Effect of cesium ions on the electrotropic actions of acetylcholine in the rabbit atrium

Cs+ (15-20 mM) decreases the electrotropic vagal effects on an isolated vagal innervated rabbit atrium. By means of investigating the action potentials and the phase plane trajectories of trabeculae from the rabbit atrium using a modified single sucrose gap technique the anomalous rectification disappears and also the effect of acetylcholine (ACh) on action potential duration. We presume that the anomalous rectification should be a necessary condition of electrotropic vagal (ACh) action on the rabbit atrium.     

Disparity between expression of transferrin receptor ligand binding and non-ligand binding domains on human lymphocytes

We compared transferrin receptor (TfR) expression on human peripheral blood lymphocytes (PBL) activated by phorbol myristate acetate (PMA) or L-phytohemagglutinin (LPHA) using two techniques: (1) 125I-iron-saturated transferrin (FeTf) binding, (2) reactivity with monoclonal anti-TfR antibodies--OKT9 and B3/25. These monoclonal antibodies do not block FeTf binding, and therefore bind to TfR domains separate from the ligand binding site. Unstimulated PBL bound fewer than 1,000 molecules of 125I-FeTf per cell, and less than 5% of cells expressed TfR antigens detected by OKT9 or B3/25. 125I-FeTf binding and antibody binding increased in parallel on LPHA-activated PBL. After exposure to LPHA for 72 hr, 125I-FeTf binding increased 100-fold to 10(5) molecules per cell and greater than 50% of cells expressed TfR antigens. By contrast, PMA activation of PBL markedly increased binding of OKT9 and B3/25 but not the binding of 125I-FeTf. Cell surface expression of TfR antigens seen by OKT9 and B3/25 did not differ between LPHA- and PMA-activated PBL. However, after 72 hr with PMA, 125I-FeTf binding increased only 6-fold and consistently remained at less than 10(4) molecules per cell. Therefore, PMA induced a disparity between expression of TfR ligand binding domains and immunological domains at the cell surface. Cell proliferation assessed by fluorescent DNA analysis was similar in cultures stimulated by LPHA or PMA. These data indicate that lymphoid cells may possess a mechanism for modulating TfR expression in which down-regulation of FeTf binding occurs without receptor internalization. Alternatively, it is possible that this observation may reflect a membrane perturbation effect of PMA.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

Binding of isolectins from red kidney bean (Phaseolus vulgaris) to purified rat brush-border membranes

Ingestion of red kidney bean phytohemagglutinin causes impaired growth and intestinal malabsorption, and facilitates bacterial colonization in the small intestine of weanling rats. We have studied interactions of the highly purified phytohemagglutinin erythroagglutinating (E4) and mitogenic (L4) isolectins with microvillous membrane vesicles prepared from rat small intestines. E4 and L4 were radioiodinated with 125I by the chloramine-T technique. E4 and L4 isolectins both bound to microvillous membrane vesicles. Binding was saturable and reversible. Each mg of membrane protein bound 744 +/- 86 micrograms E4 and 213 +/- 21 micrograms L4. The apparent Ka for E4 and L4 binding was 2.5 x 10(-6) and 13.0 x 10(-6) M-1, respectively. Binding of each 125I-labelled isolectin was abolished by 100-fold excess of unlabelled isolectin. In each case binding also was inhibited by appropriate oligosaccharide inhibitors, indicating that isolectin-microvillous membrane interactions were mediated by carbohydrate recognition. Patterns of saccharide inhibition of isolectin binding were different for E4 and L4. Competitive binding experiments demonstrated mutual noncompetitive inhibition of E4 and L4 binding consistent with steric hindrance. Therefore, E4 and L4 each bound to its own set of receptors. Based on the known saccharide specificities of E4 and L4, these data indicate that there are differences in expression of complex asparagine-linked biantennary and tri- or tetraantennary oligosaccharides at the microvillous surface. The data also provide the possibility that direct interactions of one or more phytohemagglutinin isolectins with intestinal mucosa in vivo may contribute to the antinutritional effects associated with ingestion of crude red kidney beans.     

Acrosomal status evaluation in human ejaculated sperm with monoclonal antibodies

An important question in mammalian gamete physiology concerns how capacitation and the occurrence of acrosome reactions in motile sperm relate to fertility. Evaluation of these relationships has been restricted by practical limitations because rapid, quantitative assays are unavailable. We have developed a rapid, reproducible assay for the evaluation of acrosomal status utilizing monoclonal antibodies specific to antigens localized in the acrosomal cap region of the sperm head. Mice were immunized with human ejaculated sperm preparations and the resultant hybridomas producing antisperm antibody were selected by solid-phase radioimmunoassay and indirect immunofluorescence (IIF). Two monoclonal antibodies (HS-19, HS-21) recognized target antigens restricted to the acrosomal cap by IIF, and 87 +/- 8.5% of the sperm in fresh ejaculates from 10 different sperm donors showed positive cap fluorescence with these reagents. Loss of HS-21 binding as measured by IIF was correlated with disappearance of the acrosomal cap as observed directly by transmission electron microscopy. Acrosomal disappearance, artificially induced in vitro using the calcium ionophore A23187, also resulted in a loss of HS-21 binding. The induction of acrosomal loss by ionophore was dependent upon extracellular calcium. The data presented suggest that specific monoclonal antibodies can be used for the rapid evaluation of acrosomal status in mammalian sperm.     

Properties of aconitine-modified sodium channels in single cells of mouse ventricular myocardium

Effects of the plant alkaloid Aconitine on the kinetics of sodium channels were studied in enzymatically isolated single cells of the mouse ventricular myocardium. Aconitine (1 mumol/l) induced a prolongation of the 90% repolarization of action potentials from 52.4 +/- 3.7 ms to 217.0 +/- 12.5 ms. Delayed terminal repolarization and oscillatory afterpotentials preceded spontaneous activity with high frequencies. Peak sodium currents were diminished from 28.0 +/- 9.0 to 14.0 +/- 6.0 nA. The reversal potential of the sodium current was shifted from 16.0 +/- 11.0 to -8.0 +/- 6.0 mV (52.5 mmol/l extracellular sodium concentration) suggesting a decreased selectivity of the Aconitine-modified Na channels. The m-affinity-curves were shifted 31 mV towards more negative potentials at a constant slope. The h affinity-curves were shifted in the same direction by 13 mV. The slope parameter of the h affinity-voltage relationship was enlarged from 9.1 +/- 2.2 mV to 15.6 +/- 4.4 mV. Shifts in m affinity and h affinity resulted in an increased "window". The alkaloid modified channels inactivated extremely slowly at potentials negative to -40 mV, but showed a fast and complete inactivation at potentials positive to -40 mV.     

DETERMINATION OF GUSTATORY SUCROSE THRESHOLD IN WATER BY USE OF A LINEAR SUCROSE GRADIENT

A novel experimental method was developed which allows the determination of the threshold concentration of sucrose by use of a linear sucrose gradient in water. With this method a continuous tasting of the test-liquid is possible. A panel of 15 persons experienced in taste-testing was used. Three gradients of different steepness were applied: 0 to 1.5% (w/w) sucrose in 2 min (I), 3 min (II) and 4 min (III). The results of the new method were compared with those of the standard method (DIN). With gradients I and II we found values which were significantly higher than those of the standard method (I: 0.49% (w/w); II: 0.46% (w/w); DIN: 0.31% (w/w)), whereas with gradient III the same threshold value was found as with the DIN-Method (III: 0.32% (w/w)).     

Formation and photolability of low-spin ferrous cytochrome c peroxidase at alkaline pH.

The ferrous form of native cytochrome c peroxidase (CCP) is known to undergo a reversible transition when titrated over the pH range of 7.00-9.70. This transition produces a conversion from a pentacoordinate high-spin to a hexacoordinate low-spin heme active site and is clearly apparent in the heme optical absorption spectra. Here, we report the characterization of this transition and its effect upon the local heme environment using various optical spectroscopies. The formation of hexacoordinate low-spin heme is interpreted to involve the binding of His-52 at the distal site after the perturbation of the extensive H-bonded network within and around the heme pocket of CCP(II) at alkaline pH. Interestingly, CD investigations of CCP(II) in the far-UV and Soret regions indicate the dissappearance of a single high-spin species and the existence of at least two low-spin species of CCP(II) as the pH is raised above 7.90. Furthermore, transient resonance Raman experiments demonstrate that the hexacoordinate low-spin species can be photolyzed within 10-ns laser pulses, producing a species similar to the low-pH (high-spin) form of CCP(II) at alkaline pH. However, the extent of photolysis is quite pH dependent, with a maximum photodissociation yield at pH = 8.50.