gebhart/patrepeval huggingface dataset:
Alanine-stretch scanning mutagenesis: a simple and efficient method to probe protein structure and function (1997)

We have developed a foolproof method to substitute a stretch of residues by alanines. After the introduction of aPstI site by IPCR, thus creating two alanine codons, additional codons are introduced at this site through the use of an 'alanine-stretch cartridge'. These cartridges comprise an antibiotic resistance gene flanked on both sides by alanine codons. Excision of the resistance gene byPvuII then yields the correct insertion of codons. The method is both highly reliable and flexible and should be of general use.
First-Principles Modeling of Unpassivated and Surfactant-Passivated Bulk Facets of Wurtzite CdSe:  A Model System for Studying the Anisotropic Growth of CdSe Nanocrystals (2005)

Equilibrium geometries, surface energies, and surfactant binding energies are calculated for selected bulk facets of wurtzite CdSe with a first-principles approach. Passivation of the surface Cd atoms with alkyl phosphonic acids or amines lowers the surface energy of all facets, except for the polar 000 facet. On the nonpolar facets, the most stable configuration corresponds to full coverage of surface Cd atoms with surfactants, while on the polar 0001 facet it corresponds only to a partial coverage. In addition, the passivated surface energies of the nonpolar facets are in general lower than the passivated polar 0001 facet. Therefore, the polar facets are less stable and less efficiently passivated than the nonpolar facets, and this can rationalize the observed anisotropic growth mechanism of wurtzite nanocrystals in the presence of suitable surfactants.
Identification of Non-telomeric G4-DNA Binding Proteins in Human, E. coli, Yeast, and Arabidopsis (2002)

G4-DNA binding proteins of E. coli, Saccharomyces cerevisiae, Arabidopsis, and human have been identified by a synthetic non-telomeric G4-DNA oligo 5'-d(ACTGTCGTACTTGATATGGGGGT)-3' using gel mobility shift assays. G4-DNA binding proteins are specific to G4-DNA, a four-stranded guanine-DNA structure. Bound complexes of G4-DNA and proteins were identified in nuclear extracts of all examined organisms in this study. In humans, three different G4-DNA and protein complexes were identified. However, human telomeric G-quadruplex oligo did not compete with G4-DNA oligo in the competition assays, suggesting that the identified G4-DNA binding proteins may be different from the known human telomeric G4-DNA binding proteins. We discovered two complexes of G4-DNA and protein in Arabidopsis identified in mobility shift assays. Interestingly, two complexes of G4-DNA and proteins were identified from E. coli, which have a circular genomic DNA structure. Results of this investigation suggest that non-telomeric G4-DNA structure and its binding proteins may be involved in important functional roles in both prokaryotes and eukaryotes.
Programmed Cell Death of Adult-Generated Hippocampal Neurons Is Mediated by the Proapoptotic Gene Bax (2004)

In the dentate gyrus (DG) of the adult mouse hippocampus, a substantial number of new cells are generated daily, but only a subset of these survive and differentiate into mature neurons, whereas the majority undergo programmed cell death (PCD). However, neither the intracellular machinery required for adult stem cell-derived neuronal death nor the biological implications of the significant loss of these newly generated cells have been examined. Several markers for apoptosis failed to reveal cell death in Bax-deficient mice, and this, together with a progressive increase in neuron number in the DG of the Bax knock-out, indicates that Bax is critical for the PCD of adult-generated hippocampal neurons. Whereas the proliferation of neural progenitor cells was not altered in the Bax-knock-out, there was an accumulation of doublecortin, calretinin+, and neuronal-specific nuclear protein+ postmitotic neurons, suggesting that Bax-mediated PCD of adult-generated neurons takes place during an early phase of differentiation. The absence of PCD in the adult also influenced the migration and maturation of adult-generated DG neurons. These results suggest that PCD in the adult brain plays a significant role in the regulation of multiple aspects of adult neurogenesis.
Kynurenine pathway inhibition as a therapeutic strategy for neuroprotection (2012)

The oxidative pathway for the metabolism of tryptophan along the kynurenine pathway generates quinolinic acid, an agonist at N-methyl-D-aspartate receptors, as well as kynurenic acid which is an antagonist at glutamate and nicotinic receptors. The pathway has become recognized as a key player in the mechanisms of neuronal damage and neurodegenerative disorders. As a result, manipulation of the pathway, so that the balance between the levels of components of the pathway can be modified, has become an attractive target for the development of pharmacological agents with the potential to treat those disorders. This review summarizes some of the relevant background information on the pathway itself before identifying some of the chemical strategies for its modification, with examples of their successful application in animal models of infection, stroke, traumatic brain damage, cerebral malaria and cerebral trypanosomiasis.
Maintenance of the Keratocyte Phenotype during Cell Proliferation Stimulated by Insulin (2005)

Keratocytes normally express high levels of aldehyde dehydrogenase and keratocan. They proliferate and lose their keratocyte markers when they become fibroblastic during corneal wound healing. Keratocytes cultured in fetal bovine serum also become fibroblastic, proliferate, and lose these markers. In this report, we studied the effects of three serum growth factors, fibroblast growth factor-2, insulin, and platelet-derived growth factor-BB, on keratocyte proliferation and the maintenance of the keratocyte markers in 7-day cultures in cells plated at low (5,000 cells/cm2) and high (20,000 cells/cm2) density in serum-free medium. Keratocyte proliferation was measured by [3H]thymidine incorporation and by DNA content of the cultures. Cytosolic aldehyde dehydrogenase and keratocan accumulated in the medium were quantified by Western blot. The results showed that all the growth factors stimulated proliferation, but insulin stimulated proliferation more consistently. The keratocyte markers aldehyde dehydrogenase and keratocan were maintained after 7 days in culture in all growth factors, but keratocyte cell morphology was only maintained in medium containing insulin. Most of the proteoglycans were degraded in cultures of keratocytes plated at low density and cultured in the absence of growth factors. This degradation was prevented when keratocytes were cultured in the presence of the growth factors or when keratocytes were plated at high density. The results of this study show that insulin can expand keratocytes in vitro, maintain their phenotype, and prevent proteoglycan degradation.
Recombinant interferon-alpha, -beta, and -gamma enhance the proliferative response of human B cells. (1987)

Recombinant interferons (IFN-alpha, -beta, and -gamma) were examined for their effects on B cell activation. Relatively small IgM+ B cells from human blood samples were isolated by fluorescence-activated cell sorting and were used as target cells. Although the interferons themselves were nonmitogenic, each enhanced the proliferative response induced by a mitogenic anti-mu monoclonal antibody, with IFN-beta usually showing the greatest enhancement and IFN-gamma the least. Pretreatment with the interferons primed resting B cells to undergo enhanced DNA synthesis in response to the anti-mu antibody DA4. Conversely, anti-mu pretreatment, followed by IFN treatment, did not induce B cells to enter the S phase. Time-course analysis revealed that IFN could augment the anti-mu response even when added as late as the final 24 hr of a 3-day culture interval. Combinations of IFN-gamma plus IFN-alpha or -beta were synergistic in the anti-mu response, whereas the IFN-alpha plus IFN-beta combination was not. The data suggest that interferons produced by both lymphocytes (IFN-gamma) and nonlymphoid inflammatory cells (IFN-alpha and -beta) can enhance B cell growth via different mechanisms.
Regulation of JAK3 expression in human monocytes: phosphorylation in response to interleukins 2, 4, and 7. (1995)

The Janus family of kinases (JAKs) has been shown to be involved in the signal transduction of a number of cytokine receptors. Recently, we have cloned a novel JAK family member, JAK3, that is expressed in natural killer and activated T cells and is coupled functionally and physically to the interleukin 2 (IL-2) receptor in these cells. Here we report that JAK3 was expressed at low but detectable levels in human monocytes. In contrast, JAK3 expression was strongly induced during activation by interferon gamma (IFN-gamma) or lipopolysaccharide. Moreover, JAK3 became tyrosine phosphorylated in response to IL-2, IL-4, and IL-7 but not response to IFN-gamma or granulocyte/macrophage colony-stimulating factor. Together, these findings suggest that JAK3 is functionally important in activated monocytes and cells of the myeloid lineage and is involved in signaling responses of cytokines that use the common gamma-chain of the IL-2 receptor.
Gangliosides of Human Melanoma: GM2 and Tumorigenicity2 (1987)

An increased synthesis of the ganglioside GM2 has been reported on transformed murine cells, human fetal tissues, and transformed melanocytes. This study was designed to investigate whether any correlation existed between GM2 expression and the tumorigenicity of human melanoma. Ten established human melanoma cell lines, 5 rich in GM2 (group A) and 5 poor in GM2 (group B), were selected on the basis of previous ganglioside analysis of 28 melanoma cell lines. Six athymic nude mice per cell line were given an sc injection of 10(6) human melanoma cells/mouse. Tumors were measured every 2-4 days. By day 36 after the injection, 28 of 30 mice (93%) in group A developed medium to large tumors, but only 1 of 30 (3%) in group B developed a small tumor (P less than .005). The correlation of GM2 content of individual melanomas with tumor growth rate also was very high. GM2 content was expressed as nanomoles per gram wet weight of each melanoma. The area under the log values of tumor growth curves from day 0 to day 36, which represented tumor growth rate, tumor size, and latent period, was proportional to GM2 content, with a correlation coefficient of 0.927 and with P less than .001. The same log relationship when tested with other gangliosides, GM3, GD3, and GD2, was not statistically significant. These results, combined with the fact that variations in GM2 content do not affect in vitro growth of human melanoma cells, suggest that GM2 expression may be directly related to the dedifferentiation or the tumorigenicity of human melanoma.
Ultrafast Algorithm for Designing Focused Combinational Arrays (2000)

A novel greedy algorithm for the design of focused combinatorial arrays is presented. The method is applicable when the objective function is decomposable to individual molecular contributions and makes use of a heuristic that allows the independent evaluation and ranking of candidate reagents in each variation site in the combinatorial library. The algorithm is extremely fast and convergent and produces solutions that are comparable to and often better than those derived from the substantially more elaborate and computationally intensive stochastic sampling techniques. Typical examples of design objectives that are amendable to this approach include maximum similarity to a known lead (or set of leads), maximum predicted activity according to some structure−activity or receptor binding model, containment within certain molecular property bounds, and many others.
Statistical Pools and Electoral Success in Vote-Dilution Cases (1995)

In Thornburg v Gingles,' the Supreme Court established a two-tier test for determining whether a particular electoral system violates Section 2 of the Voting Rights Act ("VRA"). 2 First, the Court delineated a three-part threshold test that all plaintiffs must meet to prove a claim of vote dilution in a multimember district. 3 Second, the Court held that plaintiffs, once they have satisfied the threshold test, must demonstrate that under the "totality of the circumstances," the practice in question deprives them of their right to participate equally in the political process by diluting their votes. 4 In some instances, Section 2 plaintiffs may challenge state or local electoral processes that narrow the field of eligible candidates by requiring that they meet certain professional criteria.Judicial elections in which candidates must be experienced lawyers represent such a situation.This Comment examines whether courts should consider the percentage of attorneys eligible to run for judicial office who are minority group members as an important factor in Section 2 judicial-election cases.In particular, this Comment will explore whether the best statistical pool to employ under the "totality of the circumstances" is: (1) the percentage of lawyers who are members of the minority group;(2) the percentage of the voting-age population in a given district that are members of the minority group; or (3) the percentage of the district's population that belongs to the minority group.While the Supreme Court has not addressed the question, the Fifth and Eleventh Circuits have split over which statistical pool to use in these judicial-election suits.Part I of this Comment outlines the Supreme Court's interpretation of the amended Section 2, the history of Section 2 t B.
<title>Atomic scale optical monitoring of the initial growth of TiO2 thin films</title> (2001)

The initial atomic-layer-chemical-vapor-deposition growth of titanium dioxide from TiCl<SUB>4</SUB> and water on quartz glass substrates is monitored in real time by incremental dielectric reflection. An interesting means for bringing the growth from the very beginning into a time-homogeneous mode is proposed and preliminarily studied. It consists in an in situ TiCl<SUB>4</SUB>-treatment procedure. The crystal structure and surface morphology of the prepared ultrathin films are characterized.
Evidence that inducible nitric oxide synthase is involved in LPS‐induced plasma leakage in rat skin through the activation of nuclear factor‐κB (1998)

1. Rats challenged with lipopolysaccharide (LPS) produce large amounts of nitric oxide (NO) following the induction of the inducible NO-synthase (iNOS) in several tissues and organs. Recent studies have shown that the expression of iNOS is regulated at the transcriptional level by a transcription nuclear factor-kappaB (NF-kappaB). In this study we investigated the role of NO in a model of LPS-induced plasma-leakage in rat skin and the involvement of NF-kappaB. 2. Plasma leakage in the rat skin was measured over a period of 30 min to 2 h as the local accumulation of intravenous (i.v.) injection of [125I]-human serum albumin ([125I]-HSA) in response to intradermal (i.d.) injection of LPS. LPS (1, 10, 100 microg/site) produced a dose-related increase in plasma extravasation (18.2+/-3.2, 27.2+/-2.9, 40.4+/-9.6 microl/site) as compared to saline control (11.4+/-2.2 microl/site). This increase was maximal after 2 h; therefore this time point and the dose of LPS 10 microg/site was used in all the successive experiments. 3. To investigate the role of NO in LPS-induced plasma leakage in rat skin, the non-selective NOS inhibitor NG-nitro-L-arginine-methyl ester (L-NAME) or the more selective iNOS inhibitor S-methyl-isothiourea (SMT) was injected i.d. with LPS. L-NAME and SMT (0.01, 0.1 and 1 micromol/site) inhibited LPS-induced plasma leakage in a dose-related fashion (L-NAME: 26.0+/-5.5, 20.2+/-1.6, 18.0+/-2.0 microl/site; SMT: 19.5+/-1.5, 17.0+/-1.6, 15.0+/-2.6 microl/site) as compared to LPS alone (27.2+/-2.9 microl/site). At the lowest concentration used (0.01 micromol/site), SMT significantly reduced plasma leakage by 30%+/-0.7 while L-NAME (0.01 micromol/site) was not effective. 4. Treatment with increasing concentrations of pyrrolidinedithyocarbamate (PDTC) (0.01, 0.1, 1 micromol/site), an inhibitor of NF-kappaB activation, injected i.d. 30 min before LPS challenge, inhibited in a concentration-dependent fashion LPS-induced plasma leakage by 9.0+/-0.6, 33+/-4.0, 51+/-2.0% respectively. Moreover, PDTC (0.1, 1 micromol/site) suppressed LPS-induced NF-kappaB DNA-binding. 5. Western blot analysis showed significant levels of iNOS proteins in the skin samples of LPS-treated rats, as compared to basal levels present in saline-injected rat skin. PDTC (0.1, 1.0 micromol/site) dose-dependently decreased the amount of iNOS protein expression induced by LPS. 6. Our results indicate that LPS-induced plasma leakage in rat skin is modulated by NO mainly produced by the inducible isoform of NOS. Furthermore, the suppression of plasma leakage by PDTC, an inhibitor of NF-kappaB activation, is correlated to the inhibition of iNOS protein expression.
The myeloperoxidase system of human phagocytes generates Nε-(carboxymethyl)lysine on proteins: a mechanism for producing advanced glycation end products at sites of inflammation (1999)

Reactive aldehydes derived from reducing sugars and peroxidation of lipids covalently modify proteins and may contribute to oxidative tissue damage. We recently described another mechanism for generating reactive aldehydes from free alpha-amino acids. The pathway begins with myeloperoxidase, a heme enzyme secreted by activated neutrophils. Conversion of alpha-amino acids to aldehydes requires hypochlorous acid (HOCl), formed from H2O2 and chloride by myeloperoxidase. When L-serine is the substrate, HOCl generates high yields of glycolaldehyde. We now demonstrate that a model protein, ribonuclease A (RNase A), exposed to free L-serine and HOCl exhibits the biochemical hallmarks of advanced glycation end (AGE) products -- browning, increased fluorescence, and cross-linking. Furthermore, Nepsilon-(carboxymethyl)lysine (CML), a chemically well-characterized AGE product, was generated on RNase A when it was exposed to reagent HOCl-serine, the myeloperoxidase-H2O2-chloride system plus L-serine, or activated human neutrophils plus L-serine. CML production by neutrophils was inhibited by the H2O2 scavenger catalase and the heme poison azide, implicating myeloperoxidase in the cell-mediated reaction. CML was also generated on RNase A by a myeloperoxidase-dependent pathway when neutrophils were activated in a mixture of amino acids. Under these conditions, we observed both L-serine-dependent and L-serine-independent pathways of CML formation. The in vivo production of glycolaldehyde and other reactive aldehydes by myeloperoxidase may thus play an important pathogenic role by generating AGE products and damaging tissues at sites of inflammation.
Targeted Ubiquitination and Degradation of G-Protein-Coupled Receptor Kinase 5 by the DDB1-CUL4 Ubiquitin Ligase Complex (2012)

The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors (GPCRs) and desensitize GPCR-mediated signaling. Recent studies indicate they also function non-catalytically via interaction with other proteins. In this study, a proteomic approach was used to screen interacting proteins of GRK5 in MDA-MB-231 cells and HUVEC cells. Mass spectrometry analysis reveals several proteins in the GRK5 immunocomplex including damaged DNA-binding protein 1 (DDB1), an adaptor subunit of the CUL4-ROC1 E3 ubiquitin ligase complex. Co-immunoprecipitation experiments confirmed the association of GRK5 with DDB1-CUL4 complex, and reveal that DDB1 acts as an adapter to link GRK5 to CUL4 to form the complex. Overexpression of DDB1 promoted, whereas knockdown of DDB1 inhibited the ubiquitination of GRK5, and the degradation of GRK5 was reduced in cells deficient of DDB1. Furthermore, the depletion of DDB1 decreased Hsp90 inhibitor-induced GRK5 destabilization and UV irradiation-induced GRK5 degradation. Thus, our study identified potential GRK5 interacting proteins, and reveals the association of GRK5 with DDB1 in cell and the regulation of GRK5 level by DDB1-CUL4 ubiquitin ligase complex-dependent proteolysis pathway.
Analysis of Protein Expression in Cell Microarrays: A Tool for Antibody-based Proteomics (2006)

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome.
Brain Derived Neurotrophic Factor (BDNF) Contributes to the Pain Hypersensitivity Following Surgical Incision in the Rats (2008)

The present study showed that incision induced the segmental upregulation of BDNF in the DRG and spinal cord through somatic afferent nerve transmission, and the upregulated BDNF contributed to the pain hypersensitivity induced by surgical incision.
N-linked oligosaccharides of murine major histocompatibility complex class II molecule. Role in antigenic peptide binding, T cell recognition, and clonal nonresponsiveness. (1992)

To evaluate the functional role of the N-linked oligosaccharides of major histocompatibility complex (MHC) class II molecules, affinity-purified murine IAs class II molecules were deglycosylated in the presence of asparagine amidase enzyme. The deglycosylated IAs molecules were characterized by 12% SDS-polyacrylamide gel analysis under reduced and native conditions and the complete enzymatic removal of all three N-linked sugar components from the alpha/beta heterodimer was confirmed by lectin-link Western blot analysis. Like the native IAs molecules, the deglycosylated IAs molecules were fully capable of binding an antigenic peptide from myelin basic protein MBP(89-101). The kinetics of dissociation of preformed complexes of IAs.MBP(89-101) and deglycosylated IAs.MBP(89-101) were compared at 4 and at 37 degrees C. Both complexes were equally stable at 4 degrees C; however, at 37 degrees C the deglycosylated IAs.MBP(89-101) complexes showed an increased rate of dissociation as compared with the native IAs.MBP(89-101) complexes. When tested for their ability to recognize the T cell receptor on T cells, both complexes bound to cloned HS-1 T cells that recognize and respond to IAs.MBP(89-101). Finally, the complexes of deglycosylated IAs.MBP(89-101) were tested for the induction of in vitro nonresponsiveness and compared with native IAs.MBP(89-101) complexes. Both complexes were capable of inducing 95-100% nonresponsiveness in a proliferation assay. These results suggest that the N-linked oligosaccharide of MHC class II molecules may not be essential for either antigenic peptide binding or T cell recognition. In addition results obtained here provide evidence that the carbohydrate moities of MHC class II molecules may not be involved in induction of T cell clonal anergy.