, constructed in vitro, exhibited an increased attenuation of neurovirulence as compared with recombinant viruses containing either of the attenuating elements alone.
A flexible peptide spacer increases the efficacy of holoricin anti-T cell immunotoxins. (1988)

Immunotoxins, constructed by chemically cross-linking an antibody and protein toxin, do not possess the high efficacy of the native toxin. Decreases in toxicity are due in part to the steric constraints imposed on the two macromolecules, which result in both decreased antibody binding and toxin function. In examining the structural features that influence efficacy in holotoxin-antibody conjugates, it was found that the incorporation of a 29-residue polypeptide, derived from the insulin B chain between the antibody and ricin moiety, resulted in an increase in both potency and efficacy. In a murine model system, potency of the peptide spacer conjugate was increased nearly 10-fold; however, when examined by the procedure used to purge bone marrow, the peptide spacer conjugate was not demonstrably more toxic to nontarget cells than the nonspacer conjugate. Thus, in addition to increases of efficacy and potency, this novel immunotoxin demonstrated increased specificity by approximately 10-fold. To test the general utility of peptide spacer inclusion, a T101-ricin conjugate was constructed with the peptide spacer. It yielded a protein synthesis inhibition rate of -0.6 log/h on MOLT-3 cells, greater than 10-fold more efficacious than a previously constructed nonspacer T101-ricin conjugate examined under similar conditions.