Biomedical subjects

D A Brown

Publications and source records attributed to D A Brown.

At least 19 recordsLinked to original sources

PubMed1992

Ablation of transplanted HTLV-I Tax-transformed tumors in mice by antisense inhibition of NF-kappa B.

Mice transgenic for the human T cell leukemia virus (HTLV-I) Tax gene develop fibroblastic tumors that express NF-kappa B-inducible early genes. In vitro inhibition of NF-kappa B expression by antisense oligodeoxynucleotides (ODNs) inhibited growth of these culture-adapted Tax-transformed fibroblasts as well as an HTLV-I-transformed human lymphocyte line. In contrast, antisense inhibition of Tax itself had no apparent effect on cell growth. Mice treated with antisense to NF-kappa B ODNs showed rapid regression of transplanted fibrosarcomas. This suggests that NF-kappa B expression may be necessary for the maintenance of the malignant phenotype and provides a therapeutic approach for HTLV-I-associated disease.

3T3 Cells↗

PubMed1992

Closure of potassium M-channels by muscarinic acetylcholine-receptor stimulants requires a diffusible messenger.

The M-current (IK(M)) is a slow voltage-gated K+ current which can be inhibited by muscarinic acetylcholine-receptor (mAChR) agonists. In the present experiments we have tested whether this inhibition results from a local (membrane-delimited) interaction between the receptor and adjacent channels, or whether channel closure is mediated by a diffusible messenger. To do this, single KM(+)-channel currents were recorded from membrane patches in dissociated rat superior cervical sympathetic neurons by using cell-attached patch electrodes. Channel activity was inhibited when muscarine was applied to the cell membrane outside the patch but persisted when channels were exposed to muscarine added to the pipette solution. We conclude that a diffusible molecule (or molecules) is (are) required to induce intrapatch channel closure following activation of extra-patch receptors.

Animals↗

PubMed1992

Neurotransmitter modulation of calcium channels is dependent on the charge carrier used in the recording of currents.

Currents through calcium channels were recorded using calcium, barium and strontium as charge carriers in NG108-15 cells. The mean normalised peak current amplitude at 0 mV was not significantly different between the charge carriers; however, the sustained component (measured at the end of the 500 ms command step) was ca. 3 times larger in barium and strontium. Further, the inhibition by acetylcholine or noradrenaline, although the same at the peak of the current envelope, was significantly greater on the sustained portion of the current for barium and strontium. Increasing internal calcium-buffering (to reduce calcium-dependent inactivation with calcium as the charge carrier) did not increase the amount of inhibition of the sustained portion of current. These results suggest a cautious approach to analysis of neurotransmitter modulation of calcium currents using other charge carriers than calcium.

Acetylcholine↗

PubMed1992

Synapse formation between dissociated basal forebrain neurones and hippocampal cells in culture.

Dissociated neurones from rat basal forebrain and hippocampus were co-cultured in vitro for 8-15 days. Patch-clamp recordings from individual presumed hippocampal pyramidal cells revealed synaptic currents following focal extracellular stimulation of single presumed basal forebrain neurones. Of 18 neurone pairs, 13 showed inward (excitatory) synaptic currents, 4 showed outward (inhibitory) synaptic currents, and 1 showed a mixed current. Latencies varied from 4 to 15 ms, suggesting both mono- and polysynaptic currents. These experiments indicate that synaptic connections can be established between basal forebrain and hippocampal cells in dissociated cell culture.

Animals↗

PubMed1992

Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface.

We show that a protein with a glycosylphosphatidyl inositol (GPI) anchor can be recovered from lysates of epithelial cells in a low density, detergent-insoluble form. Under these conditions, the protein is associated with detergent-resistant sheets and vesicles that contain other GPI-anchored proteins and are enriched in glycosphingolipids, but do not contain a basolateral marker protein. The protein is recovered in this complex only after it has been transported to the Golgi complex, suggesting that protein-sphingolipid microdomains form in the Golgi apparatus and plasma membrane and supporting the model proposed by Simons and colleagues for sorting of certain membrane proteins to the apical surface after intracellular association with glycosphingolipids.

Acid Phosphatase↗

PubMed1992

Characterization of nuclear protein binding to the interferon-gamma promoter in quiescent and activated human T cells.

Nuclear protein binding to the human interferon-gamma (IFN-gamma) promoter was investigated to determine the structural basis for the control of gene expression during T cell activation. DNase I footprinting of gel-shift complexes demonstrated that proteins bind to two downstream (-124 to -114 and -36 to -30) and one upstream (-534 to -486) element in the IFN-gamma gene promoter. Treatment of human peripheral blood lymphocytes or continuous T cell tumors with phorbol 12-myristate 13-acetate (PMA) plus phytohemagglutinin or calcium ionophore results in a pattern of response that is similar when using either the upstream or downstream elements. Upon induction of T cells, the lower mobility gel-shift band disappears. Yet the equivalent band which is also present in non-T cells is unperturbed after PMA + calcium ionophore treatment. The higher mobility band which is modified upon induction is restricted to the T cell lineage. Upstream and downstream elements share similar protein-binding motifs as indicated by the homology of footprinted sequences, the similarity of protein-binding patterns, and the ability of these elements to compete against each other in gel-shift protein-binding assays. Protein binding to the downstream elements appears to be interactive, since both sites are required for complex formation. When either of the two downstream elements is disrupted by site-directed mutagenesis, the higher mobility gel-shift band is diminished by an amount that is consistent with the reduction in reporter (chloramphenicol acetyltransferase) gene expression. Therefore, proteins in the ubiquitous gel-shift band appear to be associated with the inactive state of IFN-gamma, while the modified band is closely associated with the positive regulation of IFN-gamma gene expression.

Base Sequence↗

PubMed1992

Intracellular Mg2+ inhibits the IP3-activated IK(Ca) in NG108-15 cells. [Why intracellular citrate can be useful for recording IK(Ca)].

Receptor-mediated formation of inositol 1,4,5-trisphosphate (IP3) can induce an outward Ca(2+)-activated K+ current [IK(Ca)] in some neural cells. We have investigated IK(Ca) activated by intracellular injections of IP3 in whole-cell patch-clamped neuroblastoma x glioma hybrid cells. The current could only be recorded reliably using citrate as the anion in the pipette, but not using acetate, aspartate, chloride, fluoride, gluconate or methylsulphate. This could be attributed to buffering of intracellular Mg2+ by citrate. Theoretical calculations suggested free [Mg2+] of 1.0 and 0.07 mM respectively in the acetate- and citrate-based recording solutions. Further, IP3-activated IK(Ca) could be recorded when the free Mg2+ level in the acetate, chloride or methylsulphate solutions was lowered to the range (0.05 mM) calculated for the citrate solution. Thus, raised [Mg2+] blocks IK(Ca). This appeared to be due to inhibition of the response to released Ca2+, since high [Mg2+] also blocked the response to intracellular injections of Ca2+ ions. Mean Mg2+ levels in intact neuroblastoma x glioma hybrid cells measured by Mag-Indo-1/AM fluorescence were estimated to be less than 0.14 mM. We therefore conclude that IP3-induced IK(Ca) is expressed under normal conditions, but may be subject to regulation by intracellular Mg2+.

Anions↗

PubMed1992

Neurotransmitters inhibit the omega-conotoxin-sensitive component of Ca current in neuroblastoma x glioma hybrid (NG 108-15) cells, not the nifedipine-sensitive component.

Voltage-dependent calcium currents (ICa) in NG 108-15 cells consisted of three pharmacologically distinct components: a transient low-voltage-activated (LVA) current, sensitive to Ni2+; a high-voltage-activated (HVA) current sensitive to the dihydropyridine antagonist, nifedipine and a HVA current sensitive to omega-conotoxin GVIA (CgTx). The voltage sensitivities and decay kinetics of the two HVA currents were indistinguishable. The neurotransmitters acetylcholine (ACh) and noradrenaline inhibited ICa. This inhibition was not occluded by Ni2+ or nifedipine, but was abolished by CgTx. It is therefore concluded that the neurotransmitter-sensitive component of ICa is restricted to that component of HVA current inhibitable by omega-conotoxin.

Acetylcholine↗

PubMed1992

Goal attainment scaling as a measure of change in infants with motor delays.

Goal attainment scaling (GAS) is an individualized criterion-referenced measure of change that has several unique features, compared with the behavioral objective, and five possible levels of attainment for each goal. The validity of GAS as a measure of motor change was investigated in 65 infants, 3 to 30 months of age, with motor delays. For each infant, two goals to be attained within a 6-month period of intervention were established. After the 6-month period, the mean GAS T-score of 55.4 was significantly higher than the expected mean of 50. This finding indicated that the infants' motor change exceeded the therapists' expectations. Neither the type nor the category of goal influenced the therapists' ability to select outcomes that the infants were capable of achieving. Because moderate and low correlations were found between GAS T-scores and Peabody gross motor and fine motor change scores, the two assessments appear to measure different aspects of motor development. Selected child, family, and program variables were examined as sources of bias in GAS T-scores. Motor delay was the only variable that biased GAS T-scores; infants who were less delayed had higher GAS T-scores. The results indicate that GAS was responsive to change in individualized motor goals and support the model of GAS as an idiosyncratic measure. The unique features of GAS offer advantages for measurement of motor change compared with the behavioral objective and norm-referenced developmental scales.

Bias↗

PubMed1992

Long-term stability of hand-held dynamometric measurements in children who have myelomeningocele.

The long-term stability of hand-held dynamometric measurements was assessed in 30 muscle groups of 12 children with myelomeningocele, before and after a 23-day interval. Measurements from a majority of the muscle groups had excellent stability, based on statistical indicators of association (Pearson Product-Moment Correlation Coefficients, r =.76-.98) and agreement (intraclass correlation coefficients, ICC = .75-.99). Muscle groups with lower long-term stability were the right and left wrist extensors and flexors, the left hip adductors and extensors, the left knee flexors, and the right and left knee extensors. Upper-extremity muscle groups had higher long-term stability than did lower-extremity muscle groups. The results indicate that the dynamometric measurements were highly reliable when the test-retest interval was 23 days. Other researchers have previously shown high reliability for these measurements over shorter periods of time. Improved reliability might be obtained by supporting the lower extremity during hip extension tests; padding the dynamometer end pieces, especially when testing over bony prominences; and using a smaller, digital dynamometer. The hand-held dynamometer appears to warrant use and further investigation with pediatric populations.

Adolescent↗

PubMed1992

Cannabinoid receptor agonists inhibit Ca current in NG108-15 neuroblastoma cells via a pertussis toxin-sensitive mechanism.

Cannabinoid receptor ligands irreversibly inhibited peak voltage-activated Ca currents (44%) in NG108-15 cells; this inhibition was Pertussis toxin-sensitive. Inhibition was largely due to a reduction in the omega-conotoxin sensitive portion of high-voltage activated (HVA) current, although there was also a significant decrease in low-voltage activated current (56%) and in the nifedipine-sensitive portion of HVA current (41%).

Analgesics↗

PubMed1992

Kinetic and pharmacological properties of the M-current in rodent neuroblastoma x glioma hybrid cells.

1. The M-like current IK(M,ng) in differentiated NG108-15 mouse neuroblastoma x rat glioma hybrid cells has been studied using tight-seal, whole-cell patch-clamp recording. 2. When calculated from steady-state current-voltage curves, the conductance underlying IK(M,ng) showed a Boltzmann dependence on voltage with half-activation voltage Vo = -44 mV (in 3 mM [K+]) and slope factor (a) = 8.1 mV/e-fold increase in conductance. In 12 mM [K+] Vo = -38 mV and a = 6.9 mV. The deactivation reciprocal time constant accelerated with hyperpolarization with slope factor 17 mV/e-fold voltage change. 3. The reversal potential for deactivation tail currents varied with external [K+] as if PNa/PK were 0.005. 4. Steady-state current was increased on removing external Ca2+. In the presence of external Ca2+, reactivation of IK(M, ng) after a hyperpolarizing step was delayed. This delay was preceded by an inward Ca2+ current, and coincided with an increase in intracellular [Ca2+] as measured with Indo-1 fluorescence. Elevation of intracellular [Ca2+] with caffeine also reduced IK(M, ng). 5. IK(M, ng) was inhibited by external divalent cations in decreasing order of potency (mM IC50 in parentheses): Zn2+ (0.011) greater than Cu2+ (0.018) greater than Cd2+ (0.070) greater than Ni2+ (0.44) greater than Ba2+ (0.47) greater than Fe2+ (0.69) greater than Mn2+ (0.86) greater than Co2+ (0.92) greater than Ca2+ (5.6) greater than Mg2+ (16) greater than Sr2+ (33). This was not secondary to inhibition of ICa since: (i) inhibition persisted in Ca(2+)-free solution; (ii) La3+ did not inhibit IK(M, ng) at concentrations which inhibited ICa; and (iii) organic Ca2+ channel blockers were ineffective. Inhibition comprised both depression of the maximum conductance and a positive shift of the activation curve. Addition of Ca2+ (10 microM free [Ca2+]) or Ba2+ (1 mM total [Ba2+]) to the pipette solution did not significantly change IK(M, ng). 6. IK(M, ng) was reduced by 9-amino-1,2,3,4-tetrahydroacridine (IC50 8 microM) and quinine (30 microM) but was insensitive to tetraethylammonium (IC50 greater than 30 mM), 4-aminopyridine (greater than 10 mM), apamin (greater than 3 microM) or dendrotoxin (greater than 100 nM). 7. IK(M, ng) was inhibited by bradykinin (1-10 microM) or angiotensin II (1-10 microM), but not by the following other receptor agonists: acetylcholine (10 mM), muscarine (10 microM), noradrenaline (100 microM), adrenaline (100 microM), dopamine (100 microM), histamine (100 microM), 5-hydroxytryptamine (10 microM), Met-enkephalin (1 microM), glycine (100 microM), gamma-aminobutyric acid (100 microM) or baclofen (500 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1992

Effects of bradykinin on ion conductances in NG108-15 neuroblastoma x glioma hybrid cells recorded with patch-clamp electrodes.

Under whole-cell recording, bradykinin (BK) produced an initial outward membrane current followed by an inward current in voltage-clamped NG108-15 cells. The initial outward current was associated with a rise in intracellular Ca2+ and was accompanied by the opening of Ca(2+)-dependent K(+)-channels recorded with a cell-attached patch electrode. This current was inhibited by intracellular Mg2+. The inward current was associated with inhibition of the voltage-dependent K(+)-current IK(M). These effects accord with those previously observed in microelectrode-impaled cells, with the difference that BK produced much more pronounced and long-lasting desensitization in the patch-clamped cells.

Acetylcholine↗

PubMed1991

Potassium currents contributing to action potential repolarization in dissociated cultured rat superior cervical sympathetic neurones.

Pharmacological blocking agents were used to assess the contributions of different K(+)-currents to spike-repolarization and early spike-afterhyperpolarization recorded in dissociated, tissue-cultured rat superior cervical sympathetic neurones using both patch-clamp and impalement microelectrode techniques. Effects of 4-aminopyridine (4-AP) and tetraethylammonium (TEA), in concentrations which selectively reduced the delayed rectifier current IK(DR) and Ca(2+)-activated K(+)-current IK(Ca, fast), respectively, indicated that IK(DR) made a significant contribution to both spike repolarization and spike afterhyperpolarization under all recording conditions, whereas the contribution of IK(Ca,fast) depended on the level of intracellular Ca(2+)-buffering. No evidence for a significant role for the transient current IK(A) could be adduced in these experiments.

4-Aminopyridine↗

PubMed1991

The naphthalenesulphonamide calmodulin antagonist W7 and its 5-iodo-1-C8 analogue inhibit potassium and calcium currents in NG108-15 neuroblastoma x glioma cells in a manner possibly unrelated to their antagonism of calmodulin.

Patch clamp techniques were used to record voltage-sensitive calcium and potassium currents from NG108-15 cells. N-(6-aminohexyl)-5-chloro-1-naphthalene- sulphonamide (W7), a calmodulin (CaM) antagonist and its more potent (10 times) 5-iodo-1-C8 analogue (J8) inhibited these currents in a dose-dependent manner. The inhibition was not dependent on internal or external Ca2+. W7 was about four times more potent as an inhibitor of the transient potassium current (IC50 = 8 microM) than of the M-current or of the calcium current. J8 was also selective for the potassium currents (IC50 values: transient current 4 microM, M-current 11 microM) compared to the calcium current (IC50 36 microM). It is suggested that the inhibition does not result from an anti-CaM action of the compounds.

Animals↗

PubMed1991

T lymphocyte-synovial fibroblast interactions induced by mycobacterial proteins in rheumatoid arthritis.

An in vitro system was established in which single-cell suspensions of lymphocytes and synovial cells from the joints of patients with rheumatoid arthritis were cultured and produced an outgrowth of an organized inflammatory tissue with an extracellular matrix and capsule. The tissue outgrowth, which had histologic features of pannus, required the addition of mycobacterial antigen and interleukin-2 to the tissue culture medium and was dependent upon the presence of T lymphocytes and their interaction with synovial fibroblasts.

Arthritis, Rheumatoid↗

PubMed1991

The human interferon-gamma gene contains an inducible promoter that can be transactivated by tax I and II.

T cells infected with the HTLV (human T cell leukemia virus)-I and -II retroviruses often express the lymphokine interferon-gamma (IFN-gamma). We sought the molecular explanation for this phenomena by co-transfecting the transactivator genes of these human retroviruses together with 5' flanking sequences of the human IFN-gamma gene linked to a heterologous reporter gene. In the presence of phytohemagglutinin or anti-T cell receptor monoclonal antibodies and phorbol 12-myristate 13-acetate, the IFN-gamma promoter is activated in human T cells, which can be further enhanced by co-transfection of the tax I or II genes. Using Bal-31 deletion analysis, we have found that induction by mitogens can be mapped to a region between -284 to -260 (bp) 5' of the transcriptional start site, which is separable from the sequence(s) further upstream which transmit the synergistic transactivation by tax I or II.

Antibodies, Monoclonal↗

PubMed1991

An objective method for assessing graded electrically evoked afferent activity in humans.

A problem arises in human sensorimotor studies when attempts are made to equate the intensity of electrical stimulation of a peripheral nerve with the amount of afferent activity generated. Results presented here reveal that sural recruitment curves exhibit large session to session differences both within and among subjects. These differences hinder the prediction of afferent activation based solely upon stimulus intensity. An alternative method based upon measurement of the evoked potentials is described for improving on these predictions. Sural nerve stimulation is used to demonstrate this method, but the method should be applicable wherever a peripheral nerve is accessible for electrical stimulation and evoked recordings.

Adult↗