Dependence of ionophore- and caffeine-induced calcium release from sarcoplasmic reticulum vesicles on external and internal calcium ion concentrations.

The effects of the ionophore, X537A, and caffeine on ATP-dependent calcium transport by fragmented sarcoplasmic reticulum were studied in the absence (calcium storage) or presence (calcium uptake) of calcium-precipitating anions. The ionophore caused rapid calcium release after calcium storage, the final level of calcium storage being the same whether a given concentration of X537A was added prior to initiation of the reaction or after calcium storage had reached a steady state. Although 10 to 12 muM X537A caused approximately 90% inhibition of oxalate-supported calcium uptake when added prior to the start of the reaction, this ionophore concentration caused only a small calcium release when added after a calcium oxalate precipitate had formed within the vesicles, and only slight inhibition of calcium uptake velocity when added during the calcium uptake reaction. When low initial calcium loads limited calcium uptake to 0.4 mumol of calcium/mg of protein, subsequent calcium additions in the absence of the ionophore led to renewed calcium uptake. Uptake of the subsequent calcium additions was not significantly inhibited by 10 to 12 muM X537A. These phenomena are most readily understood in terms of constraints imposed by fixed Cai (calcium ion concentration inside the vesicles) on the pump-leak situation in sarcoplasmic reticulum vesicles containing a large amount of an insoluble calcium precipitate, where most of the calcium is within the vesicles and Cai is maintained at a relatively low level. These constraints restrict calcium loss after calcium permeability is increased because calcium release can end when the calcium pump is stimulated by the increased Cao (calcium concentration outside the vesicles) so as to compensate for the increased efflux rate. In contrast, an increased permeability in vesicles that have stored calcium in the absence of a calcium-precipitating ion causes a much larger portion of the internal calcium store to be released. Under these conditions calcium storage capacity is low so that release of stored calcium is less able to raise Cao to levels where the calcium pump can compensate for the increased efflux rate. The constraints imposed by anion-supported calcium uptake explain the finding that more calcium is released by X537A or caffeine when these agents are added at higher levels of Cao, and that more calcium leaves the vesicles in response to a given increase in calcium permeability at higher Cai. Although such calcium release is amplified by increased Cao, the amplification is attributable to the constraints described above and does not represent a "calcium-triggered calcium release."     

Hypothalamic sarcoidosis and hypopituitarism

4 patients with presumed pituitary hypothalamic sarcoidosis are described. 3 had histological diagnoses compatible with sarcoidosis and in the other this diagnosis was strongly suspected from chest X-rays. 2 patients presented with diabetes insipidus. ACTH reserve was diminished in 3 out of 4 and growth hormone reserve was diminished in the 3 who were tested. All 4 patients developed secondary amenorrhea. 3 patients had hypothalamic hypothyroidism. Prolactin dynamics were intact. Tomograms of the sella turcica in all 4 and computerized tomography of the hypothalamic area in 2 patients failed to reveal any abnormality.     

Regulation of IgE antibody production by serum molecules. IV. Complete Freund's adjuvant induces both enhancing and suppressive activities detectable in the serum of low and high responder mice.

Two biologically active serum molecules manifesting precisely opposite biologic effects, both of which are selective for IgE antibody synthesis, can be detected in the serum and ascites fluids of CFA-immune mice. One activity, described previously, is suppressive and hence termed suppressive factor of allergy (SFA); the other, reported for the first time herein, is enhancing and has been termed enhancing factor of allergy (EFA). The ability to detect one vs the other activity requires certain special manipulations such as different doses of low dose x-irradiation. Conclusive evidence for the existence of two distinct factors mediating these two opposing biologic effects was obtained in studies demonstrating that affinity chromatography on concanavalin A-Sepharose segregated the two molecular entities. Thus, SFA binds poorly or not at all to Con A-Sepharose, whereas EFA binds to Con A and can be recovered in the eluate eluted with the competitive sugar alpha-methyl-D-glucopyranoside.     

Adaptive differentiation of murine lymphocytes. III. T and B lymphocytes display reciprocal preference for one another to develop optimal interacting partner cell sets.

Responses to the synthetic terpolymer L-glutanmic acid, L-lysine, L-phenylalanine (GLphi) and hapten derivatives thereof are controlled by two complementing H-2 linked Ir genes in the mouse. F1 hybrids derived from two different nonresponder strains (one of which possesses the alpha and the other beta Ir-GLphi gene) are phenotypic responders to GLphi and 2,4-dinitrophenyl (DNP)-GLphi. Moreover, spleen cells from DNP-GLphi-primed F1 mice can adoptively transfer secondary anti-DNP antibody responses to irradiate been challenged with DNP-GLphi. When, however, GLphi-primed F1 helper T cells are transfered together with the DNP-specific F1 B cells that had been primed in separate mice altogether by DNP coupled to an unrelated protein carrier, such mixtures failed to develop adequate adoptive secondary anti-DNP responses to DNP-GLphi. This contrasted with the ability of the same GLphi-primed F1 T cells to provide helper activity for DNP-primed B cells from responder recombinant B10.A (5R) mice. More important, the apparent defect of GLphi-primed F1 T cells in providing help for DNP-primed F1 B cells (primed to a DNP-protein conjugate) could be readily overcome by using DNP-primed B cells from donor F1 mice primed with DNP-GLphi. As discussed herein, these results suggest that interacting T and B lymphocytes pair off into partner cell sets, any pair of which interact optimally when a "best fit" reciprocal self-recognition occurs between them.     

The in vitro effect of indomethacin on basal bone resorption, on prostaglandin production and on the response to added prostaglandins

Mouse calvaria were maintained in organ culture without serum additives. Basal active resorption, as measured by ⁴⁵Ca and hydroxyproline release, was significantly inhibited to 74% control levels by indomethacin (1.4 × 10⁻⁷ M). Prostaglandin F and prostaglandin E₂ production, determined by radioimmunoassay, were both significantly lowered by this concentration of indomethacin. DNA, protein and hydroxyproline synthesis, as indices of cell toxicity, were unaffected by low concentrations of indomethacin, while concentrations of 1.4 × 10⁻⁶M inhibited protein synthesis (p<0.005). In the presence of indomethacin (1.4 × 10⁻⁷M) both PGE₂ and PGF_2α stimulated resorption in a dose-dependent manner, with PGE₂ being the more potent. Neither prostaglandin affected hydroxyproline synthesis at low concentrations, but PGE₂ had a marked inhibitory action at a higher concentration (10⁻⁶M). In combination, the effects of PGE₂ and PGF_2α showed no evidence of synergism or any antagonistic action. The study shows that in vitro calcium and hydroxyproline resorption in the unstimulated mouse calvaria are inhibited by indomethacin at concentrations measured in serum during human therapy. The decreased PGF and PGE₂ production associated with this decreased bone resorption in the presence of non-toxic concentrations of indomethacin would suggest a role for these prostaglandins in maintaining the basal resorption of cultured bone.     

Prostaglandin release by normal and osteomyelitic human bones

The release of prostaglandin E (PGE) and prostacyclin (as 6-keto PGF1 alpha) by human osteomyelitic bone, compared with normal (control) bone, incubated in vitro was evaluated. Prostacyclin was the main arachidonic acid metabolite released by normal human bone, and similar quantities were released by osteomyelitic bone. However, PGE production was 5-30-fold higher in osteomyelitic bone, compared with control, thus becoming the major prostanoid in this disease. It is concluded that PGE production is probably involved in the inflammatory and/or bone resorption processes that occur in osteomyelitis.     

Constitutive and mitogen-induced production of T cell growth factor by stable T cell hybridoma lines

Stable T cell growth factor- (TCGF; IL 2) producing cloned T cell hybridoma lines were constructed by fusing murine alloantigen-activated T cells with the 8-azaguanine-resistant lymphoma line, BW5147. Many, but not all, clones of one of these hybridomas, i.e., hybridoma 24, secreted TCGF constitutively, but production was markedly enhanced by stimulation with T cell mitogens. Large numbers of TCGF-secreting hybridoma cells in a stable functional state could be obtained from histocompatible mice inoculated with cloned T cell hybridomas. Moreover, such in vivo-derived hybridoma cells could be stimulated sequentially with mitogen at least twice to secrete their biologically-active product, resulting in larger TCGF yields from the same cells. The secreted product of these T cell hybridoma lines resembled TCGF isolated from other cellular sources in that it: a) supported the growth of a TCGF-dependent T cell line; b) provided help for the induction of alloantigen-reactive cytotoxic T lymphocytes from thymocyte precursors; c) facilitated concanavalin A-induced mitogenic responses of low thymocyte numbers; d) had an apparent m.w. of 30,000 to 40,000 by gel filtration chromatography; and e) was eluted from DEAE-Sephacel ion-exchange chromatography columns by salt concentrations of 30 to 150 mM NaCl. The ability of these T cell hybridomas to grow in vivo and retain their functional characteristics in a stable form should prove useful in terms of providing large numbers of TCGF-secreting cells and studying in vivo aspects of the production of TCGF as well as other immunoregulatory mediators.     

Glucokinase in bird liver: a membrane bound enzyme

There have been numerous reports that liver of birds contain only isoenzymes of the low K_M hexokinases, but lack the high K_M glucokinase. We describe here the presence of glucokinase in livers of chicken and Japanese quail. The enzyme is membrane bound and is solubilized by vigorous mechanical disruption of the tissue. With gentle homogenization the glucokinase is recovered upon centrifugation in the 1000g pellet, from which it may be liberated by prolonged sonication. It appears to be localized in the cell plasma membrane. The activities of hexokinase and glucokinase appear to be about equal in liver parenchyma of fed chicken, but in that of Japanese quail the activity of glucokinase exceeds greatly that of hexokinase.