gebhart/patrepeval huggingface dataset:
Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism (1996)

To expand the utility of recombinant adenovirus vectors for gene therapy applications, methods to alter native viral tropism to achieve cell-specific transduction would be beneficial. To this end, we are pursuing genetic methods to alter the cell recognition domain of the adenovirus fiber. To incorporate these modified fibers into mature virions, we have developed a method based on homologous DNA recombination between two plasmids. A fiber-deleted, propagation-defective rescue plasmid has been designed for recombination with a shuttle plasmid encoding a variant fiber gene. Recombination between the two plasmids results in the derivation of recombinant viruses containing the variant fiber gene. To establish the utility of this method, we constructed a recombinant adenovirus containing a fiber gene with a silent mutation. In addition, we generated an adenovirus vector containing chimeric fibers composed of the tail and shaft domains of adenovirus serotype 5 and the knob domain of serotype 3. This modification was shown to alter the receptor recognition profile of the virus containing the fiber chimera. Thus, this two-plasmid system allows for the generation of adenovirus vectors containing variant fibers. This method provides a rapid and facile means of generating fiber-modified recombinant adenoviruses. In addition, it should be possible to use this system in the development of adenovirus vectors with modified tropism to allow cell-specific targeting.
Biochemical analysis of distinct Rab5- and Rab11-positive endosomes along the transferrin pathway (1999)

Rab GTPases are associated with distinct cellular compartments and function as specific regulators of intracellular transport. In the endocytic pathway, it is well documented that Rab5 regulates transport from plasma membrane to early (sorting) endosomes. In contrast, little is known about the precise localization and function of Rab4 and Rab11, which are believed to control endocytic recycling. In the present study we have analysed the protein composition of Rab5- and Rab11-carrying endosomes to gain further insight into the compartmental organization of the endocytic and recycling pathway. Endosome populations of this transport route were purified by immunoadsorption from endosome-enriched subcellular fractions using antibodies directed against the cytoplasmic tail of the transferrin receptor, Rab5 or Rab11. Endocytosed transferrin moved sequentially through compartments that could be immunoadsorbed with anti-Rab5 and anti-Rab11, consistent with the theory that Rab5 and Rab11 localise to sorting and recycling endosomes, respectively. These compartments exhibited morphological differences, as determined by electron microscopy. Although their overall protein compositions were very similar, some proteins were found to be selectively enriched. While Rab4 was present on all endosome populations, Rab5 and Rab11 were strikingly segregated. Furthermore, the Rab11-positive endosomes were rich in annexin II, actin and the t-SNARE syntaxin 13, compared to Rab5-containing endosomes. In an in vitro assay, the Rab5 effector protein EEA1 was preferentially recruited by Rab5-positive endosomes. Taken together, our data suggest an organization of the transferrin pathway into distinct Rab5- and Rab11-positive compartments.
Deletion of Keap1 in the Lung Attenuates Acute Cigarette Smoke–Induced Oxidative Stress and Inflammation (2009)

Exposure to cigarette smoke (CS) is the primary factor associated with the development of chronic obstructive pulmonary disease (COPD). CS increases the level of oxidants in the lungs, resulting in a depletion of antioxidants, which promotes oxidative stress and the destruction of alveolar tissue. In response to CS, pulmonary epithelial cells counteract increased levels of oxidants by activating Nrf2-dependent pathways to augment the expression of detoxification and antioxidant enzymes, thereby protecting the lung from injury. We hypothesize that increasing the pathways activated by Nrf2 will afford protection against CS-induced lung damage. To this end we have developed a novel mouse model in which the cytosolic inhibitor of Nrf2, Keap1, is genetically deleted in Clara cells, which predominate in the upper airways in mice. Deletion of Keap1 in Clara cells resulted in increased expression of Nrf2-dependent genes, such as Nqo1 and Gclm, as determined by microarray analysis and quantitative PCR. Deletion of Keap1 in airway epithelium decreased Keap1 protein levels and significantly increased the total level of glutathione in the lungs. Increased Nrf2 activation protected Clara cells against oxidative stress ex vivo and attenuated oxidative stress and CS-induced inflammation in vivo. Expression of KEAP1 was also decreased in human epithelial cells through siRNA transfection, which increased the expression of Nrf2-dependent genes and attenuated oxidative stress. In conclusion, activating Nrf2 pathways in tissue-specific Keap1 knockout mice represents an important genetic approach against oxidant-induced lung damage.
Evidence for molecular subtypes of HIV-associated lymphoma (1991)

The pathogenesis of the HIV-associated lymphomas is not well understood. In order to begin characterizing this class of lymphoma, we initiated a molecular genetic study of DNA extracted from 31 diagnostic biopsy specimens from patients diagnosed with AIDS-associated non-Hodgkin's lymphoma. Analysis of 25 peripheral lymphomas showed that 14 were monoclonal B-cell processes, while 11 appeared to be of polyclonal origin. Five of the 14 monoclonal lymphomas were found to have rearrangements of the c-myc gene. Epstein-Barr virus (EBV) genomes were found in seven out of 14 monoclonal samples, but only two out of nine polyclonal samples. The six primary central nervous system (CNS) lymphoma samples were more homogeneous than the peripheral samples and all were monoclonal, positive for EBV and lacked detectable c-myc gene rearrangements. This study allows us to subdivide the HIV-associated lymphomas into three major molecular subtypes: (1) monoclonal B-cell process frequently associated with c-myc rearrangement or detectable EBV genomes, (2) polyclonal B-cell process typically without evidence of EBV, and (3) monoclonal primary CNS process associated with EBV genomes and lacking detectable c-myc rearrangement.
Bisphosphonates in Bone Disease: From the Laboratory to the Patient (1995)

Bone and Mineral Metabolism: Bone Physiology: Morphology. Composition of bone. Modeling and remodeling. Calcium homeostasis. Bone as an organ. Assessment of bone turnover. Bisphosphonates-Preclinical: Background to the pharmacological development: Chemistry. Actions. Physicochemical effects. Biological effects. Inhibition of bone resorption. Inhibition of mineralization. Pharmocokinetics: Intestinal absorption. Distribution. Renal clearance. Other modes of application. Animal toxicology: Acute toxicology. Nonacute toxicology. Bisphosphonates-clinical: Introduction. Paget's Disease: Definition. Epidemiology. Pathophysiology. Clinical manifestations. Treatment with drugs other than bisphosphonates. Treatment with bisphosphonates. Osteolytic tumor-induced bone disease: Definition. Pathophysiology. Clinical manifestations. Treatment with drugs other than bisphosphonates. Treatment with bisphosphonates. Non-tumor induced hypercalcemia: Definition. Pathophysiology. Clinical manifestations. Treatment with drugs other than bisphosphonates. Treatment with bisphosphonates. Osteoporosis: Definition. Epidemiology. Pathophysiology. Clinical manifestations. Treatment with drugs other than bisphosphonates. Treatment with bisphosphonates. Heterotopic calcification and ossification: Definition. Pathophysiology. Clinical manifestations. Treatment with drugs other than bisphosphonates. Treatment with bisphosphonates. Other diseases: Adverse events: Bisphosphonates in general. Individual bisphosphonates. Contraindications. Future prospects. Commercially available bisphosphonates: Index.
The family of bacterial ADP-ribosylating exotoxins. (1995)

Pathogenic bacteria utilize a variety of virulence factors that contribute to the clinical manifestation of their pathogenesis. Bacterial ADP-ribosylating exotoxins (bAREs) represent one family of virulence factors that exert their toxic effects by transferring the ADP-ribose moiety of NAD onto specific eucaryotic target proteins. The observations that some bAREs ADP-ribosylate eucaryotic proteins that regulate signal transduction, like the heterotrimeric GTP-binding proteins and the low-molecular-weight GTP-binding proteins, has extended interest in bAREs beyond the bacteriology laboratory. Molecular studies have shown that bAREs possess little primary amino acid homology and have diverse quaternary structure-function organization. Underlying this apparent diversity, biochemical and crystallographic studies have shown that several bAREs have conserved active-site structures and possess a conserved glutamic acid within their active sites.
Specific stimulation of human T lymphocytes by substance P. (1983)

Substance P (SP) and SP(4-11) stimulated significantly the proliferation of human T lymphocytes in vitro at respective concentrations of 10(-11) M to 10(-7) M and 10(-9) M to 10(-7) M, as assessed by the uptake of [3H]thymidine and [3H]leucine. The proliferative response of human T lymphocytes to PHA was augmented by 10(-9) M to 10(-8) M SP and 10(-10) M to 10(-7) M SP(4-11). The inactive analogue [D-Pro2, D-Phe7, D-Trp9]-SP inhibited the effects of SP on T lymphocytes. The specific stimulation of T lymphocytes by SP suggests a unique mechanism for the regulation of immunologic responses by peripheral nerve-derived factors.
Blackboard Systems (1986)

The first blackboard system was the HEARSAY-II speech understanding system that evolved between 1971 and 1976. Subsequently, many systems have been built that have similar system organization and run-time behavior. The objectives of this document are (1) to define what is meant by blackboard systems, and (2) to show the richness and diversity of blackboard system designs. The article begins with a discussion of the underlying concept behind all blackboard systems, the blackboard model of problem solving. In order to bridge the gap between a model and working systems, the blackboard of framework, an extension of the basic blackboard model is introduced, including a detailed description of the model's component and their behavior. A model does not come into existence on its own is usually an abstraction of many examples. In Section 2, the history of ideas is traced and the designs of some application systems that helped shape the blackboard model are detailed. The author then describes and contrasts existing blackboard systems. Blackboard systems are divided into applications and and skeletal systems. In application systems the blackboard system components are integrated with the domain knowledge required to solve the problem at hand. Skeletal systems are devoid of domain knowledge, and, as the name implies, consist of the essential system components from which application systems can be built by the addition of knowledge and the specification of control (i.e. meta-knowledge). In Section 3.6, the author summarizes the features of the application systems and in Section 4 presents the author's perspective on the utility of the blackboard approach to problem solving and knowledge engineering. (Author)
Cryptic epitopes on the nicotinic acetylcholine receptor are recognized by autoreactive CD4+ cells. (1993)

Experimental autoimmune myasthenia gravis is induced in C57BL/6 mice by injection of Torpedo nicotinic acetylcholine receptor (TAChR). We investigated here the presence of cryptic CD4+ epitopes on the TAChR molecule, and their relationship with potentially autoreactive CD4+ cells, which survived clonal deletion. CD4+ cells from C57BL/6 mice immunized with native or denatured TAChR were challenged in vitro with overlapping synthetic peptides, 20-residue long, screening the sequences of TAChR alpha, gamma, and delta subunits. Only three epitopes on the alpha subunit were recognized consistently. Mice immunized with large doses (nanomoles) of TAChR clearly recognized only the immunodominant sequence T alpha 150-169. Anti-TAChR CD4+ cells did not cross-react with murine alpha subunit sequences, or with any synthetic sequence of human gamma and delta subunits, which are very similar to the corresponding murine subunits. To facilitate recognition of cryptic epitopes, we injected mice with pools of synthetic peptides corresponding to the sequences of TAChR alpha, gamma, and delta subunits. In addition to the three immunodominant alpha subunit epitopes, other epitopes were recognized by CD4+ cells within the sequences T alpha 304-322, T gamma 105-124, T gamma 120-139, T gamma 401-420, T gamma 357-376, T delta 16-35, T delta 61-80, T delta 121-140, and T delta 301-320. CD4+ cells thus sensitized cross-reacted with the mammalian sequences alpha 304-322, gamma 105-124, gamma 120-139, and delta 301-320. Mice were immunized with large doses (approximately 40 nmol) of individual TAChR synthetic cryptic epitopes. CD4+ cells sensitized to five cryptic epitopes (the ones listed above plus delta 121-140) cross-reacted with autologous sequences. We determined the dose dependence of the sensitization of CD4+ cells in vivo to the strongly immunodominant epitope peptide T alpha 150-169 and to the cryptic epitope peptides T gamma 120-139 and T delta 301-320 by immunizing mice with increasing doses of peptide (approximately 1.2 to approximately 20 nmol), and testing the in vitro anti-peptide response of the CD4+ cells. No difference was found for the epitopes tested. Doses of 3 to 10 micrograms induced a strong CD4+ sensitization, and the dose dependence of the in vitro response of the sensitized cells to the relevant peptide was comparable. Production of cryptic epitopes upon in vitro TAChR processing was investigated by testing peptide-sensitized CD4+ cells with native TAChR: only two cryptic epitopes were produced.
A Comunicação mediada por computador. O exemplo do IRC (2005)

O artigo pretende abordar um sistema de comunicação multi-utilizador em tempo real com recurso ao texto escrito (o IRC - Internet Relay Chat) cada vez mais vulgarizado e que conhece adeptos cada vez mais espalhados geograficamente e em maior número. O objecto desta análise, para além da comunicação linguística propriamente dita, deverá sobretudo considerar, através do comportamento verbal dos ¿falantes¿, o desenvolvimento de fenómenos comunicacionais e de interacções colaborativas dos interlocutores que substituam, num ambiente de comunicação verbal com recurso exclusivo ao texto escrito, o papel do contacto com os gestos ou com os olhares, por exemplo, os quais, em modelos de comunicação em presença, ora reforçam a mensagem verbal, ora a contrariam.
Molecular Cloning and Expression Analysis of the Mitochondrial Pyruvate Dehydrogenase from Maize1 (1999)

Four cDNAs, one encoding an alpha-subunit and three encoding beta-subunits of the mitochondrial pyruvate dehydrogenase, were isolated from maize (Zea mays L.) libraries. The deduced amino acid sequences of both alpha- and beta-subunits are approximately 80% identical with Arabidopsis and pea (Pisum sativum L.) homologs. The mature N terminus was determined for the beta-subunit by microsequencing the protein purified from etiolated maize shoot mitochondria and was resolved by two-dimensional gel electrophoresis. This single isoelectric species comprised multiple isoforms. Both alpha- and beta-subunits are encoded by multigene families in maize, as determined by Southern-blot analyses. RNA transcripts for both alpha- and beta-subunits were more abundant in roots than in young leaves or etiolated shoots. Pyruvate dehydrogenase activity was also higher in roots (5-fold) compared with etiolated shoots and leaves. Both subunits were present at similar levels in all tissues examined, indicating coordinated gene regulation. The protein levels were highest in heterotrophic organs and in pollen, which contained about 2-fold more protein than any other organ examined. The relative abundance of these proteins in nonphotosynthetic tissues may reflect a high cellular content of mitochondria, a high level of respiratory activity, or an extra plastidial requirement for acetate.
Different T Cell Receptor Affinity Thresholds and CD8 Coreceptor Dependence Govern Cytotoxic T Lymphocyte Activation and Tetramer Binding Properties (2007)

T cells have evolved a unique system of ligand recognition involving an antigen T cell receptor (TCR) and a coreceptor that integrate stimuli provided by the engagement of peptide-major histocompatibility complex (pMHC) antigens. Here, we use altered pMHC class I (pMHCI) molecules with impaired CD8 binding (CD8-null) to quantify the contribution of coreceptor extracellular binding to (i) the engagement of soluble tetrameric pMHCI molecules, (ii) the kinetics of TCR/pMHCI interactions on live cytotoxic T lymphocytes (CTLs), and (iii) the activation of CTLs by cell-surface antigenic determinants. Our data indicate that the CD8 coreceptor substantially enhances binding efficiency at suboptimal TCR/pMHCI affinities through effects on both association and dissociation rates. Interestingly, coreceptor requirements for efficient tetramer labeling of CTLs or for CTL activation by determinants displayed on the cell surface operated in different TCR/pMHCI affinity ranges. Wild-type and CD8-null pMHCI tetramers required monomeric affinities for cognate TCRs of KD < approximately 80 microM and approximately 35 microM, respectively, to label human CTLs at 37 degrees C. In contrast, activation by cellular pMHCI molecules was strictly dependent on CD8 binding only for TCR/pMHCI interactions with KD values >200 microM. Altogether, our data provide information on the binding interplay between CD8 and the TCR and support a model of CTL activation in which the extent of coreceptor dependence is inversely correlated to TCR/pMHCI affinity. In addition, the results reported here define the range of TCR/pMHCI affinities required for the detection of antigen-specific CTLs by flow cytometry.
Microfluidic carbon-blackened polydimethylsiloxane device with reduced ultra violet background fluorescence for simultaneous two-color ultra violet/visible-laser induced fluorescence detection in single cell analysis (2012)

In single cell analysis (SCA), individual cell-specific properties and inhomogeneous cellular responses are being investigated that is not subjected to ensemble-averaging or heterogeneous cell population effects. For proteomic single cell analysis, ultra-sensitive and reproducible separation and detection techniques are essential. Microfluidic devices combined with UV laser induced fluorescence (UV-LIF) detection have been proposed to fulfill these requirements. Here, we report on a novel microfluidic chip fabrication procedure that combines straightforward production of polydimethylsiloxane (PDMS) chips with a reduced UV fluorescence background (83%-reduction) by using PDMS droplets with carbon black pigments (CBP) as additives. The CBP-droplet is placed at the point of detection, whereas the rest of the chip remains transparent, ensuring full optical control of the chip. We systematically studied the relation of the UV background fluorescence at CBP to PDMS ratios (varying from 1:10 to 1:1000) for different UV laser powers. Using a CBP/PDMS ratio of 1:20, detection of a 100 nM tryptophan solution (S/N = 3.5) was possible, providing a theoretical limit of detection of 86 nM (with S/N = 3). Via simultaneous two color UV/VIS-LIF detection, we were able to demonstrate the electrophoretic separation of an analyte mixture of 500 nM tryptophan (UV) and 5 nM fluorescein (VIS) within 30 s. As an application, two color LIF detection was also used for the electrophoretic separation of the protein content from a GFP-labeled single Spodoptera frugiperda (Sf9) insect cell. Thereby just one single peak could be measured in the visible spectral range that could be correlated with one single peak among others in the ultraviolet spectra. This indicates an identification of the labeled protein γ-PKC and envisions a further feasible identification of more than one single protein in the future.
Regulation of protein function by native metastability (2000)

In common globular proteins, the native form is in its most stable state. In contrast, each native form exists in a metastable state in inhibitory serpins (serine protease inhibitors) and some viral membrane fusion proteins. Metastability in these proteins is critical to their biological functions. Mutational analyses and structural examination have previously revealed unusual interactions, such as side-chain overpacking, buried polar groups, and cavities as the structural basis of the native metastability. However, the mechanism by which these structural defects regulate protein functions has not been elucidated. We report here characterization of cavity-filling mutations of alpha(1)-antitrypsin, a prototype serpin. Conformational stability of the molecule increased linearly with the van der Waals volume of the side chains. Increasing conformational stability is correlated with decreasing inhibitory activity. Moreover, the activity loss appears to correlate with the decrease in the rate of the conformational switch during complex formation with a target protease. These results strongly suggest that the native metastability of proteins is indeed a structural design that regulates protein functions.
Biofilms: Microbial Life on Surfaces (2002)

Microorganisms attach to surfaces and develop biofilms. Biofilm-associated cells can be differentiated from their suspended counterparts by generation of an extracellular polymeric substance (EPS) matrix, reduced growth rates, and the up- and down- regulation of specific genes. Attachment is a complex process regulated by diverse characteristics of the growth medium, substratum, and cell surface. An established biofilm structure comprises microbial cells and EPS, has a defined architecture, and provides an optimal environment for the exchange of genetic material between cells. Cells may also communicate via quorum sensing, which may in turn affect biofilm processes such as detachment. Biofilms have great importance for public health because of their role in certain infectious diseases and importance in a variety of device-related infections. A greater understanding of biofilm processes should lead to novel, effective control strategies for biofilm control and a resulting improvement in patient management.
Near Infrared Calibrations for Goat's Milk Components: Protein, Total Casein, αs-, β- and k-caseins, Fat and Lactose (1993)

A fast and inexpensive method to quantify the main constituents of goat's milk using near infrared (NIR) spectroscopy, is proposed. Since the efficiency of milk-to-cheese transformation depends on the total amount of casein, as well as on the relative proportions of the different types of caseins, it is desirable to quantify not only protein, fat and lactose, as is normally done routinely in the cheese industry and milk recording schemes, but also total casein, α s -, β- and k-caseins. Quantities of these main components have been measured in milk samples of three Spanish breeds of goats, using traditional chemical, electrophoretical and densitometric laboratory methods in order to produce NIR calibrations for total protein, total casein, casein fractions (α s -, β- and k-caseins), fat and lactose. Low standard errors and high coefficients of determinations have been obtained for the calibrations and validations. Goat's milk spectra are described, and the contribution of different components to the observed pattern is discussed. Due to the low cost, facility and rapidity of the analyses after the spectrophotometer has been calibrated for each milk component, the use of near infrared spectroscopy becomes a suitable tool to analyse large numbers of samples and, therefore, very suitable for dairy goat recording schemes.