Open letter plea for financial assistance.
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Education, Medical↗
Biomedical subjects
J H Robinson
Publications and source records attributed to J H Robinson.
A limiting dilution assay to determine the frequency of antigen presenting cells.
A limiting dilution assay to measure the functional frequency of antigen presenting cells (APC) has been developed and used to measure the frequency of mouse lymphoid cells which stimulated the mixed leucocyte response. The experiments utilised measurements of proliferation in 20 microliter hanging drop microcultures of activated T lymphocytes. When the data supported a model of single-hit kinetics, the APC frequency in the titrated stimulating cell population was calculated by the method of maximum likelihood. The assay was validated by measuring the frequency of unpurified spleen cells and enriched spleen dendritic cells which presented alloantigens in mixed leucocyte culture. APC frequency in unpurified spleen cells was 1:7254 (SE +/- 2134, 8 experiments) and was increased markedly to 1:15 (SE +/- 7, 7 experiments) when enriched populations consisting 70-95% of dendritic cells were used as stimulators.
Animals↗
Delayed type hypersensitivity responses to the Qa-Tla region of the mouse major histocompatibility complex.
We have elicited a small but significant delayed-type hypersensitivity response against the Qa-Tla region of H-2 following immunisation of C57BL/6 mice with cells from the congenic strain C57BL/6.Tlaa. Optimal responses were detected 14 days after immunisation with 10-40 X 10(6) cells. The significance of delayed type hypersensitivity to Qa-Tla is discussed in relation to the genetic loci which map to this region.
Animals↗
Large scale, rapid purification of recombinant tissue-type plasminogen activator.
Recombinant tissue-type plasminogen activator (rt-PA) from cultures of a genetically manipulated Bowes melanoma cell line (TRBM6) was purified in batches of average volume 451 using an autoclavable, reusable, continuous chromatography system comprising zinc chelate-Sepharose CL4B and lysine-Sepharose CL4B. After eight successive purifications the rt-PA was ultrafiltered to yield a preparation containing 4.9 mg protein/ml and 2.7 X 10(6) IU/ml. Analysis by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue R250 showed major protein bands at Mr = 63,000 and 65,000; most of the material was in the 1-chain form. The potential usefulness of a simple, rapid continuous chromatography system that can be operated under aseptic conditions is discussed.
Animals↗
Isolation and preliminary characterisation of active B-chain of recombinant tissue-type plasminogen activator.
Purified 2-chain recombinant tissue-type plasminogen activator (t-PA) was reduced under mild conditions - 10 mM dithiothreitol/5 degrees C/1.5 h - and the two chains were separated by chromatography on lysine Sepharose. The t-PA B chain was fully active as determined by its activity towards the chromogenic substrate S-2288 (H-D-ile-pro-arg p-nitroanilide). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing or non-reducing conditions revealed a single polypeptide at Mr = 35,000 or 29,000 respectively. In addition, under non-reducing conditions a fibrinolytic band at apparent Mr = 29,000 was present after fibrin zymography. The N-terminal sequence was confirmed as ile-lys-gly. The t-PA B chain had a specific amidolytic activity, using S-2288, of 170,000 to 210,000 SU/mg protein. (This compares to a specific activity of the native 2-chain t-PA of 170,000 SU/mg). It resembles urokinase-type plasminogen activator in its inability to be stimulated by fibrin and its dose response on human fibrin plates. However, t-PA B-chain was stimulated to almost the same extent as t-PA by poly-D-lysine. The isoelectric points, at pH 5.6 and 5.7, fall outside the range generally quoted for t-PA preparations (pH 7.8-8.8).
Cell Line↗
Identification of the calcium-inducible plasminogen activators secreted by a human diploid fibroblast cell line (MRC-5).
Human embryonic lung fibroblast (MRC5) cells secreted small amounts of plasminogen activators into normal, serum-free harvest medium. Stimulation with calcium led to markedly enhanced levels of activator. The major species of plasminogen activator in the harvest medium of the stimulated cultures resembled u-PA when analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by fibrin zymography. This activator was partially-purified using controlled-pore glass and Blue Sepharose CL6B. Characterisation by fibrin zymography in the presence of specific antibody, by 3H-DFP labelling and by fibrin-binding ability indicated that the activator was indistinguishable from high molecular weight u-PA. The possible physiological role of the production of relatively large amounts of u-PA by a lung cell capable of producing both u-PA and t-PA is discussed.
Calcium↗
Increased yield of human tissue-type plasminogen activator obtained by means of recombinant DNA technology.
Extra copies of the human tissue-type plasminogen activator (t-PA) gene were introduced into the Bowes melanoma cell line. We obtained a recombinant cell line (TRBM6) which secretes approximately ten-fold more t-PA than the parent cell line. The identity of the plasminogen activator made by the new cell line was confirmed by sizing on sodium dodecyl sulphate polyacrylamide gels and by specific quenching using anti-t-PA antibody. We estimate that the recombinant line produces t-PA at a rate of approximately 3 pg/cell/24 hr and that t-PA accumulates in the harvest medium at a rate of approximately 4000 International t-PA Units/ml/24 hr.
Cell Line↗
Studies on the role of the thymus in the selection and clonal expansion of T-cell precursors.
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Animals↗
Protein synthesis in 3T3 cells stimulated to proliferate at various rates.
Reproducible conditions were defined for using rates of leucine incorporation as a valid measure of rates of de novo protein synthesis in mouse 3T3 cells. Upon stimulation of quiescent cultures, rates of de novo synthesis of proteins increased and pool levels of amino acids decreased in proportion to the concentration of serum in the stimulating medium. Rates of de novo protein synthesis (per cell) exhibited a biphasic pattern of increase. These rates approached a plateau value at the end of the lag phase and increased again as cells entered S phase. This pattern of behaviour helps to explain the observed relationships between cell growth (increase in mass) and cell proliferation (increase in cell number).
Amino Acids↗
Isolation of a human tissue-type plasminogen-activator genomic DNA clone and its expression in mouse L cells.
We have isolated a cDNA clone corresponding to a substantial portion of the human tissue-type plasminogen activator (t-PA) protein. It encodes almost all of the protein B chain and part of the 3' untranslated region. We have used this clone to screen bacteriophage lambda and cosmid libraries of human genomic DNA. Several related genomic clones were isolated. One of these, a cosmid clone, carried approx. 40 kb of human DNA. Mapping experiments indicate that the region containing the protein-coding exons is approx. 20 kb in length. The cosmid, containing the t-PA gene and the aminoglycosyl-3'-phosphotransferase dominant-selection marker, was introduced into mouse L cells. Approximately half of the transformants were shown to produce human t-PA. We demonstrated that the fibrinolytic t-PA activity could be specifically quenched by anti-t-PA antibody and that the recombinant t-PA was of similar size (by SDS-polyacrylamide gel electrophoresis) to the t-PA produced by the human Bowes melanoma cell line. Our results suggest that the cosmid clone carries the whole t-PA coding region together with the regulatory elements necessary for its expression.
Amino Acid Sequence↗
The ontogeny of thymic macrophages: thymic macrophages express Ia from 15 days gestation onwards in the mouse.
This study reports the isolation and enrichment of murine foetal thymic macrophages. The majority of enriched adherent cells of thymus from 15 days gestation onwards were classical macrophages as judged by phagocytosis as well as expression of F4.80 and Fc receptors. In addition, a proportion of foetal thymic macrophages was shown to be Ia+ by double labelling with latex particles and indirect immunofluorescence for surface Ia antigens, and using monoclonal antibodies, it was shown that the Ia+ cells expressed both I-A- and I-E-region-coded determinants. The function of Ia+ macrophages in the developing thymus is discussed.
Animals↗
The ontogeny of antigen-presenting cells in fetal thymus evaluated by MLR stimulation.
This study demonstrates that cells with the characteristics of antigen-presenting cells (APC) are present in murine thymus from as early as 14 days gestation, around the time that T lymphopoiesis is initiated in the embryo. Thymic APC were identified as la+ adherent cells capable of presenting alloantigens to T cells in MLC. The possible role of these cells in thymocyte differentiation is discussed.
Animals↗
Evoked potentials in the dentate gyrus reflect the retention of past sensory events.
Averaged sensory evoked potentials were recorded from the outer molecular layer of the dentate gyrus (OM AEP) in freely moving rats trained to criterion performance in a two-tone differential discrimination paradigm. OM AEPs showed consistent fluctuations in amplitude based on the acquired significance of the tone through their association with reward (CS+) or non-reward (CS-). However, the relationship between tone significance and OM AEP configuration was dependent upon the sequence of trials preceding the evoked potential, irrespective of whether the potential was evoked by a CS+ or CS-. Specifically, when the preceding sequence contained an increased number of CS- trials, the amplitude of the initial negative component (N1) of the OM AEP was increased, while that of the slower (N2) component was reduced; conversely, when the preceding sequence contained an increased number of CS+ trials, the amplitude of N1 was reduced, while that of N2 was increased. The results suggest that the transmission of sensory information into the dentate gyrus is biased by prior experience.
Animals↗
Morphological assessment of immunologically relevant cells in the thymus.
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Animals↗
Differences in opiate-induced synaptic excitability of hippocampal slices prepared from tolerant and nontolerant rats.
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Animals↗
Intracellular correlates of morphine excitation in the hippocampal slice preparation.
The influences of morphine and opioid peptides on hippocampal CA1 pyramidal cells were investigated using intracellular recordings from the in vitro slice preparation. Morphine applied to somal and basal dendritic areas of CA1 cells via a pressure ejection system confirmed a number of excitatory actions of opiates and opioid peptides in this brain region. These included an increase in the amplitude and duration of orthodromically (radiatum) elicited EPSPs and a decrease in amplitude of the following IPSP. The increase in EPSP amplitude was accompanied by a reduction in stimulus intensity necessary for eliciting the action potential. Morphine delivered to the slice in this manner induced synaptically elicited and spontaneous multiple spike burst discharges. In slices maintained in 10(-4) M pentobarbital, morphine reversed the presumably GABA mediated long-duration depolarization following orthodromic stimulation. Finally, depending on the specific site of application (apical or basal dendritic region) of the opiate, morphine produced two different effects on the resting membrane potential and input resistance of CA1 pyramidal cells. These findings are discussed as to whether opiates act directly upon excitatory influences in the hippocampus, or via blockade of GABA mediated inhibitory mechanisms.
Action Potentials↗
Kainic acid produces depolarization of CA3 pyramidal cells in the vitro hippocampal slice.
Kainic acid (KA) (10(-6)-10(-8) M) reversibly depolarized CA3 pyramidal cells when applied topically to the apical dendritic area of these cells in the hippocampal slice. The magnitude of membrane depolarization and the time to recovery of resting membrane potential were concentration-related. Application of 10(-5) M KA produced complete membrane depolarization which did not recover in baseline levels. Unlike CA3 neurons cells from the CA1 region were unaffected by KA (10(-6)-10(-8) M). However, 10(-5) M KA also proved effective in depolarizing CA1 cells.
Animals↗
Entorhinal and septal inputs differentially control sensory-evoked responses in the rat dentate gyrus.
Averaged sensory-evoked potentials were recorded from the outer molecular layer of the dentate gyrus in naïve rats and in rats conditioned to respond in the presence of an auditory stimulus. Two components (negative peaks) of the potentials were functionally distinguished in terms of responsiveness to unique or conditioned auditory stimuli. Each component was independently generated by a separate input pathway to the dentate gyrus: The perforant path provided an "insignificance" or "unexpected" feature of the sensory stimulus when appropriate, and the septum controlled the development of a second component as a function of the behavioral significance of the stimulus during the acquisition of auditory discrimination behavior. A reciprocal relationship between the peak amplitudes of both components of the average evoked potentials dependent on the relative behavioral significance of the sensory stimulus was observed in all animals during extinction and reconditioning of the sensory discrimination task. The findings indicate that the entorhinal and septal projections to the dentate granule cells are activated differentially by sensory stimuli as a function of their acquired behavioral significance to the animal.
Animals↗