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Biomedical subjects

R Eckert

Publications and source records attributed to R Eckert.

At least 19 recordsLinked to original sources
PubMed1992

High-resolution measurements of gap-junctional conductance during perfusion with anti-connexin antibodies in pairs of cultured mammalian cells.

Antibodies against the main proteins in hepatic gap junctions--connexin-26 and connexin-32--have been used in conjunction with high-resolution patch-clamp techniques to investigate whether a relation exists between connexin type and conductance of single gap-junctional channels. Two different cell lines, BRL cells, derived from rat liver, and FL cells a human amniotic cell line exhibited the same single-channel conductances in double whole-cell recordings, but reacted differently upon dialysis with antibodies. Preliminary results indicate that both cell lines express mainly connexin-43. Thus, in spite of the inhibitory action of anti-cx26 and anti-cx32 antibodies observed, the data question the reliability of these antibodies for the functional characterization of gap-junction proteins in electrophysiological experiments.

Animals↗
PubMed1992

Immunogenicity of recombinant core particles of hepatitis B virus containing epitopes of human immunodeficiency virus 1 core antigen.

A Gag protein segment of human immunodeficiency virus 1 (HIV-1) has been fused to a C terminally truncated core antigen of hepatitis B virus (HBcAg) using an E. coli expression system. Fusion of 90 amino acids of HIV-1 Gag protein to HBcAg still allowed the formation of capsids presenting on their surface epitopes of HIV-1 core protein, whereas fusion of 317, 189, or 100 amino acids of Gag prevented self-assembly of chimeric particles. Mice immunized with recombinant particles emulsified with Freund's complete adjuvant (CFA) or aluminium hydroxide developed high anti-HBcAg titers. However, anti-HIVp24 antibodies were detected only in mice inoculated with immunogen emulsified with CFA.

AIDS Vaccines↗
PubMed1992

Cis-urocanic acid as a mediator of ultraviolet-light-induced immunosuppression.

Treatment of an organism with UVB light or PUVA (8-methoxypsoralen + UVA light) not only leads to alterations in the irradiated skin but also to systemic immunomodulation, due to the release of several chemical mediators of immunosuppression like prostaglandins, acute-phase proteins, IL-1 inhibitor, alpha-melanocyte-stimulating hormone, propiomelanocorticotropin or other cytokines. A recently described mediator is urocanic acid, which is transformed by UV light in the skin from the trans- to the cis-isomer and that exerts a systemic immunomodulatory effect. In our experiments, treatment with PUVA or with cis-urocanic acid prevents the rejection of rat heart allografts in 50% and 40% of cases, respectively. Control grafts are rejected in fewer than 10 days. PUVA treatment of donor leukocytes before transfusion into the prospective recipient inhibits only their sensitizing, not their graft-protecting, effect on subsequent skin grafts in mice. PUVA treatment also prevents acute lethal GVH disease in mice after irradiation with a sublethal dose of x-rays and transfusion of semiallogeneic spleen cells. Treatment of recipient mice with cis-urocanic acid has the same effect. The humoral immune response to sheep erythrocytes is not influenced by cis-urocanic acid. These results demonstrate that PUVA treatment or its chemical mediator, cis-urocanic acid, may be used in transplantation and hematology as naturally occurring immunosuppressive agents, especially for the control and manipulation of GVH leukemia reaction.

Animals↗
PubMed1991

Inhibitory modulation of fast and slow Ca(2+)-currents in neuroblastoma x glioma cells during differentiation.

Mouse neuroblastoma x rat glioma hybrid cells (N x G, 108CC15) were used to study the inhibitory effects of the synthetic opioid D-Ala2-D-Leu5-enkephalin (DADLE), somatostatin, adrenaline-alpha 2 and angiotensin II on voltage-dependent Ca(2+)-currents (ICa) using the patch-clamp technique in the whole-cell configuration mode. The inhibitory effects could be abolished by pretreatment of N x G cells with pertussis toxin or intracellular infusion of GDP beta S indicating an involvement of a pertussis toxin sensitive GTP-binding protein (G-protein), presumably Go. The effect of DADLE, the strongest inhibitor of ICa, was studied during dibutyryl cyclic AMP (dBcAMP) induced differentiation. Using omega-conotoxin GVIA (omega-CTX) and methoxyverapamil (D600) as specific Ca(2+)-channel blockers of the N- and L-type Ca(2+)-channels, it was found that in N x G cells DADLE predominantly induces inhibition of T- and N-type Ca(2+)-channels.

Animals↗
PubMed1991

Inhibition of Langerhans cell ATPase and contact sensitization by lanthanides--role of T-suppressor cells.

Lanthanides are rare earths, elements 55-71 in the periodic table, that are of interest in biologic systems as isomorphic competitors for calcium binding sites. Lanthanides were tested for their inhibitory influence on the Ca++/Mg(++)-dependent ATPase of epidermal langerhans cells in vitro, and on the immunologic function of Langerhans cells in vivo. The trivalent ions of lanthanides, lanthanum, and cerium completely inhibited the ATPase staining of Langerhans cells in vitro. When mice were sensitized with dinitrofluorobenzene on skin sites pretreated with topical lanthanum chloride, and challenged on untreated ear skin, a markedly reduced contact hypersensitivity response was observed. This hyporesponsiveness was found to be antigen specific, and could be passively transferred to naive syngeneic animals recipients by CD4-CD8+ spleen cells. These results suggest that inhibition of the epidermal Langerhans cell surface ATPase by application of topical lanthanum and the induction of antigen-specific immunologic tolerance may be related events.

Adenosine Triphosphatases↗
PubMed1991

Isolation, detection, and amplification of intact mRNA from dermatome strips, epidermal sheets, and sorted epidermal cells.

Three different strategies for isolating RNA from epidermal cells were compared. Starting with dermatome sections frozen or disaggregated epidermal cells purified by fluorescence activated cell sorting (FACS), RNA was isolated with a guanidinium thiocyanate technique. Specific mRNA were detected by Northern blot analysis (involucrin, keratin 5, actin), or by reverse transcription and amplification with the polymerase chain reaction (PCR), using primers specific for keratinocyte products (keratins 1 and 14) and Langerhans cells (CD1a). Messenger RNA's characteristic of Langerhans cells and of keratinocytes at different stages of differentiation were detected in dermatome and epidermal sheet preparations as well as in FACS-separated cells. The use of snap-frozen dermatome sections allows the isolation of RNA from epidermis that has undergone minimal trauma and is very close to its in vivo state, but that includes RNA from some dermal cells. Extraction of RNA from Dispase-separated sheets involves slightly more manipulation of the epidermis but provides a sample free from dermal contaminants. PCR analysis of sorted epidermal cells is both sensitive and specific, but involves still greater manipulation. This final technique, however, allows the investigation of mRNA produced by small groups of epidermal cells that are still much closer to their in vivo state than if they had been cultured. By combining these techniques it is possible to determine the baseline production of specific mRNA in the skin in vivo and to assign their production to specific groups of cells with a sensitivity and specificity greater than any approach previously described.

Base Sequence↗
PubMed1991

[Immunorestitutive action of hydrolysates and ultrafiltrates of bovine spleen].

Investigations with different spleen extracts of varied composition and purity have shown immunomodulatory effects. In the present study the in vivo effect of a bovine spleen hydrolysate (Prosplen) on a radiation-induced immunodeficiency model is demonstrated. Intraperitoneal application of 1 mg/g bw hydrolysate 3/times a week caused a highly significant acceleration of the restitution of the humoral and cell-mediated immunoreactivity towards sheep erythrocytes in sublethally (600 cGy) radiated mice: plaque-forming cells in the spleen, hemagglutination, hemolysistiter of the serum, delayed type of hypersensitivity = DTH reaction. The immunoreactivity of the treated groups reached normal levels within 6-8 weeks, whereas in the groups receiving placebo this was observed after 10 weeks at the earliest. Cytofluorometric investigations revealed an accelerated restitution of the B-lymphocyte fraction in the spleen. Mice that was not irradiated showed no effect after treatment with the hydrolysate. Similar results were obtained using an ultrafiltrate of the hydrolysate.

Adjuvants, Immunologic↗
PubMed1978

Calcium entry leads to inactivation of calcium channel in Paramecium.

Under depolarizing voltage clamp of Paramecium an inward calcium current developed and subsequently relaxed within 10 milliseconds. The relaxation was substantially slowed when most of the extracellular calcium was replaced by either strontium or barium. Evidence is presented that the relaxation is not accounted for by a drop in electromotive force acting on calcium, or by activation of a delayed potassium current. Relaxation of the current must, therefore, result from an inactivation of the calcium channel. This inactivation persisted after a pulse, as manifested by a reduced calcium current during subsequent depolarization. Inactivation was retarded by procedures that reduce net entry of calcium, and was independent of membrane potential. The calcium channel undergoes inactivation as a consequence of calcium entry during depolarization. In this respect, inactivation of the calcium channel departs qualitatively from the behavior described in the Hodgkin-Huxley model of the sodium channel.

Animals↗
PubMed1978

Potassium activation associated with intraneuronal free calcium.

Relations between calcium entry and activation of a calcium-dependent outward current during depolarization were examined under voltage clamp in dorid giant neurons injected with the calcium-sensitive photoprotein aequorin. Activation kinetics and amplitude of the slow calcium-dependent component were both found to be related to the rate and extent of free calcium accumulation and to the electromotive force acting on potassium ions, independent of the calcium activation kinetics. This indicates that the activation of the calcium-dependent outward current is more closely related to the transient intracellular accumulation of free calcium ions than to the movement of calcium through the plasma membrane during depolarization.

Animals↗
PubMed1978

Calcium-dependent repolarization in Paramecium.

1. Intracellular injection, recording and current-passing methods were used to investigate the role of intracellular Ca in the modulation of electrical behaviour in the ciliate Paramecium caudatum.2. Injection of EGTA converted graded regenerative responses ascribed to Ca inward current to all-or-none action potentials. The EGTA injection also caused a discontinuity in the steady state I-V relations to outward current, but had little effect on hyperpolarizing current-voltage responses.3. The overshoot of the all-or-none spike produced by the EGTA-injected cell followed an approximate 29 mV increase for a tenfold increase in external Ca concentration and was independent of changes in external K and Na concentrations.4. The EGTA-induced all-or-none action potential tended to produce plateaus that could last up to 20 sec. During the plateau the membrane slowly repolarized to a critical potential, upon which repolarization occurred precipitously.5. Injection of 10(-6)M-free Ca(2+) as a Ca-EGTA buffer hyperpolarized the membrane and decreased the potential shifts to inward current pulses. These responses are consistent with an increase in K conductance.6. During EGTA plateaus reversed beating of the cilia indicated a rise in intracellular Ca, and thus an inability of the EGTA to complex the Ca as rapidly as it entered the cilia. Reversal of the motile apparatus thus appears to be activated at lower concentrations of intracellular Ca than are required to activate the inferred Ca-dependent K system.7. In uninjected cells removal of the cilia, which results in a loss of the voltage-activated Ca channels (Dunlap, 1977), or addition of extracellular Ba both tended to linearize the steady state I-V relations.8. Injections of Cs and TEA tended to linearize the steady state I-V relations, but did not result in either a conversion to an all-or-none spike or a discontinuity in the depolarizing steady-state I-V relations.9. It is concluded that in Paramecium a Ca-activated K conductance short-circuits the inward current of the regenerative Ca response, preventing all-or-none behaviour. The occurrence of plateau spikes following EGTA injection indicates that the Ca conductance inactivates very slowly in face of a maintained depolarization. Such slow Ca-inactivation is consistent with the slow relaxation of Ca-dependent ciliary reversal that occurs during maintained depolarization.10. The possibility is discussed that injection of EGTA may also enhance the Ca conductance.

Action Potentials↗
PubMed1978

An electrophysiological study of the regulation of ciliary beating frequency in Paramecium.

1. The role of the surface membrane in the control of ciliary beat frequency in Paramecium was examined by intracellular electrophysiological techniques and pressure injection of Ca2+ and EGTA. Experiments were done on wild type P. caudatum and on both the wild type and a pawn mutant of P. tetraurelia. 2. The increased frequency of beating that accompanies reversal of power stroke orientation in response to depolarization in the wild type fails to occur during depolarization in the mutant pawn, which also fails to exhibit ciliary reversal upon depolarization. 3. Injection of moderate amounts of EGTA blocked the frequency increase without interfering with reversal of the beat in response to depolarization of the wild type. Larger injection of EGTA also prevented reversed beating. 4. The beat frequency in the normal (forward-swimming) direction increased during hyperpolarization in pawn. The hyperpolarizing frequency-voltage relations were quantitatively similar to those of the wild type. 5. Injection of EGTA to a final concentration of 10 mM into wild type cells neither modified the resting frequency nor blocked the frequency increase which normally accompanies hyperpolarization. 6. Transient ciliary reversal in both pawn and wild type produced by injection of Ca2+ could be terminated by the passage of inward current. The power stroke returned to the normal forward-swimming direction and the ciliary beating frequency increased. Upon termination of the inward current the cilia of Ca2+-injected cells again beat in reverse for many seconds. 7. The results support previous reports that increased frequency of beating and ciliary reversal seen in response to depolarization both require the entry of Ca2+ through the surface membrane. On the other hand, the results indicate that frequency increase with hyperpolarization is independent of an altered rate of Ca2+ entry. 8. Increased frequency during hyperpolarization appears to be related more closely to electrotonic membrane current than to membrane potential. It is proposed that inward current might activate high frequency beating by altering the ionic environment of the axoneme within the restricted volume of the cilium by electrophoretic means.

Animals↗
PubMed1978

Studies on column fractionation of immune cells. VII. Electrophoretic mobility and functional behavior of mouse spleen cells after fractionation on a weakly basic ion exchanger.

After passing normal mouse spleen cells through columns of a weakly basic ion-exchanger, the percentage of cells with B cell-typical electrophoretic mobility (EPM) is 90% after fractionation at pH 8.0 and 10--20% at pH 5.0. However, the characterization of fractionated cells with regard to theta-antigen as well as graft-versus-host reaction shows that separation of T- and B-lymphocytes had not occurred. After incubation of the separated cells for 2 h at 37 degrees C the EPM returned to that of unseparated cells. It appears that the net charge of T- and B-lymphocytes is changeable depending on pH, temperature and separation material.

Animals↗
PubMed1977

Calcium-dependent depression of a late outward current in snail neurons.

Neuron cell bodies of Helix pomatia were voltage-clamped with a 300-millisecond depolarizing test pulse (pulse II) delivered I second after a depolarizing conditioning pulse (pulse I). The outward current, measured 200 milliseconds after the onset of pulse. II, exhibited a strong depression that was dependent on the presence of pulse. I. The maximum depression of the pulse II outward current occurred when pulse I voltages lay in the range over which calcium influx is inferred to be greatest; depression of the pulse II current subsided as pulse I potentials approached the putative calcium equilibrium potential. In the presence of extracellular [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) or D600, the intensity of the pulse II current became largely independent of pulse I, approaching the values of maximal depression seen in normal Ringer solution. On the other hand, lowering the intracellular pH with extracellular carbon dioxide-carbonate buffer had no measurable effect on the outward currents. Other experiments showed that it is primarily the calcium-dependent, outward-current hump of the N-shaped late current-voltage curve that is depressed by presentation of the conditioning pulse. It was concluded that distinct from an early potassium-activating role, calcium entering during a depolarization leads, during a subsequent depolarization, to a depression of the calcium-activated potassium system that persists for many seconds.

Action Potentials↗
PubMed1977

Voltage-dependent facilitation of Ca2+ entry in voltage-clamped, aequorin-injected molluscan neurons.

Voltage-clamp experiments were performed on giant neurons of the nudibranch Anisodoris nobilis injected with the Ca-sensitive photoprotein, aequorin. Depolarization beyond -10 to +5 m V produced an aequorin signal, the amplitude of which depended on the extracellular Ca2+ concentration, the amplitude of the depolarization, and its duration. In paired pulse experiments, the amplitude of the aequorin signal produced in response to the second of two identical depolarizing pulses was larger than that produced during the first, resulting from an increased entry of Ca2+ during the second pulse. The increment in Ca conductance inferred from the augmented signal during the second pulse was independent of Ca2+ influx during the first pulse but, instead, was related to the amplitude and duration of the first pulse.

Aequorin↗
PubMed1977

Lymphokines of T and B cells. I. Demonstration that T and B cells of tumor antigensensitised mice produce different lymphokines changing surface charge of target cells.

Lymphocytes from MC sarcoma-bearing mice were exposed to a soluble tumor antigen. The lymphokine-containing supernatant thus obtained was fractionated on Sephadex G-75. Fractions were tested for their activity to reduce the surface charge of indicator macrophages by cell electrophoresis technique. When unseparated spleen or lymph node cells were used the charge-reducing activity of the supernatant was found in three regions according to molecular weights of about 100,000, 60,000 - 35,000 and 13,000 Daltons. The appearance of activity was found to be time-dependent. As indicated by tests with separated lymphocytes both B and T cells appear to release charge-reducing activity.

Animals↗