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PubMed1994

Generation of Chinese hamster ovary cell glycosylation mutants by retroviral insertional mutagenesis. Integration into a discrete locus generates mutants expressing high levels of N-glycolylneuraminic acid.

Retroviral insertional mutagenesis can both generate somatic cell mutants and pinpoint the genomic locus associated with a mutant phenotype. In the present study, this approach was applied to Chinese hamster ovary cells (CHO) made susceptible to Moloney murine leukemia virus (MoMuLV) infection by stable expression of an ecotropic retrovirus receptor. These CHO cells were infected with a replication incompetent MoMuLV construct with a promoterless hygromycin phosphotransferase (hygro) gene inserted into the U3 region of the long terminal repeat and a second selectable marker, neomycin phosphotransferase (neo), expressed from an internal promoter. CHO clones containing integrated proviruses were selected with hygromycin or G418, and the subset of these with reduced cell surface Neu5Ac were then selected with wheat germ agglutinin (WGA). The majority of the resulting clones had a phenotype not previously described for WGA-resistant CHO mutants arising spontaneously or from chemical mutagenesis: Neu5Ac was almost completely replaced by Neu5Gc. We have provisionally termed these clones SAP mutants, for sialic acid phenotype. Southern analysis of HindIII digested DNA from four SAP mutants revealed that the MoMuLV provirus is present in a 10.4-kilobase (kb) fragment. Probing with a flanking CHO sequence resulted in equivalent hybridization to a 4.6-kb fragment and the 10.4-kb provirus-containing fragment in all four cases, while uninfected parental cells and non-SAP glycosylation mutants generated in the same retrovirus insertional mutagenesis experiments yielded only the 4.6-kb fragment. Sequencing of the 3'-flanking DNA revealed that each of the four SAP mutants had a unique provirus integration site falling within a 796 bp region of the CHO genome. The frequency with which SAP mutants arise suggests that this may be a preferred site for retrovirus integration.

Animals↗
PubMed1998

Proteasomes can either generate or destroy MHC class I epitopes: evidence for nonproteasomal epitope generation in the cytosol.

Proteasomes have been implicated in the production of the majority of peptides that associate with MHC class I molecules. We used two different proteasome inhibitors, the peptide aldehyde N-acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL) and the highly specific inhibitor lactacystin, to examine the role of proteasomes in generating peptide epitopes associated with HLA-A*0201. Neither LLnL nor lactacystin was able to completely block the expression of the HLA-A*0201. Furthermore, the effects of LLnL and lactacystin on the expression of different categories of specific epitopes, TAP independent vs TAP dependent and derived from either cytosolic or membrane proteins, were assessed. As predicted, presentation of two TAP-dependent epitopes was blocked by LLnL and lactacystin, while a TAP-independent epitope that is processed in the endoplasmic reticulum was unaffected by either inhibitor. Surprisingly, both LLnL and lactacystin increased rather than inhibited the expression of a cytosolically transcribed and TAP-dependent peptide from the influenza A virus M1 protein. Mass spectrometric analyses of in vitro proteasome digests of a synthetic 24 mer containing this epitope revealed no digestion products of any length that included the intact epitope. Instead, the major species resulted from cleavage sites within the epitope. Although cleavage at these sites was inhibitable by LLnL and lactacystin, epitope-containing species were still not produced. We conclude that proteasomes may in some cases actually destroy epitopes that would otherwise be destined for presentation by class I molecules. These results suggest that some epitopes are generated by nonproteasomal proteases in the cytosol.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

MCM generator: a Java-based tool for generating medical metadata.

In a previous paper we introduced the need to implement a mechanism to facilitate the discovery of relevant Web medical documents. We maintained that the use of META tags, specifically ones that define the medical subject and resource type of a document, help towards this goal. We have now developed a tool to facilitate the generation of these tags for the authors of medical documents. Written entirely in Java, this tool makes use of the SAPHIRE server, and helps the author identify the Medical Subject Heading terms that most appropriately describe the subject of the document. Furthermore, it allows the author to generate metadata tags for the 15 elements that the Dublin Core considers as core elements in the description of a document. This paper describes the use of this tool in the cataloguing of Web and non-Web medical documents, such as images, movie, and sound files.

Cataloging↗
PubMed1997

Presentation of tetanus toxoid to autologous T cells by dendritic cells generated from human blood. Improved specificity with dendritic cells generated without fetal calf serum.

Dendritic cells (DC) are highly specialised to initiate primary immune responses and may therefore serve as natural adjuvant in future strategies for specific immunotherapy, e. g. with tumor antigens. The originally developed culture system to generate DC from peripheral human blood with GM-CSF and IL-4 was dependent on the use of fetal calf serum. We employed such DC as antigen presenting cells in a modified lymphocyte proliferation assay to measure the response of autologous T cells to tetanus toxoid. However, a substantial proliferative response of T cells was also observed in control wells without antigen, i.e. in the setting of a syngeneic mixed leukocyte reaction. This makes it difficult, if not impossible, to monitor antigen-specific responses in vitro. In a recently developed improved method fetal calf serum was replaced by 1% autologous human plasma. Using such DC in our lymphocyte proliferation assay background proliferation was markedly reduced. T cell responses to tetanus toxoid were strongest when the antigen was added to DC three days before cocultivation with T cells. We conclude that DC cultured in FCS-free autologous systems, suitable for clinical use, can process and present tetanus protein to autologous T cells. Using such DC in a lymphocyte proliferation assay may facilitate the measurement of antigen-specific T cell responses.

Animals↗
PubMed1991

Adsorption and expression of penicillin G acylase immobilized onto methacrylate polymers generated with varying pore generating solvent volume.

Adsorption and expression of penicillin G acylase was studied on macroporous methacrylate polymer beads of differing pore volume, generated with kerosene. The absorption and expression of the penicillin G acylase was dependent on pore volume. Maximum expression of 57% of adsorbed enzyme was obtained on beads synthesized with 40 mL of kerosene, indicating minimum pore-diffusion limitations.

Enzymes, Immobilized↗
PubMed1983

Two late response components in on-off ganglion cells of the frog retina: the delayed response-generated by red rods; the second off-response-generated by green rods.

Frog on-off retinal ganglion cells react to diffuse, large-sized stimuli not only with an "early response" and a "delayed response" but also with a "second off-response". The intensity at which the delayed response becomes visible coincides with the quantum amount necessary for saturating the red rod response. The spectral sensitivity of the delayed response fits the nomogram for the 502 nm pigment. Unlike the delayed response, the second off-response appears earlier with increasing stimulus intensity and follows the 433 nm nomogram. Therefore, it is most probably generated by the green rods.

Animals↗
PubMedDate not supplied

Size calculation of restriction enzyme HaeIII-generated fragments detected by probe YNH24 by comparison of data from two laboratories: the generation of fragment-size frequencies.

Restriction fragment-length polymorphism of locus D2S44 detected by the highly polymorphic probe YNH24 and restriction endonuclease HaeIII can be used to improve parentage testing when representative fragment-size frequencies can be obtained. By joining the results of different laboratories, it is possible to set up a meaningful databank. Therefore, the same randomly chosen samples were tested for the HaeIII RFLP detected by probe YNH24 in Düsseldorf (DUS) and Amsterdam (AMS). The results of the different fragment-size calculations obtained by using internal markers and a computerized system (DUS-cad and AMS-cad), and by using external markers and manual calculations (DUS-man), were analyzed. Comparing these results, no statistically significant differences were seen. The results obtained with probe YNH24 and enzyme HaeIII in Düsseldorf and Amsterdam can be used to attain a sufficient number of samples to generate relevant fragment-size frequencies.

DNA↗
PubMed2005

Are generalised scalar implicatures generated by default? An on-line investigation into the role of context in generating pragmatic inferences.

Recent research in semantics and pragmatics has revived the debate about whether there are two cognitively distinct categories of conversational implicatures: generalised and particularised. Generalised conversational implicatures are so-called because they seem to arise more or less independently of contextual support. Particularised implicatures are more context-bound. The Default view is that generalised implicatures are default inferences and that their computation is relatively autonomous--being computed by some default mechanism and only being open to cancellation at a second stage when contextual assumptions are taken into consideration (i.a.). It is at that second stage where contextual assumptions are considered that particularised implications are computed. By contrast, Context-Driven theorists claim that both generalised and particularised implicatures are generated by the same process and only where there is contextual support (Chierchia, 2004; Horn, 1984; Levinson, 2000 i.a.). In this paper, we present three on-line studies of the prototypical cases of generalised implicatures: the scalar implicatures 'some of the Fs' > 'not all the Fs' and 'X or Y' > 'either X or Y but not both'. These studies were designed to test the context-dependence and autonomy of the implicatures. Our results suggest that these scalar implicatures are dependent on the conversational context and that they show none of the autonomy predicted by the Default view. We conclude with a discussion of the degree to which such implicatures are purely context-driven and whether an interactionist default position may also be plausible.

Adult↗
PubMed2004

Generation of active fragments from human zymogens in the bradykinin-generating cascade by extracellular proteases from Vibrio vulnificus and V. parahaemolyticus.

Vibrio vulnificus is an opportunistic human pathogen causing septicemia, and the infection is characterized by formation of the edematous skin lesions on limbs. This pathogenic species secretes a thermolysin-like metalloprotease as a virulence determinant. The metalloprotease was confirmed to activate human factor XII-plasma kallikrein-kinin cascade that results in liberation of bradykinin, a chemical mediator enhancing the vascular permeability, from high-molecular weight kininogen. Namely, the metalloprotease showed to generate active fragments by cleavage of Arg-Ile, Arg-Val or Gly-Leu peptide bond in human zymogens (plasma prekallikrein and factor XII). In spite of induction of the sufficient vascular permeability-enhancing and edema-forming reaction in the guinea pig model, a serine protease from V. parahaemolyticus, a human pathogen causing primarily watery diarrhea, showed far less ability to activate and to cleave the human zymogens. These results in part may explain why only V. vulnificus often causes serious edematous skin damages in humans.

Amino Acid Sequence↗
PubMed1988

Associative neural network model for the generation of temporal patterns. Theory and application to central pattern generators.

Cyclic patterns of motor neuron activity are involved in the production of many rhythmic movements, such as walking, swimming, and scratching. These movements are controlled by neural circuits referred to as central pattern generators (CPGs). Some of these circuits function in the absence of both internal pacemakers and external feedback. We describe an associative neural network model whose dynamic behavior is similar to that of CPGs. The theory predicts the strength of all possible connections between pairs of neurons on the basis of the outputs of the CPG. It also allows the mean operating levels of the neurons to be deduced from the measured synaptic strengths between the pairs of neurons. We apply our theory to the CPG controlling escape swimming in the mollusk Tritonia diomedea. The basic rhythmic behavior is shown to be consistent with a simplified model that approximates neurons as threshold units and slow synaptic responses as elementary time delays. The model we describe may have relevance to other fixed action behaviors, as well as to the learning, recall, and recognition of temporally ordered information.

Animals↗
PubMed1975

The triple pulse generator: Clinical evaluation of a new multipurpose external pulse generator.

This report describes our experience with a new, versatile battery operated, multi-purpose pulse generator especially designed for the electrophysiological investigation and treatment of cardiac arrhythmias in man. The unit was constructed according to our specifications and has been clinically in 75 patients over a period of 18 months. We have found the triple pulse pacemaker safe, reliable and functioning precisely according to specifications both clinically and when repeatedly checked with a storage oscilloscope.

Arrhythmias, Cardiac↗
PubMed2003

Indium-mediated beta-allylation, beta-propargylation, and beta-allenylation onto alpha,beta-unsaturated ketones: reactions of in-situ-generated 3-tert-butyldimethylsilyloxyalk-2-enylsulfonium salts with in-situ-generated organoindium reagents.

3-tert-Butyldimethylsilyloxyalk-2-enylsulfonium salts, generated in situ from the reaction of alpha,beta-enones with dimethyl sulfide in the presence of TBSOTf, underwent a novel nucleophilic substitution with allylindiums to give silyl enol ethers of delta,epsilon-alkenyl ketones in good yields, which correspond to formal Michael addition products. In a similar manner, 1,4-propargylation of propargylindiums onto the sulfonium salts produced the corresponding silyl enol ethers of delta,epsilon-alkynyl ketones in good yields. Organoindium reagents derived from gamma-substituted propargyl bromide and indium afforded the corresponding silyl enol ethers of beta-allenyl ketones in good yields. The reaction proceeds via an addition-substitution mechanism involving the formation of allylic sulfonium salts. The presence of the intermediate sulfonium salt was confirmed by observation of the low-temperature (1)H NMR spectra.

Journal Article↗
PubMed2004

Arrays of horizontally-oriented mini-reservoirs generate steady microfluidic flows for continuous perfusion cell culture and gradient generation.

This paper describes the use of arrays of horizontally-oriented reservoirs to deliver liquids through microchannels at a constant flow rate over extended periods of time (hours to days). The horizontal orientation maintains a constant hydraulic pressure drop across microfluidic channels even as the volumes of liquids within the reservoirs change over time. For a given channel-reservoir system, the magnitude of the flow velocity depends linearly on the height difference between reservoirs. The simple structure and operation mechanism make this pumping system versatile. A one-inlet-one-outlet system was used to continuously deliver media for perfusion cell culture, and an array of inlet reservoirs coupled to an outlet reservoir via microchannels was used to drive flows of multiple laminar streams. The parallel pumping scheme conveniently generated various smooth and step concentration gradients, and allowed evaluation of the effect of colchicine on myoblasts. Since the reservoir arrays are configured to be compatible with commercialized multichannel pipettors designed for 96 well plate handling, this simple pumping scheme is envisioned to be broadly useful for medium to high throughput microfluidic perfusion cell culture assays, cell migration assays, multiple laminar flow drug tests, and any other applications needing multiple microfluidic streams.

Cell Culture Techniques↗
PubMed1998

A study of the prevalence of hydrogen peroxide generating Lactobacilli in bacterial vaginosis: the determination of H2O2 concentrations generated, in vitro , by isolated strains and the levels found in vaginal secretions of women with and without infection.

The concentration of hydrogen peroxide in the vaginal secretions of normal women were compared with levels found in women infected with bacterial vaginosis (BV). The mean concentration found in 90 normal samples was 0.171 microg per ml, whereas in women with BV, the mean level was 0.04 microg per ml (P < 0.001). In vitro culture experiments using TMB agar showed that in normal women, 75% of Lactobacillus strains produced H2O2 and 25% did not, whereas in women 'infected' with BV. 14% of the isolates produced H2O2 and 86% did not (P < 0.001). Quantitative culture tests showed that both anaerobic and facultative strains of Lactobacilli isolated from normal women generated significantly higher levels of H2O2 than strains cultured from women with BV. Anaerobic strains isolated from normal women produced a mean level of 6.8 microg per ml, compared with the mean level of 1.4 microg per ml from women with BV. Facultative strains from normal women produced mean levels of 18.3 microg per ml compared with 2.4 microg per ml produced by strains from BV-infected women, which had been incubated under the same conditions (P < 0.001). There results indicate that Lactobacilli which produce H2O2 probably have a significant role in protecting the vaginal ecosystem from BV infection.

Journal Article↗
PubMed2003

Student generated learning objectives: extent of congruence with faculty set objectives and factors influencing their generation.

CONTEXT: A problem-based learning strategy is used for curriculum planning and implementation at the Arabian Gulf University, Bahrain. Problems are constructed in a way that faculty-set objectives are expected to be identified by students during tutorials. Students in small groups, along with a tutor functioning as a facilitator, identify learning issues and define their learning objectives. PURPOSE: We compared objectives identified by student groups with faculty-set objectives to determine extent of congruence, and identified factors that influenced students' ability at identifying faculty-set objectives. METHOD: Male and female students were segregated and randomly grouped. A faculty tutor was allocated for each group. This study was based on 13 problems given to entry-level medical students. Pooled objectives of these problems were classified into four categories: structural, functional, clinical and psychosocial. Univariate analysis of variance was used for comparison, and a p > 0.05 was considered significant. RESULTS: The mean of overall objectives generated by the students was 54.2%, for each problem. Students identified psychosocial learning objectives more readily than structural ones. Female students identified more psychosocial objectives, whereas male students identified more of structural objectives. Tutor characteristics such as medical/non-medical background, and the years of teaching were correlated with categories of learning issues identified. CONCLUSION: Students identify part of the faculty-set learning objectives during tutorials with a faculty tutor acting as a facilitator. Students' gender influences types of learning issues identified. Content expertise of tutors does not influence identification of learning needs by students.

Bahrain↗
PubMed1981

Capacity of genetically different T lymphocytes to induce lethal graft-versus-host disease correlates with their capacity to generate suppression but not with their capacity to generate anti-F1 killer cells. A non-H-2 locus determines the inability to induce lethal graft-versus-host disease.

When comparing, in a murine model, the kind of graft-versus-host (GVH) disease (GVHD) induced by the donor strain DBA/2 on the one hand and several H-2-congenic resistant B10 donor strains on the other, we found that strain DBA/2 was a universal nonkilling GVH donor for H-2-incompatible nonirradiated F1 hybrid recipients. In this respect, DBA/2 T cells differed from those of the H-2-identical donor strain B10.D2 as well as those of other b10 donor strains. The inability of strain DBA/2 to kill by GVH reaction was not limited to certain H-2 incompatibilities in the F1 recipients, but was nonspecific. The inability to kill is determined by a dominant locus not linked to H-2. DBA/2 T cells were also incapable of inducing the severe suppression of hematocrit values, bone marrow erythropoiesis, thymic cell proliferation, and splenic IgG production in the F1 recipients that was observed after the injection of T cell from the B10 strains. However, DBA/2 T cells, in contrast with those of the B10 donor strains, were vigorous stimulators of IgG production in H-2-incompatible F1 hybrid recipients. Surprisingly, strain DBA/2 as well as the B10 donor strains had good capacity to generate anti-F1 TK cells. Taken together, these findings raise the possibility that lethal GVHD disease is not caused, or not caused exclusively, by donor killer T cells, but by those donor T cells that directly or indirectly induce a suppression of cell proliferation in certain vital organs of the recipient.

Animals↗
PubMed2003

Electric and magnetic field generation and target heating by laser-generated fast electrons.

The electric and magnetic fields generated by a beam of fast electrons in a conductor are calculated analytically, including the change in resistivity due to Ohmic heating. It is assumed that the resistivity has an arbitrary power law dependence on temperature, the fast electron current density is fixed (rigid beam), charge neutralization is instantaneous, and that magnetic diffusion is negligible. The implications for high-intensity laser-solid interactions are discussed. The minimum fast electron density for fast ignition by Ohmic heating is given, and found to be unrealistically high.

Journal Article↗