The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization.

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.     

Batrachotoxin-modified sodium channels in planar lipid bilayers. Characterization of saxitoxin- and tetrodotoxin-induced channel closures

The guanidinium toxin-induced inhibition of the current through voltage-dependent sodium channels was examined for batrachotoxin-modified channels incorporated into planar lipid bilayers that carry no net charge. To ascertain whether a net negative charge exists in the vicinity of the toxin-binding site, we studied the channel closures induced by tetrodotoxin (TTX) and saxitoxin (STX) over a wide range of [Na+]. These toxins carry charges of +1 and +2, respectively. The frequency and duration of the toxin-induced closures are voltage dependent. The voltage dependence was similar for STX and TTX, independent of [Na+], which indicates that the binding site is located superficially at the extracellular surface of the sodium channel. The toxin dissociation constant, KD, and the rate constant for the toxin-induced closures, kc, varied as a function of [Na+]. The Na+ dependence was larger for STX than for TTX. Similarly, the addition of tetraethylammonium (TEA+) or Zn++ increased KD and decreased kc more for STX than for TTX. These differential effects are interpreted to arise from changes in the electrostatic potential near the toxin-binding site. The charges giving rise to this potential must reside on the channel since the bilayers had no net charge. The Na+ dependence of the ratios KDSTX/KDTTX and kcSTX/kcTTX was used to estimate an apparent charge density near the toxin-binding site of about -0.33 e X nm-2. Zn++ causes a voltage-dependent block of the single-channel current, as if Zn++ bound at a site within the permeation path, thereby blocking Na+ movement. There was no measurable interaction between Zn++ at its blocking site and STX or TTX at their binding site, which suggests that the toxin-binding site is separate from the channel entrance. The separation between the toxin-binding site and the Zn++ blocking site was estimated to be at least 1.5 nm. A model for toxin-induced channel closures is proposed, based on conformational changes in the channel subsequent to toxin binding.     

Cell surface T3 expression requires the presence of both alpha- and beta-chains of the T cell receptor

We have identified a surface T3- Jurkat variant which has a defective alpha-chain but which possesses an intact beta-chain. The transfection of a functional mouse alpha-chain into this human T cell induces the expression of surface T3 molecules associated with mouse alpha-human beta-heterodimers detected by anticlonotypic antibodies. Treatment of the transfectant with anti-T3, anti-mouse Ti-alpha, or anti-human Ti-beta antibodies clears all Ti-T3 complexes from the surface. These results demonstrate that functional alpha- and beta-chains are both required for expression of T3 on the cell membrane, and that the Ti heterodimers present and associated with T3 on Jurkat cells involve only alpha- and beta-chains.     

Evidence from nitrogen-15 and solvent deuterium isotope effects on the chemical mechanism of adenosine deaminase

We have determined 15N isotope effects and solvent deuterium isotope effects for adenosine deaminase using both adenosine and the slow alternate substrate 7,8-dihydro-8-oxoadenosine. With adenosine, 15N isotope effects were 1.0040 in H2O and 1.0023 in D2O, and the solvent deuterium isotope effect was 0.77. With 7,8-dihydro-8-oxoadenosine, 15N isotope effects were 1.015 in H2O and 1.0131 in D2O, and the solvent deuterium isotope effect was 0.45. The inverse solvent deuterium isotope effect shows that the fractionation factor of a proton, which is originally less than 0.6, increases to near unity during formation of the tetrahedral intermediate from which ammonia is released. Proton inventories for 1/V and 1/(V/K) vs percent D2O are linear, indicating that a single proton has its fractionation factor altered during the reaction. We conclude that a sulfhydryl group on the enzyme donates its proton to oxygen or nitrogen during this step. pH profiles with 7,8-dihydro-8-oxoadenosine suggest that the pK of this sulfhydryl group is 8.45. The inhibition of adenosine deaminase by cadmium also shows a pK of approximately 9 from the pKi profile. Quantitative analysis of the isotope effects suggests an intrinsic 15N isotope effect for the release of ammonia from the tetrahedral intermediate of approximately 1.03 for both substrates; however, the partition ratio of this intermediate for release of ammonia as opposed to back-reaction is 14 times greater for adenosine (1.4) than for 7,8-dihydro-8-oxoadenosine (0.1).(ABSTRACT TRUNCATED AT 250 WORDS)     

The hemodynamic destruction of circulating cancer cells

The blood-stream is the major disseminative route for metastasizing cancer cells, and metastases are generated when the cancer "microemboli" are trapped in the microcirculation. However, most circulating cancer cells are rapidly destroyed shortly before and/or after arrest. Traditionally, destruction is attributed to the cellular or humoral response of the host defense systems. A novel, non-exclusive mechanism for cancer cell destruction has been proposed by Weiss and Dimitrov in which friction or adhesion between circulating cancer cells and capillary walls causes local vascular blockage, and the blood-pressure differentials normally existing over the entire length of a capillary are consequently applied over the length of the cancer cell. In a simple model, this pressure differential is expected to cause expansion of the cancer cell membrane, resulting in increases in tension above a critical level, with consequent membrane rupture and cell death. In vivo and in vitro experimental tests of this hypothesis are outlined.     

Behavioral endocrinology of male rats with periodic sexual contacts with exclusive or varied females

Sexual contact keys a profound series of acute and chronic changes in males that, presumably, are orchestrated by acute pulsatile release of hormones. An experimental paradigm is reported in which male rats were paired periodically with either the same or different estrous females to receive identical amounts of copulatory experiences. Results confirmed the hypothesis that exposure to an unfamiliar female would induce a different endocrine response which would be reflected in various androgen-sensitive systems. The "successively polygynous" males showed more sexual behavior than "monogamous" males, and their respective females solicited the males differently, as well. Circulating levels of testosterone were higher immediately after sexual contact with an unfamiliar than with a familiar female partner. There were no differences in testosterone titers among the groups when the animals were killed at either 2 or 7 weeks after the final copulatory experiences. Yet, necropsies at 2 weeks postcopulation revealed that primary and secondary sex structures from polygynous males clearly were larger. Differences between the two experimental groups were reduced after 7 weeks of sexual rest, yet, polygnous males continued to show a different structural profile than the other groups. Conclusions were that males may experience greater activation of androgen-sensitive behavior and physiology following qualitatively different sexual contacts.     

Transgenic HLA-DR alpha faithfully reconstitutes IE-controlled immune functions and induces cross-tolerance to E alpha in E alpha 0 mutant mice

We have constructed transgenic mice that express the human class II MHC molecule HLA-DR alpha on a genetic background in which the equivalent endogenous gene, H-2 IE alpha, is not expressed. In these mice, DR alpha complemented the E beta chain such that tissue-specific expression of an interspecies hybrid DR alpha-E beta heterodimer was obtained. Despite 25% amino acid differences between DR alpha and E alpha, immune responsiveness to IE-controlled antigens, clonal deletion of IE-reactive T cells, and alloantigenicity were quantitatively and qualitatively indistinguishable in IE-positive mice and in mice that had integrated at least four copies of the transgene. These results demonstrate a remarkable degree of structural, regulatory, and functional conservation. They also suggest that tolerance induction involves only discrete portions of MHC molecules.     

The fine structure of the newly discovered propodial ganglia of the veliger larva of the nudibranch Onchidoris bilamellata

Summary Two pairs of ganglia are found in the propodial region of the veliger of Onchidoris bilamellata: the anterolateral pair is located at the foremost corners of the propodium, and the frontal pair is located beside the propodial midline. Both sets of ganglia are positioned below the epidermis, and they are joined to the cerebral ganglia by large, common connectives. Each ganglion possesses sensory cells, nerve cells and sheath cells, and the frontal pair contains a complement of secretory cells. Externally, the propodial ganglia are manifested as sensory fields. The fields of the anterolateral pair are elliptical in shape, and each appears as a band of cilia bordering an unciliated zone. The region devoid of cilia is composed of ordinary epidermal cells, whereas the ciliated portion is comprised of dendritic endings originating from cells in the ganglion. Dendrites arise from one type of sensory cell and pass through the epidermis in bundles. Each dendrite terminates as a single cilium at the epidermal surface. Sensory fields of the frontal ganglia are key-shaped and oppose one another on the anterior end of the foot. Each field appears as a flat, circular, unciliated region which extends into a ciliated groove that runs dorsally toward the mouth. The groove contains the terminals of secretory cells, ciliated sensory cells, and the cell bodies of nonciliated sensory cells. The nonciliated sensory cells, characterized by a microvillous apex, are the dominant cells in the flattened circular zone. The space between the frontal ganglia and the epidermis is bridged by bundles of processes which are similar to those of the anterolateral ganglia. However, these tracts contain collections of the apical processes of secretory cells, the dendrites of ciliated sensory cells, and the axons of nonciliated sensory cells. Morphological and behavioral evidence indicates that the propodial ganglia serve a chemosensory function during settlement and metamorphosis.     

Reduced neuroexcitatory effect of domoic acid following mossy fiber denervation of the rat dorsal hippocampus: further evidence that toxicity of domoic acid involves kainate receptor activation

Domoic acid, an excitatory amino acid structurally related to kainic acid, has been shown to be responsible for the severe intoxication presented, in 1987, by more than one hundred and fifty people having eaten mussels grown in Prince Edward Island (Canada). Unitary extracellular recordings were obtained from pyramidal neurons of the CA3 region of the rat dorsal hippocampus. The excitatory effects of microiontophoretic applications of domoic acid and of the agonists of the two other subtypes of glutamatergic receptors, quisqualate and N-methyl-D-aspartate, were compared on intact and colchicine-lesioned sides. Similar to what has been previously found for kainate, the colchicine lesion of the mossy fiber projections induced a 95% decrease of the neuronal responsiveness to domoic acid, whereas the effect of quisqualate was unchanged and that of N-methyl-D-aspartate was only slightly decreased. These results provide further electrophysiological evidence that domoic acid is a potent agonist of kainate receptors and that it may produce its neuroexcitatory and neurotoxic effects, in the hippocampal CA3 region, through activation of kainate receptors located on the mossy fiber terminals.