Interleukin 2 receptors on cultured murine epidermal Langerhans cells

Rat monoclonal antibodies 3C7 and 7D4 detect two distinct functional regions of the murine interleukin 2 (IL 2) receptor. When studying the emergence kinetics of IL 2 receptors in mixed epidermal cell (EC)-lymphocyte cultures by using 3C7 and 7D4 in an indirect immunofluorescence assay, we regularly encountered a distinctive membrane fluorescence not only on lymphocytes, but also on a subpopulation of cells exhibiting a dendritic morphology. Reasoning that these 3C7/7D4-reactive dendritic cells might represent a subpopulation of epidermal dendritic cells, we studied mouse EC for the presence of 3C7/7D4- reactive cells. Although 3C7/7D4 reactivity was never detected on freshly isolated EC or on epidermal sheets, a small number of 3C7/7D4+ cells was encountered after 24 to 48 hr of culture. These cells exhibited a dendritic shape, expressed Ia antigens, lacked Thy-1 antigens, and displayed the ultrastructural features of Langerhans cells (LC) with the notable exception of Birbeck granules. Although after 24 hr, only 20% of Ia+ EC were 3C7/7D4+, the vast majority of LC displayed 3C7/7D4 binding sites after 4 to 5 days of culture. Preincubation of cultured LC-enriched EC with recombinant human IL 2 prevented subsequent 3C7-but not 7D4-binding to these cells. Western blot analysis of 7D4-reactive material of detergent extracts from LC-enriched EC revealed three bands in the same m.w. range as reported for CTLL cells. These results demonstrate that cultured LC express IL 2 receptors and may bear important implications for a better understanding of growth regulation, differentiation, and immunologic functions of LC.     

The photocycle of the chloride pump halorhodopsin. I: Azide-catalyzed deprotonation of the chromophore is a side reaction of photocycle intermediates inactivating the pump

Halorhodopsin, the light-driven chloride pump of halobacteria, undergoes a photochemical cycle in the 10 ms range. Two intermediates, HR640 and HR520, accumulate in the photosteady state after short times (within 100 ms) of illumination. Upon prolonged illumination a third species, HRL410 accumulates, which is formed from HR520/HR640 by deprotonation of the chromophore in a side reaction of the photocycle. In the dark, HRL410 requires several minutes to reconvert thermally to HR478. Thus, molecules in the HRL410 state must be inactive pumps since their maximal turnover number could only be a few per hour. Inorganic bases, such as azide, catalyze the deprotonation of HR520/HR640 as well as the reprotonation of HRL410. Both reactions are accelerated several hundred times by azide but the photosteady-state concentration of HRL410 remains unchanged.     

Uridine phosphorylase activity among the class mollicutes

Uridine phosphorylase activity has been used to detect mycoplasmas in cell cultures by measuring formation of¹⁴C-uracil from¹⁴C-uridine. In this report we show that all species ofMycoplasma, Acholeplasma, andUreaplasma tested exhibited uridine phorphorylase activity. Among the genusSpiroplasma, serogroups I-1, I-3, I-5, I-7, I-8, IV, XIII, and XIV lacked uridine phosphorylase activity.Present address: Ciba-Geigy, Basel, Switzerland.     

Fluorescent probes as a measure of conformational alterations induced by nucleophilic modification and proteolysis of bovine alpha 2-macroglobulin

Conformational alterations occurring in bovine alpha 2-macroglobulin (alpha 2M) resulting from proteolysis and nucleophilic modification have been monitored by UV difference spectra, circular dichroism, and changes in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonate (TNS) and bis(8-anilino-1-naphthalenesulfonate) (Bis-ANS). The results of this study indicate that these two dyes appear capable of differentiating between conformational changes induced by proteolysis and those induced by methylamine treatment. It appears that TNS is a sensitive probe for monitoring protease-induced but not methylamine-induced conformational changes in bovine alpha 2M. Bis-ANS, on the other hand, appears suitable for monitoring conformational changes induced by methylamine treatment or proteolysis of the molecule and was used as a probe to monitor the kinetics of the conformational change induced by methylamine treatment. It was found that the conformational change did not occur simultaneously with cleavage of the thiol ester bonds by the nucleophile, measured by titration of free sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoate). The data are consistent with a model in which initial nucleophilic attack results in exposure of sulfhydryl groups, resulting in a conformational change measured by an increase in fluorescence. This event is followed by a unimolecular step representing a conformational change in the protein that results in a further increase in the fluorescence signal. The second-order rate constant for hydrolysis of the thiol ester bonds was determined to be 3.4 +/- 1.0 M-1 s-1, while the rate constant for the conformational change was (4.4 +/- 0.8) X 10(-4) s-1.     

Lack of effect of somatostatin on the stimulation of hepatic glycogenolysis by epinephrine in isolated canine hepatocytes

The effects of somatostatin on epinephrine's ability to stimulate glucose output have been examined in hepatocytes isolated from dogs fasted overnight. Half-maximal stimulation of phosphorylase a activity and glucose output occurred at an epinephrine concentration of approx. 5 X 10(-9) M. Somatostatin at 10, 100 or 1000 ng/ml had no effect on the ability of a maximal (1 X 10(-7) M) and a submaximal (1 X 10(-8) M) dose of epinephrine to activate phosphorylase at 2 min, or to stimulate glucose output over 20 min. Since the doses of somatostatin used in the present study are up to 50-fold higher than the blood concentrations commonly found when somatostatin is used in vivo to inhibit pancreatic hormone secretion, it seems unlikely that use of somatostatin in this way would affect stimulation of hepatic glycogenolysis by epinephrine in vivo.     

Halide binding by the purified halorhodopsin chromoprotein. I. Effects on the chromophore

The halorhodopsin chromoprotein, a retinal-protein complex with an apparent molecular mass of 20 kilo-daltons, exhibits all of the halide-dependent effects found for the chromophore of functional halorhodopsin in cell envelope vesicles. With increasing halide concentration (a) an alkali-dependent 580/410 nm chromophore equilibrium (attributed to reversible deprotonation of the retinal Schiff's base) is shifted toward the 580-nm chromophore and (b) the flash-induced photocycle proceeds increasingly via P520, rather than via P660. The halide-binding site(s) responsible for these effects must reside, therefore, in the chromoprotein. Chloride and bromide are about equivalent, but iodide is much less effective in these effects and in being transported. Several other anions, i.e. thiocyanate, nitrate, phosphate, and acetate, affect the absorption maximum of the chromophore but do not allow the production of P520 upon flash illumination and are not transported. However, these ions appear to compete with chloride in the flash experiments. These observations suggest that binding of anions to a relatively nonspecific site affects the protonation state of the Schiff's base in the chromophore. Either this site directly or a more specific site, connected to the first one by a sequential pathway, is involved with the photocycle intermediates and with chloride transport by halorhodopsin.     

Translational control of insulin biosynthesis. Evidence for regulation of elongation, initiation and signal-recognition-particle-mediated translational arrest by glucose.

The biosynthesis of insulin in the islets of Langerhans is strongly controlled at the translational level by glucose. We have used a variety of experimental approaches in efforts to dissect the mechanisms underlying the stimulatory effect of glucose. To assess its effects on rates of peptide-chain elongation, isolated rat islets were labelled with [3H]leucine at different glucose concentrations in the presence or absence of low concentrations of cycloheximide. Under these conditions, at glucose concentrations up to 5.6 mM, endogenous insulin mRNA did not become rate-limiting for the synthesis of insulin, whereas stimulation of non-insulin protein synthesis was abolished by cycloheximide at all glucose concentrations, indicating either that insulin synthesis is selectively regulated at the level of elongation at glucose concentrations up to 5.6 mM, or that at these concentrations inactive insulin mRNA is transferred to an actively translating pool. Glucose-induced changes in the intracellular distribution of insulin mRNA in cultured islets were assessed by subcellular fractionation and blot-hybridization using insulin cDNA probes. At glucose concentrations above 3.3 mM, cytoplasmic insulin mRNA was increasingly transferred to fractions co-sedimenting with ribosomes, and relatively more of the ribosome-associated insulin mRNA became membrane-associated, consistent with effects of glucose above 3.3 mM on both the initiation of insulin mRNA and SRP (signal recognition particle)-mediated transfer of cytosolic nascent preproinsulin to the endoplasmic reticulum. When freshly isolated islets were homogenized and incubated with 125I-Tyr-tRNA, run-off incorporation of 125I into preproinsulin was increased by prior incubation of the islets at 16.7 mM-glucose. The addition of purified SRP receptor increased the run-off incorporation of [125I]iodotyrosine into preproinsulin, especially when the islets had been preincubated at 16.7 mM-glucose. These findings taken together suggest that glucose may stimulate elongation rates of nascent preproinsulin at concentrations up to 5.6 mM, stimulates initiation of protein synthesis involving both insulin and non-insulin mRNA at concentrations above 3.3 mM, and increases the transfer of initiated insulin mRNA molecules from the cytoplasm to microsomal membranes by an SRP-mediated mechanism that involves the modification of interactions between SRP and its receptor.     

Periodate-induced lymphocyte transformation. 3. Effects of temperature and pH

The optimum conditions for sodium periodate induced lymphocyte transformation occur at low concentrations of NaIO₄, low temperature, pH between 6 and 7.5, and with short periods of exposure to the NaIO₄. Exposures for prolonged times at elevated temperatures or to NaIO₄ at pH above 7.5 result in diminished responses. The finding of maximum lymphocyte response under these conditions is compatible with the proposal that the triggering event in NaIO₄ transformation of lymphocytes is the oxidation and cleavage of the two terminal carbon atoms of sialic acid. Exposure of lymphocytes to NaIO₄ under conditions of higher temperature, higher pH or longer exposure times does not significantly reduce their ability to respond to phytohemagglutinin (PHA) or pokeweed mitogen (PWM). This finding suggests that these mitogens have different target sites from that of NaIO₄ or that they are acting on different populations of lymphocytes.     

Action spectrum for polarotropism of the germ tube of the liverwort Sphaerocarpos Donnellii aust.

Summary A series of dose response curves was worked out in the polarotropically active blue and UV spectral region. These dose response curves show changes in slope as a function of wavelength. In blue the slope values are higher than in UV. As a consequence, both the relative height of the peaks in blue and UV and the fine characteristics in blue of the action spectrum calculated on the basis of these dose response curves change decisively with different response levels taken for calculation. Therefore no decision can be made as to what photoreceptor(s) might be involved. Though at medium response levels the action spectra show similarity with action spectra of other blue-UV-mediated photoresponses, which generally are believed as being indicative of some flavin.