Anti-Ig induces release of inositol 1,4,5-trisphosphate, which mediates mobilization of intracellular Ca++ stores in B lymphocytes

Evidence from a variety of laboratories indicates that crosslinking of B cell mIg induces a rapid increase in intracellular free calcium (Ca++i). This mobilized Ca++ appears to act in concert with diacylglycerol (DAG; also released upon mIg cross-linking) to optimally activate Ca++/phospholipid-dependent protein kinase C, which plays a pivotal role in B cell activation. Here we report analysis of the source of this mobilized calcium and the mechanism responsible for its release into the cytosol. We observed the cross-linking of mIg induces the release of inositol 1,4,5-trisphosphate (InsP3), presumably as a result of action of phospholipase C on plasma membrane phosphatidylinositol 4,5-bisphosphate (PtdInsP2). The release of InsP3 and the elevation of Ca++i are coincidental, suggesting that they may be causally related. Finally, we demonstrate that submicromolar doses of InsP3 induce release of Ca++ from permeabilized cells that had preaccumulated 45Ca++ in the endoplasmic reticulum. On the basis of these findings we suggest that mIg cross-linking leads to mobilization of Ca++, in part by causing hydrolysis of PtdInsP2, yielding InsP3, which in turn causes release of calcium from the endoplasmic reticulum.     

Mannose metabolism in corn and its impact on leaf metabolites, photosynthetic gas exchange, and chlorophyll fluorescence

When intact corn leaves were provided millimolar concentrations of d-mannose through the transpiration stream photosynthesis was inhibited; 5.7 millimolar resulted in a 50% inhibition of the carbon exchange rate. This inhibition was partially reversible by the addition of orthophosphate to the feeding solution. Mannose metabolism by corn leaves was limited in that it did not act as a resource for sucrose or starch synthesis. Mannose 6-phosphate accumulated in the leaf tissues and was slowly metabolized by a pathway involving mannose 1-phosphate. Correlated with the mannose-6-phosphate accumulation were decreases in ATP, orthophosphate, sucrose, and phosphoenolpyruvate and increases in starch and maltose. When provided in the transpiration stream mannose had access to both mesophyll and bundle sheath cells. Mannose feeding led to oscillations in steady state chlorophyll fluorescence emission (680 nanometers) and an elimination of the Kautsky effect during fluorescence induction. Pyridoxal 5-phosphate and 2,4-dinitrophenol were found to be inhibitors of CO₂ exchange when provided in the transpiration stream of intact corn leaves. However, Pyridoxal 5-phosphate induced a quenching of steady state fluorescence while 2,4-dinitrophenol led to an increase in fluorescence emission.     

Levels of Free and Conjugated Abscisic Acid in Developing Floral Organs of the Navel Orange (Citrus sinensi [L.] Osbeck cv Washington)

The levels of abscisic acid (ABA) and alkaline-hydrolyzable ABA-conjugate (putatively identified as the glucosyl ester, abscisyl-β-d-glucopyranoside) were determined by enzyme immunoassay in the organs of developing navel orange (Citrus sinensis [L.] Osbeck cv Washington) flowers. Although both compounds were detected in every tissue, developmentally related differences between organs in the total and relative contents were observed. The highest ABA levels were observed in the stigma/style shortly after anthesis (11.5 ± 0.6 nanomoles ABA per gram fresh weight and 4.8 ± 0.6 nanomoles ABA-conjugate per gram fresh weight); whereas, the highest ABA-conjugate levels were observed at the same time in the floral disc (hypogynous disc plus calyx; 3.5 ± 0.1 nmol nanomols ABA per gram fresh weight and 11.8 ± 0.9 nanomoles ABA-conjugate per gram fresh weight). These results suggest that differences in ABA content reflect tissue-specific variation in the facility for ABA conjugation. Increased ABA levels were observed in the stigma/style near anthesis; however, a relationship with pollination is discounted, since `Washington' navel orange flowers are male sterile and devoid of pollen.     

Sensitivity to X-irradiation of peripheral blood lymphocytes from ageing donors

The proliferation of human blood lymphocytes from ageing donors, responding to concanavalin A, showed greater sensitivity to inhibition by X-rays than similar cells from younger donors. This increased sensitivity was associated with deficiency in repair of X-ray-induced damage to nuclear material, as measured by density in sucrose gradients, and with increased incidence of chromosomal damage following exposure of freshly isolated lymphocytes. There was also an increased frequency of spontaneous chromosomal aberrations in ageing subjects whose lymphocytes were deficient in repair of DNA damage.     

A short-chain aldehyde is a major lipoxygenase product in arachidonic acid-stimulated porcine leukocytes

Porcine leukocytes convert exogenous arachidonic acid to a complex array of products derived via the 5-, 12-, and 15-lipoxygenase pathways of metabolism. The major monohydroxylated metabolite following addition of 100 microM arachidonic acid is 12-hydroxyeicosatetraenoic acid. Of the more polar compounds on reverse-phase high pressure liquid chromatography, the most prominent is a previously uncharacterized arachidonate product which chromatographs near to the omega-oxidized metabolites of leukotriene B4. The structure of this new product was examined by high pressure liquid chromatography, UV, NMR, and also by gas chromatography-mass spectrometry of several derivatives; it was identified as 12-oxododeca-5,8,10-(Z,Z,E)-trienoic acid. It is proposed that this C-12 trienal acid is formed from 12-hydroperoxyeicosatetraenoic acid by a cleavage reaction catalyzed by the leukocyte 12-lipoxygenase in the presence of excess arachidonic acid and under anaerobic conditions. These conditions are satisfied by addition of 100 microM arachidonic acid to the leukocyte suspension (3 X 10(7) cells/ml); 12-hydroperoxyeicosatetraenoic acid is formed as the major product, excess arachidonic acid is available, and the concomitant leukocyte respiratory burst quickly depletes the solution of oxygen. Preliminary experiments indicate that this aldehyde product has significant biological activity in the activation of leukocytes. In the course of an intense inflammatory reaction it is conceivable that the conditions for synthesis of this C-12 trienal acid and related aldehydes could prevail; such aldehydes would constitute an additional class of lipoxygenase product which exacerbates the process of inflammation.     

Mutagenicity of drinking water detected by the Tradescantia micronucleus test

Spring Lake reservoir of Macomb, Illinois, is a typical model of the drinking water supply of some midwestern towns of the United States. Water samples collected periodically in 1980 and 1981 from this lake were tested for mutagenicity using the Tradescantia micronucleus (Trad-MCN) test, a highly sensitive mutagen-detecting bioassay. Water samples from 1981 were also analyzed chemically. The micronucleus (MCN) frequency peaked (12-14 MCN/100 tetrads) in mid-July in both years, as compared with the average frequency (5 MCN/100 tetrads) of the base-line control that was maintained in nutrient solution (prepared with distilled water and pure chemicals). Drinking water from the tap was tested in parallel with lake water, and its mutagenicity tended to fluctuate with the mutagenicity of the lake water.     

Reduction potential of iron in transferrin

The reduction potential of Fe3+ in transferrin was measured spectrophotometrically by equilibration with methyl viologen in the presence of sodium dithionite. For an ionic strength near 0.1 M at 25 degrees C and pH 7.3 under 0.048 atm. CO2, half of the iron is reduced at a potential near -0.40 V (vs. standard hydrogen electrode). At least one disulfide bond of the protein is partially reduced at a potential of -0.44 V, as evidenced by reaction with [14C]iodoacetate.     

Radiosensitivity of peripheral blood lymphocytes in autoimmune disease

The proliferation of peripheral blood lymphocytes, cultured with Con A, can be inhibited by ionizing radiation. Lymphocytes from patients with conditions associated with autoimmunity, such as rheumatoid arthritis, systemic lupus erythematosus and polymyositis, are more radiosensitive than those from healthy volunteers or patients with conditions not associated with autoimmunity. The nuclear material isolated from the lymphocytes of patients with autoimmune diseases is, on average, lighter in density than the nuclear material from most healthy controls. This difference in density is not related to increased sensitivity to ionizing radiation but the degree of post-irradiation change in density (lightening) is proportional to the initial density, i.e. more dense nuclear material always shows a greater upward shift after radiation. The recovery of preirradiation density of nuclear material, 1 h after radiation exposure, taken as an indication of DNA repair, correlates with the radiosensitivity of lymphocyte proliferation (Con A response); failure to return to pre-irradiation density being associated with increased sensitivity of proliferative response. These results require extension but, taken with previously reported studies of the effects of DNA methylating agents, support the idea that DNA damage and its defective repair could be important in the aetio-pathogenesis of autoimmune disease.     

Microsequence analysis of peptides and proteins. V. Design and performance of a novel gas-liquid-solid phase instrument

We describe the construction and performance of a novel, automated, Edman chemistry-based microsequencer. The reagent and solvent delivery system, the reaction cartridge for coupling and cleavage, and the conversion flask are all constructed from chemically inert perfluoroelastomers. The delivery valves are of a new design incorporating the use of electromagnetically actuated solenoids and zero-dead-volume construction, and may be connected in a modular fashion resulting in multiple inputs with a single output line which can be flushed with inert gas. The bottle closures are of a new design based on an all-Teflon compression fitting. The reaction cartridge and conversion flask are thermostated by solid-state heaters in an aluminum block. The overall size of the instrument is 25 X 34 X 14 in. The chemistry utilizes 2% aqueous triethylamine as the coupling base which is delivered to the reaction cartridge via a stream of nitrogen. The "gas-phase" delivery of the coupling base and the cleavage acid (trifluoroacetic acid) is modeled after the method described by R. M. Hewick et al. (J. Biol. Chem. 256, 7990-7997,1981). The instrument has performed well over a period of 3 years in terms of low background peaks, sensitivity in the picomole range, and reliability of operation. The use of economical components, ease of construction and operation, and sensitive analytical capability make this instrument a useful tool for microsequence analysis of peptides and proteins.     

Random association of Epstein-Barr virus genomes with host cell metaphase chromosomes in Burkitt''s lymphoma-derived cell lines.

The random association of Epstein-Barr virus DNA with host cell metaphase chromosomes of all sizes in Burkitt's lymphoma-derived cell lines was demonstrated by two substantially different techniques, namely fluorescence-activated chromosome sorting and in situ hybridization. The nature and potential importance of this association are discussed.