Zeaxanthin Formation and Energy-Dependent Fluorescence Quenching in Pea Chloroplasts under Artificially Mediated Linear and Cyclic Electron Transport

Artificially mediated linear (methylviologen) and cyclic (phenazine methosulfate) electron transport induced zeaxanthin-dependent and independent (constitutive) nonphotochemical quenching in osmotically shocked chloroplasts of pea (Pisum sativum L. cv Oregon). Nonphotochemical quenching was quantitated as Stern-Volmer quenching (SV_N) calculated as (F_m/F′_m)-1 where F_m is the fluorescence intensity with all PSII reaction centers closed in a nonenergized, dark-adapted state and F′_m is the fluorescence intensity with all PSII reaction centers closed in an energized state. Reversal of quenching by nigericin and electron-transport inhibitors showed that both quenching types were energy-dependent SV_N. Under light-induced saturating ΔpH, constitutive-SV_N reached steady-state in about 1 minute whereas zeaxanthin-SV_N continued to develop for several minutes in parallel with the slow kinetics of violaxanthin deepoxidation. SV_N above the constitutive level and relative zeaxanthin concentration showed high linear correlations at steady-state and during induction. Furthermore, F_o quenching, also treated as Stern-Volmer quenching (SV_O) and calculated as (F_o/F′_o)-1, showed high correlation with zeaxanthin and consequently with SV_N (F_o and F′_o are fluorescence intensities with all PSII reaction centers in nonenergized and energized states, respectively). These results support the view that zeaxanthin increases SV_N above the constitutive level in a concentration-dependent manner and that zeaxanthin-dependent SV_N occurs in the pigment bed. Preforming zeaxanthin increased the rate and extent of SV_N, indicating that slow events other than the amount of zeaxanthin also affect final zeaxanthin-SV_N expression. The redox state of the primary electron acceptor of photosystem II did not appear to determine SV_N. Antimycin, when added while chloroplasts were in a dark-adapted or nonenergized state, inhibited both zeaxanthin-SV_N and constitutive-SV_N induced by linear and cyclic electron transport. These similarities, including possible constitutive F_o quenching, suggest that zeaxanthin-dependent and constitutive SV_N are mechanistically related.     

Bromo-eudistomin D, a novel inducer of calcium release from fragmented sarcoplasmic reticulum that causes contractions of skinned muscle fibers

Bromo-eudistomin D induced a contraction of the chemically skinned fibers from skeletal muscle at concentrations of 10 microM or more. This contractile response to bromo-eudistomin D was completely blocked by 10 mM procaine. The extravascular Ca2+ concentrations of the heavy fractions of the fragmented sarcoplasmic reticulum (HSR) were measured directly by a Ca2+ electrode to examine the effect of bromo-eudistomin D on the sarcoplasmic reticulum. After the HSR was loaded with Ca2+ by the ATP-dependent Ca2+ pump, the addition of 10 microM bromo-eudistomin D caused Ca2+ release that was followed by spontaneous Ca2+ reuptake. In the presence of 2 microM ruthenium red or 4 mM MgCl2, no Ca2+ release was induced by 20 microM bromo-eudistomin D. The rate of 45Ca2+ efflux from HSR, which had been passively preloaded with 45Ca2+, was accelerated 7 times by 10 microM bromo-eudistomin D. The concentration of bromo-eudistomin D for half-maximum effect on the apparent efflux rate was 1.5 microM, while that of caffeine was 0.6 mM. The bromo-eudistomin D-evoked efflux of 45Ca2+ was abolished by 2 microM ruthenium red or 0.5 mM MgCl2. Bromo-eudistomin D was found to be 400 times more potent than caffeine in its Ca2+-releasing action but was similar in its action in other respects. These results indicate that bromo-eudistomin D may induce Ca2+ release from the sarcoplasmic reticulum through physiologically relevant Ca2+ channels.     

Pinealectomy delays puberty in ewe lambs

Fifteen pinealectomized and 15 unoperated ewes were exposed to constant light for 3 weeks before and 10 weeks after lambing. Fourteen pinealectomized and 15 unoperated ewes were allowed to lamb outdoors. Five ewe lambs born in constant light to the 2 groups of dams were pinealectomized at 10 weeks of age. Ewes and lambs were then returned to the field. Puberty (determined by weekly progesterone analysis) was significantly delayed (P less than 0.05) in the pinealectomized ewe lambs. Median pubertal age in pineal-intact ewe lambs was 37 weeks compared to 49 weeks in pinealectomized lambs. Constant light during the first 10 weeks of life had no effect upon puberty onset nor did the pineal status of the dam. Control lambs entered seasonal anoestrus at the time pinealectomized ewe lambs were entering puberty. Pinealectomized lambs entered anoestrus at the same time as control lambs were beginning their second breeding season. These results confirm a key role of pineal-mediated hormonal signals in the control of puberty in the sheep.     

A simple method using alizarin red S for the detection of calcium in epoxy resin embedded tissue

This report presents a simple procedure for staining 1-2 microns epoxy plastic sections of cells and mineralizing matrix present in fetal bovine bone tissue cultures. A 0.3% aqueous toluidine blue O solution was used as a cellular stain and was followed with 2% alizarin red S for the detection of calcium at sites of mineralization. Effects of concentration and pH of alizarin red S on the penetration of epon embedded thick sections were investigated. Optimal staining was achieved with a 2% aqueous alizarin red S solution adjusted to a pH of 5.5-6.5. This staining procedure provides unusually clear contrast between mineral and bone cells in plastic sections for light microscopy.     

Induction of intra- and extra-cellular phospholipids in the lungs of rats exposed to silica.

Intracellular and extracellular compartments of phospholipids in the lungs of rats were examined 28 days after intratracheal injection of silica (200 mg/kg). All compartments containing phospholipids were elevated, but the largest increases were seen in the intracellular and extracellular pulmonary surfactant. Intracellular pulmonary surfactant increased 123-fold from 1.18 +/- 0.65 to 144.9 +/- 53.8 and the extracellular surfactant increased 22-fold from 1.17 +/- 0.04 to 25.1 +/- 7.1 mg per pair of rat lungs respectively. The phospholipid composition of intracellular and extracellular surfactant did not change in response to silica, except for an almost 2-fold increase in the percentage of total phosphatidylinositol in both compartments. The phospholipid content of the lungs increased from 24.9 +/- 4.6 to 268.6 +/- 20.8 mg, with the intracellular and extracellular surfactant accounting for 59.1 and 24.6% of this total increase respectively. These data demonstrate that the major increases in the phospholipid content of the lungs induced by silica is associated with the pulmonary-surfactant system.     

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.