On the biophysics of transmembrane signalling.

Transmembrane signalling involves a number of physical translocations, changes in proximity of membrane elements like receptor subunits, or sequestration of proteins from the membrane. The monitoring of such changes with flow cytometric energy transfer revealed a new putative subunit of the IL-2 receptor and a possible intermolecular interaction between HLA class I and class II antigens. Lateral diffusion of the components of the multi-subunit IL-2 receptor was also followed. Changes in the intracellular pH were considered as a measure of efficient signal transfer in a number of cases. An overview and critical comparison of data is presented in the paper.

Immune dysfunction in rats fed a diet deficient in copper.

Rat pups maintained on copper (Cu)-adequate (6ppm), Cu-deficient (2 ppm) or Cu-depleted (0 ppm) diets from parturition were killed at 8-wk. Liver Cu and serum-ceruloplasmin levels confirmed that on the 0- and 2-ppm diets, a Cu-deficient state was induced. Although body weight was unaffected by the deficiency, the liver, heart, and thymus weights (% body weight) were altered. Hepatomegaly occurred in females fed 0-ppm Cu and males fed 2-ppm Cu. Heart weights increased in both sexes fed 0-ppm Cu. Thymus weights decreased in male rats fed 0-ppm Cu. Antibody titers and natural killer-cell cytotoxicity were markedly suppressed in the animals fed 0-ppm Cu. Male rats given 2-ppm Cu showed reduced antibody titer. Delayed-type hypersensitivity and prostaglandin E2 levels were not significantly affected. These studies suggest that certain components of the immune system are Cu dependent.

Dietary copper deficiency and autoimmunity in the NZB mouse.

NZB mice were exposed from birth to a diet either adequate or deficient in copper. By age 6 wk the mice exposed to the copper-deficient diet showed symptoms characteristic of copper deficiency (anemia, hypoceruloplasminemia, and achromatrichia). The splenic lymphocytes from the copper-deficient group had reduced numbers of cells expressing the following surface markers: Ly-5, Ly-1, B-220, and sIg. Less than 10% of the splenic lymphocytes in this group were cycling, as determined by flow cytometry analysis. The spontaneous 96-h anti-ss-DNA levels in the copper-deficient group were lower than those in the control group. The exogenous colony-forming units (CFUs) were significantly enhanced in the copper-deficient mice. The decreased splenic lymphoid populations, decreased anti-ss-DNA titers, and increased exogenous CFUs in the copper-deficient mice appear to be due to an increase in erythropoiesis at the expense of lymphopoiesis.

Severe or marginal copper deficiency results in a graded reduction in immune status in mice.

From birth mice received diets containing copper at 0.5, 1, 2 or 6 mg/kg diet. At 8 wk of age they were killed and copper status and immune responsiveness were determined. Only the groups that received copper at 0.5 or 1 mg/kg showed signs of copper deficiency, such as reduced serum ceruloplasmin, hemoglobin, hematocrit and red blood cell counts and characteristic changes in organ pathology. Body and lymphoid organ weights were altered in the groups that received copper at 0.5 or 1 mg/kg. Males were more severely affected than females. A dose-related reduction in splenic T-cell subpopulations was noted in the 0.5 and 1 mg/kg groups. Responses to lipopolysaccharide challenge were reduced, and an increase in spontaneous cycling cells was noted in the groups receiving copper at 0.5 or 1 mg/kg. Only the group receiving copper at 0.5 mg/kg had increased stem cell activity; this increase was probably due to increased erythropoiesis to meet increased demands for red blood cells in this group. These data indicate that only groups receiving copper at 0.5 or 1 mg/kg in the diet were depleted and marginally depleted in copper, respectively, and that immune hyporesponsiveness differs between the depleted and marginally depleted groups.

Further studies on the interaction of actin with heavy meromyosin and subfragment 1 in the presence of ATP.

It has been postulated that, during the hydrolysis of ATP, both normal and SH1-blocked heavy meromyosin undergo a rate-limiting transition from a refractory state which cannot bind to actin to a nonrefractory state which can bind to actin. This model leads to several predictions which were studied in the present work. First, the fraction of heavy meromysin or subfragment 1 which remains unbound to actin when the ATPase equals Vmax should have the same properties as the original protein. In the present study it was determined that the unbound protein has normal ATPase activity which suggests that it is unbound to actin for a kinetic reason rather than because it is a permanently altered form of the myosin. Second, if the heavy meromyosin heads act independently half as much subfragment 1 as heavy meromyosin should bind to actin. Experiments in the ultracentrifuge demonstrate that about half as much subfragment 1 as heavy meromyosin sediments with the actin at Vmax. Third, the ATP turnover rate per actin monomer at infinite heavy meromyosin concentration should be much higher than the ATP turnover rate per heavy meromyosin head at infinite actin concentration. This was found to be the case for SH1-blocked heavy meromyosin since, even at very high concentrations of SH1-blocked heavy meromyosin, in the presence of a fixed actin concentration, the actin-activated ATPase rate remained proportional to the SH1-blocked heavy meromyosin concentration. All of these results tend to confirm the refractory state model for both SH1-blocked heavy meromyosin and unmodified heavy meromyosin and subfragment 1. However, the nature of the small amount of heavy meromyosin which does bind to actin in the presence of ATP at high actin concentration remains unclear.

Alopecia induced in young mice by exposure to excess dietary zinc.

Second generation mice were exposed to normal (50 ppm, Group I) or excess (2000 ppm, Group II) zinc in the maternal diet during gestation and lactation, then weaned and continued on the mother's diet until sacrifice at 8 weeks. Tibia zinc reflected dietary intake. Group II had reduced plasma copper, body weight, and hematocrit; the second coat of hair appeared late and was lighter in color than Group I, possibly as an effect of copper and pigmentation development and hair growth.

Interaction of the B subunit of cholera toxin with endogenous ganglioside GM1 causes changes in membrane potential of rat thymocytes.

The fluorescent anionic dye, bisoxonol, and flow cytometry have been used to monitor changes in the membrane potential of rat thymocytes exposed to the B subunit of cholera toxin. The B subunit induced a rapid hyperpolarization, which was due to activation of a Ca2+-sensitive K+ channel. Reduction of extracellular Ca2+ to less than 1 microM by the addition of [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid immediately abolished the hyperpolarization caused by the B subunit. Cells treated with quinine and tetraethylammonium lost their ability to respond to the B subunit, whereas 4-aminopyridine did not have any effect. Thus, calcium-sensitive and not voltage-gated K+ channels appeared to be responsible for the hyperpolarization. The results of ion substitution experiments indicated that extracellular Na+ was not essential for changes in membrane potential. Further studies with ouabain, amiloride and furosemide demonstrated that electrogenic Na+/K+ ATPase, Na+/H+ antiporter and Na+/K+/Cl- cotransporter, respectively, were not involved in the hyperpolarization process induced by the B subunit. Thus, crosslinking of several molecules of ganglioside GM1 on the cell surface of rat thymocytes by the pentavalent B subunit of cholera toxin modulated plasma membrane permeability to K+ by triggering the opening of Ca2+-sensitive K+ channels. A role for gangliosides in regulating ion permeability would have important implications for the function of gangliosides in various cellular phenomena.

Procainamide induces a transitory impairment of B cell mitogenesis in beagle dogs.

Beagle dogs (3 to 6 years old) were treated with 100-150 mg procainamide HC1/kg/day. After 2, 5, and 9 months of treatment, peripheral blood lymphocytes were isolated and stimulated with pokeweed mitogen. The data demonstrated a suppression of mitogenesis only at 2 and 5 months after procainamide treatment. The lymphocytes from dogs treated for 9 months had a normal response to pokeweed mitogen. At no time during this experiment were any significant levels of serum antinuclear antibodies detected nor was any change in the number of cycling lymphocytes apparent in the experimental versus control groups. The resting membrane potential of both control and experimental groups was similar and pokeweed mitogen depolarized the cells from both groups.

Suppression of antibody response by excess dietary zinc exposure during certain stages of ontogeny.

A study was made of the effects of excess dietary zinc on the antibody response to sheep red blood cells (SRBC) in mice. C57BL/6J mice were divided into 10 different dietary groups and exposed to diets containing zinc in normal (50 ppm) or excess (2000 ppm) concentrations during gestation/lactation/postweaning development in the sequences (1) 50/50/50; (2) 50/50/2000; (3) 2000/50/50; (4) 2000/2000/50; (5) 2000/50/2000; (6) 50/2000/50; (7) 50/2000/2000; (8) 2000/2000/2000; (9) 50/50/50 (pair-fed); and (10) chow/chow/chow. Mice in group 8 had severe signs of copper deficiency at 8 weeks of age, such as reduced plasma copper, lowered plasma hematocrit, and achromotrichia. Mice receiving 2000 ppm zinc during gestation had fewer offspring per litter (measured at 2 weeks of age) and more nonviable births than mice given 50 ppm zinc during gestation. The growth curve of mice exposed to excess zinc in the 50/50/2000 group was identical to that of the control (50/50/50) group. Growth curves for all other groups were reduced by varying amounts. The plaque-forming cell response to SRBC was reduced only in the groups receiving 50/2000/2000 and 2000/2000/2000 ppm zinc (P less than 0.05); this reduced response was not associated with atrophy of the lymphoid organs. Splenic cell surface markers and mitogenic responsiveness were similar in the 50/50/50 and 2000/2000/2000 groups. These results suggest that the immune response is more susceptible to dietary manipulation during development than after the immune response has been developed.

Methemoglobinemia from perineal application of an anesthetic cream.

A 34-year-old woman presented with cyanosis and a methemoglobin level of 23.2% after perineal application of a topical anesthetic cream containing 20% benzocaine. Many commonly used products contain high levels of benzocaine, and their use can lead to life-threatening methemoglobin levels. This case reinforces the need for stricter guidelines for product use and warning labels to alert consumers to this potential side effect of topical benzocaine-containing products sold over the counter.

Cyclosporin depolarizes human lymphocytes: earliest observed effect on cell metabolism.

Cyclosporin A (CsA) produced dose-dependent membrane depolarization of human peripheral blood lymphocytes. The phenomenon was investigated applying the membrane potential probe dihexyloxacarbocyanine iodide in a flow cytometer in combination with ionophores, hormones and monoclonal antibodies binding to different subclasses of lymphocytes and the anti-interleukin 2 receptor antibody. Human interferon-gamma abolished the depolarizing effect of cyclosporin on lymphocytes. Interleukin 2 caused depolarization and also enhanced the effect of CsA. OKT4 and OKT8 monoclonal antibodies slightly hindered depolarization by CsA while OKT3, OKT11 and OKIa1 antibodies had no such effect. Valinomycin decreased CsA's effect on the membrane potential while the ionophore A-23187 and ionomycin caused depolarizations that were additive with CsA's. CsA treatment released the isotope from 42K-loaded human lymphocytes in a dose-dependent fashion. CsA addition increased intracellular calcium content. CsA decreased the motional freedom of a spin probe in the membrane, but did not hinder the binding of fluoresceinated antibodies to the cell surface. These results suggest immediate alteration in membrane structure upon CsA treatment, causing potassium leakage and calcium ion uptake. These are the earliest detected effects of CsA on cells so far.