gebhart/patrepeval huggingface dataset:
Hepatitis C Virus RNA Replication Occurs on a Detergent-Resistant Membrane That Cofractionates with Caveolin-2 (2003)

The mechanism and machinery of hepatitis C virus (HCV) RNA replication are still poorly understood. In this study, we labeled de novo-synthesized viral RNA in situ with bromouridine triphosphate (BrUTP) in Huh7 cells expressing an HCV subgenomic replicon. By immunofluorescence staining using an anti-BrUTP antibody and confocal microscopy, we showed that the newly synthesized HCV RNA was localized to distinct speckle-like structures, which also contain all of the HCV nonstructural (NS) proteins. These speckles are distinct from lipid droplets and are separated from the endoplasmic reticulum (ER), where some HCV NS proteins also reside. Membrane flotation analysis demonstrated that almost all of the NS5A and part of the NS5B proteins and all of the viral RNA were present in membrane fractions which are resistant to treatment with 1% NP-40 at 4 degrees C. They were cofractionated with caveolin-2, a lipid-raft-associated intracellular membrane protein, in the presence or absence of the detergent. In contrast, the ER-resident proteins were detergent soluble. These properties suggest that the membranes on which HCV RNA replication occurs are lipid rafts recruited from the intracellular membranes. The protein synthesis inhibitors cycloheximide and puromycin did not inhibit viral RNA synthesis, indicating that HCV RNA replication does not require continuous protein synthesis. We suggest that HCV RNA synthesis occurs on a lipid raft membrane structure.
The Allergen-Induced Airway Hyperresponsiveness in a Human-Mouse Chimera Model of Asthma Is T Cell and IL-4 and IL-5 Dependent (2001)

The cellular and molecular mechanisms involved in the airway hyperresponsiveness (AHR) of patients with allergic asthma remain unclear. A role for Th2 inflammatory cells was suggested based on murine asthma models. No direct evidence exists on the role of these cells in human asthma. The development of a mouse-human chimera might be useful, allowing the in vivo study of the components of the human immune system relevant to asthma. We investigated the role of allergen-reactive T lymphocytes in a human-mouse SCID model. SCID mice were reconstituted intratracheally with human PBMC from healthy, nonallergic, nonasthmatic donors and exposed to an aerosol of house dust mite allergen after i.p. injection with Dermatophagoides pteronyssinus I Ag and alum. The donor T lymphocytes had a Th1 cytokine phenotype. The reconstituted and allergen-challenged mice developed AHR to carbachol. The mouse airways and lungs were infiltrated with human T lymphocytes. No eosinophils or increases in human IgE were observed. The intrapulmonary human T lymphocytes demonstrated an increase in intracytoplasmic IL-4 and IL-5 and a decrease in IFN-gamma after exposure to allergen adjuvant. Antagonizing human IL-4/IL-13 or IL-5 resulted in a normalization of the airway responsiveness, despite a sustained intracellular Th2 cytokine production. These results provide evidence that the activated human allergen-reactive Th2 cells producing IL-4 or IL-5 are pivotal in the induction of AHR, whereas no critical role for eosinophils or IgE could be demonstrated. They also demonstrate that human allergen-specific Th1 lymphocytes can be driven to a Th2 phenotype.
Contrasting Effects on Discrimination Learning after Hippocampal Lesions and Conjoint Hippocampal–Caudate Lesions in Monkeys (2000)

Eighteen monkeys with lesions of the hippocampal region (the hippocampus proper, the dentate gyrus, and the subiculum) made by an ischemic procedure, radio frequency, or ibotenic acid were tested on a simple, two-choice object discrimination learning task that has been shown to be sensitive to large lesions of the medial temporal lobe. The monkeys were also tested on two other discrimination tasks (pattern discrimination and eight-pair concurrent discrimination) that can be learned normally by monkeys with large medial temporal lobe lesions. All of the lesion groups were impaired at learning the simple object discrimination task. Seven of the monkeys who had sustained damage to the hippocampal region also sustained damage to the tail of the caudate nucleus. These seven monkeys, but not the other 11 monkeys with hippocampal lesions, were impaired on pattern discrimination and concurrent discrimination learning. The results suggest that the hippocampal region is important for learning easy, two-choice discriminations, whereas the caudate nucleus is necessary for the normal learning of more difficult, gradually acquired discrimination tasks. The findings support the distinction between declarative memory, which depends on the hippocampus and related medial temporal lobe structures, and habit learning, which depends on the caudate nucleus.
Accumulation of Memory Precursor CD8 T Cells in Regressing Tumors following Combination Therapy with Vaccine and Anti-PD-1 Antibody (2014)

Immunosuppression in the tumor microenvironment blunts vaccine-induced immune effectors. PD-1/B7-H1 is an important inhibitory axis in the tumor microenvironment. Our goal in this study was to determine the effect of blocking this inhibitory axis during and following vaccination against breast cancer. We observed that using anti-PD-1 antibody and a multipeptide vaccine (consisting of immunogenic peptides derived from breast cancer antigens, neu, legumain, and β-catenin) as a combination therapy regimen for the treatment of breast cancer-bearing mice prolonged the vaccine-induced progression-free survival period. This prolonged survival was associated with increase in number of Tc1 and Tc2 CD8 T cells with memory precursor phenotype, CD27+IL-7RhiT-betlo, and decrease in number of PD-1+ dendritic cells (DC) in regressing tumors and enhanced antigen reactivity of tumor-infiltrating CD8 T cells. It was also observed that blockade of PD-1 on tumor DCs enhanced IL-7R expression on CD8 T cells. Taken together, our results suggest that PD-1 blockade enhances breast cancer vaccine efficacy by altering both CD8 T cell and DC components of the tumor microenvironment. Given the recent success of anti-PD-1 monotherapy, our results are encouraging for developing combination therapies for the treatment of patients with cancer in which anti-PD-1 monotherapy alone may be ineffective (i.e., PD-L1-negative tumors).
Synthesis and antibacterial activity of 1-(arylamino)-1H-pyrroles and 4-(1H-pyrrol-1-ylimino)-2,5-cyclohexadienes (1981)

The syntheses of 1-(arylamino)-1H-pyrroles and 4-(1H-pyrrol-1-ylimino)-2-5-cyclohexadienes are described. Several of these compounds express in vitro antibacterial activity or can be metabolized to show in vitro antibacterial activity, and a few examples have shown efficacy against tuberculosis in mice. One compound, N,N'-(2,5-cyclohexadiene-1,4-diylidene)bis-1H-pyrrol-1-amine, is completely effective at 6.25 mg/kg against Mycobacterium tuberculosis H37Rv.
Inhibition and stimulation of photoreceptor phosphodiesterases by dipyridamole and M&B 22,948. (1989)

Few high affinity inhibitors of the photoreceptor phosphodiesterases have been identified. We show here that dipyridamole and M&B 22,948 (Zaprinast), potent inhibitors of the cGMP-binding, cGMP-specific phosphodiesterase (PDE), also inhibit trypsin- or transducin-activated bovine rod and cone photoreceptor phosphodiesterases at submicromolar concentrations. Dixon plots demonstrated that the inhibition of trypsin-activated rod PDE was competitive, with Ki values of 140 nM for M&B 22,948 and 380 nM for dipyridamole. Both of these drugs were much more potent than other PDE inhibitors, including isobutylmethylxanthine (IBMX). These results reinforce the suggestion that the photoreceptor and the cGMP-binding, cGMP-specific PDE are closely related. In addition, the high affinity and selectivity of these agents should make them useful for probing the regulation and function of PDE in the photoreceptor. At low substrate concentrations, the effects of these drugs on basal unactivated PDE activity were similar to those seen with trypsin- or transducin-activated PDE. At millimolar substrate concentrations, however, the effects of the drugs were biphasic; PDE activity was stimulated at drug concentrations from 1 to 10 microM and inhibited at higher concentrations. Stimulation was not observed with IBMX. This stimulation of activity apparently was not an allosteric effect caused by direct binding of the dipyridamole and M&B 22,948 to the high affinity noncatalytic cGMP binding sites on the PDEs; whereas no cooperativity of cGMP binding to this site has been demonstrated, the drugs actually stimulated the binding of low concentrations of cGMP to this site. In addition, whereas preincubation with cGMP and cGMP analogs blocked the stimulation exerted by the drugs, they did so only at much higher concentrations than those necessary for saturation of the high affinity noncatalytic cGMP site. Because the stimulation can only be seen at higher substrate levels than are thought to exist in the photoreceptor, only the inhibitory effects of the drugs are likely to be pharmacologically relevant. However, the stimulation exerted by these drugs may point to a hitherto unknown allosteric interaction between the catalytic and regulatory sites on the PDE or to a previously unrecognized regulatory site.
[Prognostic value of atrial fibrillation in patients--survivors of acute coronary syndrome]. (2009)

For the study of contribution of atrial fibrillation (AF) during acute coronary syndrome (ACS) in long-term prognosis after clinical stabilization we examined 453 patients admitted to Moscow hospitals and followed them for 2.07 +/- 0.48 years. The following events were registered: fatal and nonfatal myocardial infarction (MI), unstable angina (UA), fatal and nonfatal stroke, death of other causes. At ACS onset sinus rhythm was noted in 419 (92.5%), permanent or persistent AF-in 16 (3.5%), attack (paroxysm) of AF - in 18 (4.0%) patients. Mean length of life before end point was 884.9 +/- 23.4, 827.3 +/- 123.3 and 514.0 +/- 111.3 days in patients with sinus rhythm, permanent/persistent AF, and attack of AF during first 10 days of ACS, respectively (p<0.001). Compared with patients in sinus rhythm in patients with attack of AF relative risk (RR) of occurrence of any end point was 1.75 (95% confidence interval [CI] 1.284 to 2.873, p< 0.001), of fatal MI - 1.72 (95% CI 1.026 to 2.873, p=0.040), of UA - 2.116 (95% CI 1.249 to 3.585, p=0.005), of stroke - 2.863 (95% CI 1.300 to 6.301, p=0.009). Multifactorial analysis selected history of MI and attack of AF during first 10 days of ACS as independent predictors of unfavorable outcome. Thus paroxysmal form of AF during hospital stay because of ACS is associated with higher probability of development of unfavorable events in the next 1-2 years.
Monoclonal antibodies that recognize calcium-dependent structures of human thrombospondin. Characterization and mapping of their epitopes. (1986)

Monoclonal antibodies (mAbs) raised against reduced and alkylated thrombospondin (TSP) were screened for the ability to react with Ca2+-replete TSP versus EDTA-treated TSP. Two mAbs designated A6.1 and D4.6 were found to react much more strongly with TSP after EDTA treatment. The dissociation constants for these mAbs were measured in 5 mM EDTA and found to be 6 X 10(-10) M for A6.1 and 7 X 10(-9) M for D4.6. Binding to A6.1 was undetectable in the presence of 1 mM Ca2+ while binding of D4.6 occurred with about 100-fold lower affinity. The Ca2+ concentration dependence of A6.1 binding was broad with a midpoint near 50 microM free Ca2+ while that of D4.6 showed a sharp transition below 0.1 microM. Upon dialysis of EDTA-treated TSP into Ca2+ containing buffer, the binding of the mAbs was prevented or decreased, indicating reversibility of the conformational transition induced by the initial removal of Ca2+ . Mg2+ can compete with the Ca2+ binding sites involved in mAb binding, but TSP dialyzed from Ca2+ into Mg2+ binds the two mAbs as well as EDTA-treated TSP, indicating that Mg2+ cannot maintain the Ca2+-replete structure of TSP. The proteolytic fragments of TSP with which the two mAbs react were determined by probing Western blots of digests of TSP with the mAbs. A6.1 reacts with the 70-kDa fragment generated by chymotrypsin in EDTA which contains the interchain disulfide bonds of TSP and the binding site(s) for type V collagen (Mumby, S. M., Raugi, G. J., and Bornstein, P. (1984) J. Cell Biol. 98, 646-652). D4.6 reacts with fragments of 140 and 120 kDa found in digests of Ca2+-replete TSP which are absent from digests in EDTA. Electron microscopy of rotary shadowed, carbon-coated replicas of TSP mAb complexes confirms the Ca2+ sensitivity of mAb binding and has been used to localize the epitopes for both mAbs on the three-dimensional structure of TSP.
Some new aspects of isoprenoid biosynthesis in plants—A review (1995)

Plants are capable of synthesizing a myriad of isoprenoids and prenyl lipids. Much attention has been focused on 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the enzyme that synthesizes mevalonate and is generally considered responsible for the regulation of substrate flux to isoprenoids. In contrast to vertebrates, where there seems to exist only one HMGR gene, in plants a small family of isogenes appears differentially expressed in regard to location and time. Much less is known in plants about the preceding steps, viz. the conversion of acetyl-CoA to HMG-CoA. An enzyme system has been isolated from radish that can catalyze this transformation, and which shows some unusual properties in vitro. The intracellular localization of the early steps of isoprenoid biosynthesis in plant cells is still a matter of debate. The various observations and hypotheses derived from incorporation and inhibition studies are somewhat contradictory, and an attempt is being made to rationalize various findings that do not at first seem compatible. There are good arguments in favor of an exclusively cytoplasmic formation of isopentenyl pyrophosphate (IPP) via mevalonic acid, but other studies and observations suggest an independent formation in plastids. Other possibilities are being considered, such as the existence of independent (compartmentalized) biosynthetic pathways of IPP formation via the so-called Rohmer pathway. Substrate channeling through the formation of end product-specific multienzyme complexes (metabolons) with no release of substrate intermediates will also be discussed.
Mass spectrometry of single-stranded restriction fragments captured by an undigested complementary sequence (2000)

In this report, we describe a simple and accurate method to analyze restriction fragments using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The two complementary strands of restriction fragments are separated through hybridization to a capture probe, which is a single-stranded undigested fragment. Using the biotin-streptavidin linkage, the hybrid is immobilized on streptavidin-coated magnetic beads. After conditioning the captured restriction fragments, they are eluted from the probe and their molecular weights are determined. The proposed method greatly improves the quality, and reduces the complexity of the mass spectrum by analyzing only one of the complementary strands of restriction fragments.
On the Origins of Cyanuric Acid Hydrolase: Purification, Substrates, and Prevalence of AtzD from Pseudomonas sp. Strain ADP (2003)

Cyanuric acid hydrolase (AtzD) from Pseudomonas sp. strain ADP was purified to homogeneity. Of 22 cyclic amides and triazine compounds tested, only cyanuric acid and N-methylisocyanuric acid were substrates. Other cyclic amidases were found not to hydrolyze cyanuric acid. Ten bacteria that use cyanuric acid as a sole nitrogen source for growth were found to contain either atzD or trzD, but not both genes.
Fluorescent SiC as a new material for white LEDs (2012)

Current III–V-based white light-emitting diodes (LEDs) are available. However, their yellow phosphor converter is not efficient at high currents and includes rare-earth metals, which are becoming scarce. In this paper, we present the growth of a fluorescent silicon carbide material that is obtained by nitrogen and boron doping and that acts as a converter using a semiconductor. The luminescence is obtained at room temperature, and shows a broad luminescence band characteristic of donor-to-acceptor pair recombination. Photoluminescence intensities and carrier lifetimes reflect a sensitivity to nitrogen and boron concentrations. For an LED device, the growth needs to apply low-off-axis substrates. We show by ultra-high-resolution analytical transmission electron microscopy using aberration-corrected electrons that the growth mechanism can be stable and that there is a perfect epitaxial relation from the low-off-axis substrate and the doped layer even when there is step-bunching.
CPT-11 Converting Enzyme from Rat Serum: Purification and Some Properties. (1991)

A rat serum enzyme that catalyzes the conversion of a pro-drug, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11), to an anticancer drug, 7-ethyl-10-hydroxycamptothecin (SN-38), was purified and its properties were characterized. The enzyme was purified by column chromatography on diethylaminoethyl Toyopearl 650M, QAE-Sephadex, Sephadex G-150, Con A-Sepharose and high performance liquid chromatography with an ion-exchanger column. It was most active at pH 7.5 and was stable at pH 4-9 for 1 h at 30 degrees C. The molecular weight was estimated to be 60 and 57 kDa by gel filtration and sodium dodecylsulfate-polyacrylamide gel electrophoresis methods, respectively, and the isoelectric point was 4.6, as determined by isoelectric focusing. The Km value for CPT-11 was 0.28 microM. This enzyme was inhibited by diisopropyl phosphorofluoridate (DFP) and phenylmethanesulfonyl fluoride (PMSF) but insensitive to eserine, p-chloromercuribenzoate (PCMB) and ethylenediaminetetraacetate (EDTA). The enzyme also hydrolyzed p-nitrophenylacetate (p-NPA), a commonly used substrate for esterases, but was not active toward acetylcholine, suggesting that the enzyme is a carboxylesterase[EC 3.1.1.1]. During the hydrolyses of CPT-11 and p-NPA, an initial burst phenomenon similar to that found in the alpha-chymotrypsin-catalyzed hydrolysis of p-NPA was observed. Kinetic analysis revealed that the deacylation of the enzyme is the rate-limiting step in substrate hydrolysis. This enzyme was found to also split other ester derivatives of SN-38 besides CPT-11.
Oral maintenance clinical trial with miglustat for type I Gaucher disease: switch from or combination with intravenous enzyme replacement (2007)

Enzyme replacement therapy (ERT) with imiglucerase reduces hepatosplenomegaly and improves hematologic parameters in Gaucher disease type 1 within 6-24 months. Miglustat reduces organomegaly, improves hematologic parameters, and reverses bone marrow infiltration. This trial evaluates miglustat in patients clinically stable on ERT. Tolerability of miglustat and imiglucerase, alone and in combination, pharmacokinetic profile, organ reduction, and chitotriosidase activity were assessed. Thirty-six patients stable on imiglucerase were randomized into this phase II, open-label trial. Statistically significant changes from baseline were assessed (paired t test) on primary objectives with secondary analyses on biochemical and safety parameters. Liver and spleen volume were unchanged in switched patients. No significant differences were seen between groups regarding mean change in hemoglobin. Mean change in platelet counts was only significant between miglustat and imiglucerase groups (P = .035). Chitotriosidase activity remained stable. In trial extension, clinical endpoints were generally maintained. Miglustat was well tolerated alone or in combination. Miglustat's safety profile was consistent with previous trials; moreover, no new cases of peripheral neuropathy were observed. Gaucher disease type 1 (GD1) parameters were stable in most switched patients. Combination therapy did not show benefit. Findings suggest miglustat could be an effective maintenance therapy in stabilized patients with GD1.
Transfection Using DEAE‐Dextran (1997)

Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)-dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE-dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE-dextran transfection, as well as two more specific protocols for typical experimental applications. The Basic Protocol is suitable for transfection of anchorage-dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.
Rapid determination of gemcitabine in plasma and serum using reversed‐phase HPLC (2007)

Gemcitabine (2'2'-difluorodeoxycytidine) is a pyrimidine analog used in the treatment of a variety of solid tumors. After intravenous (i.v.) administration, it is rapidly inactivated to 2'-deoxy-2',2'-difluorouridine (dFdU). A sensitive analytical method for the quantitation of gemcitabine is required for the assessment of alternative dosage and treatment schemes. A rapid and robust RP-HPLC assay for analysis of gemcitabine in human and animal plasma and serum was developed and validated using 2'-deoxyuridine (dU) and 5-fluoro-2'-deoxyuridine (5FdU) as internal standards. It is based on protein precipitation, the use of an Atlantis dC18 column of 100 mm length (inner diameter, 4.6 mm; particle size, 3 microm) and isocratic elution using a 10 mM phosphate buffer, pH 3.0, followed by isocratic elution with the same buffer containing 3% of ACN. For gemcitabine, RSD values for intraday and interday precision were < 4.4 and 5.3%, respectively, the LOQ was 20 ng/mL, and the assay was linear in the range of 0.020-20 microg/mL with an accuracy of > or =89%. The recovery for gemcitabine, dU and 5FdU was 86-98%. The assay was applied to determine gemcitabine levels in plasma samples of patients collected during and shortly after conventional infusion of 25-30 mg/kg body mass (levels: 2.0-18.9 microg/mL) and rats that received lower doses (1.5 mg/kg) via i.v., subcutaneous and oral drug administration (levels: 0.20-2.60 microg/mL). It could also be applied to estimate dFdU levels in human plasma.