Aspirin-triggered lipoxins (15-epi-LX) are generated by the human lung adenocarcinoma cell line (A549)-neutrophil interactions and are potent inhibitors of cell proliferation.

BACKGROUND: The mechanism by which aspirin (ASA) acts to protect against human cancer is not yet known. We recently showed that ASA triggers the formation of a new series of potent bioactive eicosanoids, 15-epi-lipoxins (15-epi-LXs or ASA-triggered LX [ATL]), during interactions between prostaglandin endoperoxide synthase-2 (PGHS-2) in endothelial cells and 5-lipoxygenase (LO) in leukocytes. Here, we investigated the transcellular biosynthesis of these eicosanoids during costimulation of the human tumor A549 cell line (alveolar type II epithelial cells) and neutrophils, and evaluated their impact on cell proliferation. MATERIALS AND METHODS: A549 cells and isolated neutrophils were coincubated and mRNA expression levels of key enzymes in eicosanoid biosynthesis were measured. In addition, product formation was analysed by physical methods. The effect of LX on cell proliferation was determined by using a soluble microculture tetrazolium (MTT) assay and by measuring [3H]-thymidine incorporation. RESULTS: Interleukin-1 beta (IL-1 beta)-primed A549 cells showed selective elevation in the levels of PGHS-2 mRNA and generated 15-hydroxyeicosatetraenoic acid (15-HETE). ASA markedly increased 15-HETE formation by A549 cells, while treatment with an inhibitor of cytochrome P450 reduced by approximately 50%, implicating both PGHS-2- and cytochrome P450-initiated routes in 15-HETE biosynthesis in these cells. Maximal production of 15-HETE from endogenous sources occurred within 24 hr of cytokine (IL-1 beta) exposure and declined thereafter. Chiral analysis revealed that approximately 85% of ASA-triggered epithelial-derived 15-HETE carries its carbon 15 alcohol group in the R configuration. Costimulation of ASA-treated A549 cells and polymorphonuclear neutrophilic leukocytes (PMN) led to production of both LXA4 and LXB4, as well as 15-epi-LXA4 and 15-epi-LXB4 (9.5 +/- 0.5 ng LX/10(7) A549 cells). 15-epi-LXA4 accounted for approximately 88% of the total amount of LXA4 produced. In addition to LXs, stimulation of A549 cells and PMN also liberated substantial amounts (77.2 +/- 8.2 ng/10(7) A549 cells) of peptidoleukotrienes (pLTs), which were not generated by either cell type alone. Addition of ASA to these co-incubations led to an increase in the amounts of LXs generated that was paralleled by a decrease in pLTs. LXA4, LXB4, 15-epi-LXA4 and 15-epi-LXB4, as well as dexamethasone, inhibited cell proliferation at 100 nM range with a rank order of activity of 15-epi-LXB4 >>> LXB4 > dexamethasone > or = 15-epi-LXA4 > LXA4. CONCLUSIONS: These results indicate that ASA promotes the formation of antiproliferative 15-epi-LXs by epithelial cell-leukocyte interactions. Moreover, they suggest that these novel eicosanoids, when generated within the microenvironment of tissues, may contribute to ASA's therapeutic role in decreasing the risk of human cancer.     

15(S)-HETE modulates LTB(4) production and neutrophil chemotaxis in chronic bronchitis

We evaluated the levels of15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE] and the expression of15-lipoxygenase (15-LO) mRNA in induced sputum obtained from 10 controland 15 chronic bronchitis subjects. 15(S)-HETE was evaluated by reversephase high-performance liquid chromatography separationfollowed by specific RIA. 15-LO mRNA expression was determined byprimed in situ labeling. The levels of both soluble and cell-associated 15(S)-HETE resulted significantly higher in chronic bronchitis than incontrol subjects. The percentage of cells expressing 15-LO mRNA wassignificantly higher in chronic bronchitis than in control subjects(P < 0.01). Double staining for specific cell typemarkers and 15-LO mRNA showed macrophages and neutrophils positive for 15-LO, whereas similar staining of peripheral blood neutrophils did notshow evidence for 15-LO expression, suggesting that expression of 15-LOin neutrophils takes place on migration into the airways. Because15(S)-HETE inversely correlated with the percentage of neutrophils insputum of chronic bronchitis subjects, we studied the effect of15(S)-HETE on leukotriene B₄ (LTB₄) productionin vitro and evaluated the concentration of LTB₄ in inducedsputum and the contribution of LTB₄ to the chemotacticactivity of induced sputum samples ex vivo. The results obtainedindicate that macrophages and neutrophils present within the airways ofchronic bronchitis subjects express 15-LO mRNA; increased basal levelsof 15(S)-HETE may contribute to modulate, through the inhibition of5-lipoxygenase metabolites production, neutrophil infiltration andairway inflammation associated with chronic bronchitis.

     

Differential subcellular localization of COX-2 in macrophages phagocytosing heat-killed Mycobacterium bovis BCG

Cyclooxygenase-2 (COX-2)-mediated prostaglandin E₂ (PGE₂) biosynthesis by macrophages downregulates microbicidal activities in innate and acquired immune responses against intracellular bacteria. Previous studies in mice showed that intraperitoneal administration of heat-killed Mycobacterium bovis bacillus Calmette-Guérin (HK-BCG) resulted in induction of splenic PGE₂-releasing macrophages in 7–14 days. In contrast, HK-BCG induced catalytically inactive COX-2 at relatively high levels in the macrophages within 1 day. In the present study, we found that COX-2 was localized subcellularly in the nuclear envelope (NE) 7 and 14 days after HK-BCG treatment, whereas COX-2 was dissociated from the NE 1 day after treatment. At 1 day after treatment, the majority of COX-2-positive macrophages had phagocytosed HK-BCG. In contrast, no intracellular HK-BCG was detected 7 and 14 days after treatment in COX-2-positive macrophages, where COX-2 was associated with the NE. However, when macrophages phagocytosed HK-BCG in vitro, all COX-2 was associated with the NE. Thus the administration of HK-BCG induces the biphasic COX-2 expression of an NE-dissociated catalytically inactive or an NE-associated catalytically active form in splenic macrophages. The catalytically inactive COX-2-positive macrophages develop microbicidal activities effectively, since they lack PGE₂ biosynthesis. nuclear envelope; autoimmune disease; prostaglandin E₂     

Limitations to net photosynthesis as affected by nitrogen status in jack pine (Pinus banksiana Lamb.) seedlings

Relative limitations of nitrogen (N) status on the processescontributing to photosynthetic rate (A) were investigated. Jackpine {Pinus banksiana Lamb.) seedlings from seeds grown in sandculture were supplied with four different N treatments for 6weeks, which resulted in a needle N content ranging from 50–85mmol m^–2 (14–32 mg g^–1 dry weight). Leaf gasexchange at varying CO₂ levels was measured and limitationson A₃₅₀ (A at ambient CO₂ level) caused by finite, limitingcarboxylation efficiency (c.e.), maximum A (A_max)and stomatalconductance were estimated from an analysis of the responseof A to internal CO₂ concentration. Although c.e. and A_max decreasedlinearly with the decline in needle N, the magnitudes of theirchanges relative to A₃₅₀ differed. A_max varied with A₃₅₀ andalways exceeded A₃₅₀ by 37–38% c.e., however, declinedfaster than A₃₅₀, as needle N level decreased. Consequently,relative limitation on A₃₅₀ caused by inefficient A_max remainedconstant, but limitations caused by c.e. increased by 10–15%at low N levels. In contrast, the limitation by stomatal conductancedeclined initially, but remained stable when N content droppedbelow 75 mmol m^–2. The results suggest: (1) a decreasein biochemical capacity, but not stomatal conductance, contributedto the reduction of A₃₅₀ induced by N-deficiency in jack pineseedlings; and (2) the capacity of carboxylation appeared tobe impaired more than that of electron transport and/or photophosphorylationand its reduction may be the major reason for the reductionin A₃₅₀. Key words: A–C_i analysis, carboxylation efficiency, electron transport, nitrogen deficiency, stomatal conductance     

Regulation of K+ current in human airway epithelial cells by exogenous and autocrine adenosine

The regulatory actions ofadenosine on ion channel function are mediated by four distinctmembrane receptors. The concentration of adenosine in the vicinity ofthese receptors is controlled, in part, by inwardly directed nucleosidetransport. The purpose of this study was to characterize the effects ofadenosine on ion channels in A549 cells and the role of nucleosidetransporters in this regulation. Ion replacement and pharmacologicalstudies showed that adenosine and an inhibitor of human equilibrative nucleoside transporter (hENT)-1, nitrobenzylthioinosine, activated K⁺ channels, most likely Ca²⁺-dependentintermediate-conductance K⁺ (I_K)channels. A₁ but not A₂ receptor antagonistsblocked the effects of adenosine. RT-PCR studies showed that A549 cellsexpressed mRNA for I_K-1 channels as well asA₁, A_2A, and A_2B but notA₃ receptors. Similarly, mRNA for equilibrative (hENT1 andhENT2) but not concentrative (hCNT1, hCNT2, and hCNT3) nucleosidetransporters was detected, a result confirmed in functional uptakestudies. These studies showed that adenosine controls the function ofK⁺ channels in A549 cells and that hENTs play a crucialrole in this process.

     

Cyclooxygenase-2-mediated DNA damage

Rat intestinal epithelial cells that express the cyclooxygenase-2 (COX-2) gene permanently (RIES cells) were used as a model of in vivo oxidative stress. A targeted lipidomics approach showed that 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) was the major hydroxylated non-esterified lipid formed in unstimulated intact cells. The corresponding hydroperoxide, 15(S)-hydroperoxyeicosatetraenoic acid (15(S)-HPETE) undergoes homolytic decomposition to the DNA-reactive bifunctional electrophile 4-oxo-2(E)-nonenal, a precursor of heptanone-etheno-2'-deoxyguanosine. This etheno adduct was identified in the DNA of RIES cells. A dose-dependent increase in adduct levels was observed in the presence of vitamin C. This suggested that vitamin C increased lipid hydroperoxide-mediated 4-oxo-2(E)-nonenal formation in the cells. The selective COX-2 inhibitor NS-398 was protective against cellular DNA damage but was less effective if vitamin C was present. Prostaglandin E(2) and 15(S)-HETE biosynthesis were completely inhibited by 110 mum NS-398 in the intact RIES cells. No inhibition of COX-1 was detected in the wild-type RIE cells at this concentration of NS-398. Arachidonic acid treatment of RIES cell lysates and ionophore stimulation of intact RIES cells produced significantly more 15(R)-HETE than the untreated intact cells. These preparations also both produced 11(R)-HETE, which was not detected in the intact cells. Aspirin treatment of the intact unstimulated RIES cells resulted in the exclusive formation of 15(R)-HETE in amounts that were slightly higher than the original 15(S)-HETE observed in the absence of aspirin, implying that significant amounts of 15(R)-HPETE had also been formed. 15(R)-HPETE should give exactly the same amount of heptanone-etheno-2'-deoxyguanosine as its 15(S)-enantiomer. However, no increase in heptanone-etheno adduct formation occurred in the aspirin-treated cells. The present study suggests a potential mechanism of tumorigenesis that involves DNA adduct formation from COX-2-mediated lipid peroxidation rather than prostaglandin formation. Therefore, inhibition of COX-2-mediated lipid hydroperoxide formation offers a potential therapeutic alternative to COX-2 inhibitors in chemoprevention strategies.     

COX-2-dependent and -independent biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes

Biosynthesis of 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) in leukocytes involves consecutive oxygenation of arachidonic acid by 5-lipoxygenase (LOX) and 15-LOX in either order. Here, we analyzed the contribution of cyclooxygenase (COX)-2 to the biosynthesis of 5,15-diHETE and 5,11-diHETE in isolated human leukocytes activated with lipopolysaccharide and calcium ionophore A23187. Transformation of arachidonic acid was initiated by 5-LOX providing 5S-HETE as a substrate for COX-2 forming 5S,15S-diHETE, 5S,15R-diHETE, and 5S,11R-diHETE as shown by LC/MS and chiral phase HPLC analyses. The levels of 5,15-diHETE were 0.45 ± 0.2 ng/10⁶ cells (mean ± SEM, n = 6), reaching about half the level of LTB₄ (1.3 ± 0.5 ng/10⁶ cells, n = 6). The COX-2 specific inhibitor NS-398 reduced the levels of 5,15-diHETE to below 0.02 ng/10⁶ cells in four of six samples. Similar reduction was achieved by MK-886, an inhibitor of 5-LOX activating protein but the above differences were not statistically significant. Aspirin treatment of the activated cells allowed formation of 5,15-diHETE (0.1 ± 0.05 ng/10⁶ cells, n = 6) but, as expected, abolished formation of 5,11-diHETE. The mixture of activated cells also produced 5S,12S-diHETE with the unusual 6E,8Z,10E double bond configuration, implicating biosynthesis by 5-LOX and 12-LOX activity rather than by hydrolysis of the leukotriene A₄-epoxide. Exogenous octadeuterated 5S-HETE and 15S-HETE were converted to 5,15-diHETE, implicating that multiple oxygenation pathways of arachidonic acid occur in activated leukocytes. The contribution of COX-2 to the biosynthesis of dihydroxylated derivatives of arachidonic acid provides evidence for functional coupling with 5-LOX in activated human leukocytes.     

Variations with Age in Net Assimilation Rate and other Growth Attributes of Sugar-beet, Potato, and Barley in a Controlled Environment: With two Figures in the Text

Sugar-beet, potato, and barley plants were grown in a controlledenvironment, for periods of up to 10 weeks from sowing, witha light intensity of 1,8oo f.c. (4·9 cal./cm.²/hr.) anda temperature of 20° C. during the 18-hour photoperiod and15° C. during the dark period, to test whether net assimilationrate varied with age and differed between the three species. Net assimilation rate of all species based on leaf area (E_A)fell approximately linearly with time. During 5 weeks E_A ofsugar-beet decreased by only about 20 per cent. and E_A of potatodecreased by 50 per cent. E_A of barley remained approximatelyconstant for 4 weeks after sowing and was halved during thesubsequent 4 weeks. The average value of E_A for all times wasgreatest for sugarbeet and least for barley. Net assimilation rates based on leaf weight (E_W) and leaf N(E_N) decreased at about 15 per cent. of the initial value perweek for all species; this was similar to the mean rate of decreaseof E_A of potato and barley, but greater than that of E_A of sugar-beet.Mean values of E_W or E_N for potato and barley were similar andless than for sugar-beet. Relative growth rate (R_W), relative leaf growth-rate (R_A), andleaf-area ratio (F) fell with time at similar rates for allspecies. Average values of R_W decreased and of F increased inthe order sugar-beet, potato, barley. R_A was greatest for potatoand least for barley.     

Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts

Osteoblasts subjected to fluid shearincrease the expression of the early response gene, c-fos, andthe inducible isoform of cyclooxygenase, COX-2, two proteins linked tothe anabolic response of bone to mechanical stimulation, in vivo. Theseincreases in gene expression are dependent on shear-induced actinstress fiber formation. Here, we demonstrate that MC3T3-E1osteoblast-like cells respond to shear with a rapid increase inintracellular Ca²⁺ concentration([Ca²⁺]_i) that wepostulate is important to subsequent cellular responses to shear. Totest this hypothesis, MC3T3-E1 cells were grown on glass slides coatedwith fibronectin and subjected to laminar fluid flow (12 dyn/cm²). Before application of shear, cells were treatedwith two Ca²⁺ channel inhibitors or various blockers ofintracellular Ca²⁺ release for 0.5-1 h. Althoughgadolinium, a mechanosensitive channel blocker, significantly reducedthe [Ca²⁺]_i response, neithergadolinium nor nifedipine, an L-type channel Ca²⁺ channelblocker, were able to block shear-induced stress fiber formation andincrease in c-fos and COX-2 in MC3T3-E1 cells. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid-AM, an intracellular Ca²⁺ chelator, or thapsigargin,which empties intracellular Ca²⁺ stores, completelyinhibited stress fiber formation and c-fos/COX-2 production in shearedosteoblasts. Neomycin or U-73122 inhibition of phospholipase C, whichmediates D-myo-inositol 1,4,5-trisphosphate (IP₃)-induced intracellular Ca²⁺ release, alsocompletely suppressed actin reorganization and c-fos/COX-2 production.Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform ofU-73122, did not inhibit these shear-induced responses. These resultssuggest that IP₃-mediated intracellular Ca²⁺release is required for modulating flow-induced responses in MC3T3-E1 cells.

     

A3 adenosine receptors regulate Cl- channels of nonpigmented ciliary epithelial cells

Adenosinestimulates Cl channels ofthe nonpigmented (NPE) cells of the ciliary epithelium. We sought toidentify the specific adenosine receptors mediating this action.Cl channel activity inimmortalized human (HCE) NPE cells was determined by monitoring cellvolume in isotonic suspensions with the cationic ionophore gramicidinpresent. The A₃-selective agonistN⁶-(3-iodobenzyl)-adenosine-5'-N-methyluronamide(IB-MECA) triggered shrinkage (apparentK_d = 55 ± 10 nM). A₃-selective antagonists blocked IB-MECA-triggered shrinkage, andA₃-antagonists (MRS-1097, MRS-1191, and MRS-1523) also abolished shrinkage produced by 10 µMadenosine when all four known receptor subtypes are occupied. TheA₁-selective agonistN⁶-cyclopentyladenosineexerted a small effect at 100 nM but not at higher or lowerconcentrations. The A_2A agonistCGS-21680 triggered shrinkage only at high concentration (3 µM), aneffect blocked by MRS-1191. IB-MECA increased intracellularCa²⁺ in HCE cells and alsostimulated short-circuit current across rabbit ciliary epithelium.A₃ message was detected in bothHCE cells and rabbit ciliary processes using RT-PCR. We conclude that human HCE cells and rabbit ciliary processes possessA₃ receptors and that adenosinecan activate Cl channels inNPE cells by stimulating these A₃ receptors.     

COX-1 dependent biosynthesis of 15-hydroxyeicosatetraenoic acid in human mast cells

15-hydroxyeicosatetraenoic acid (15-HETE) is an arachidonic acid derived lipid mediator which can originate both from 15-lipoxygenase (15-LOX) activity and cyclooxygenase (COX) activity. The enzymatic source determines the enantiomeric profile of the 15-HETE formed. 15-HETE is the most abundant arachidonic acid metabolite in the human lung and has been suggested to influence the pathophysiology of asthma. Mast cells are central effectors in asthma, but there are contradictory reports on whether 15-HETE originates from 15-LOX or COX in human mast cells. This prompted the current study where the pathway of 15-HETE biosynthesis was examined in three human mast cell models; the cell line LAD2, cord blood derived mast cells (CBMC) and tissue isolated human lung mast cells (HLMC). Levels and enantiomeric profiles of 15-HETE and levels of the downstream metabolite 15-KETE, were analyzed by UPLC-MS/MS after stimulation with anti-IgE or calcium ionophore A23187 in the presence and absence of inhibitors of COX isoenzymes. We found that 15-HETE was produced by COX-1 in human mast cells under these experimental conditions. Unexpectedly, chiral analysis showed that the 15(R) isomer was predominant and gradually accumulated, whereas the 15(S) isomer was metabolized by the 15-hydroxyprostaglandin dehydrogenase. We conclude that during physiological conditions, i.e., without addition of exogenous arachidonic acid, both enantiomers of 15-HETE are produced by COX-1 in human mast cells but that the 15(S) isomer is selectively depleted by undergoing further metabolism. The study highlights that 15-HETE cannot be used as an indicator of 15-LOX activity for cellular studies, unless chirality and sensitivity to pharmacologic inhibition is determined.     

Variation in the DNA Content of Glycine Species

Nuclei were isolated from cotyledons of a range of accessionsfrom 14 species of Glycine. These were stained with ethidiumbromide and the relative fluorescence for each genotype wasmeasured by flow cytometry. The DNA content was estimated bycomparison of relative fluorescence with that from nuclei fromseedling leaves of Allium cepa, whose DNA content has been calculatedpreviously by chemical assay. The 4C amounts for diploid Glycineranged from 3.80 to 6.59 pg. Two groups of diploid species appearedfrom the analysis. The first consisted of species with amountsranging from 3.80 to 5.16 pg and included G. canescens (AA),G. argyrea (A₁ A₁), G. clandestina (A²A²), G. microphylla(BB),G. latifolia (B₁B₁), G. tabacina 2n=40 (B²B²), G. tomentella2n=38 (EE) and 2n=40 (DD), G. max and G. soja (GG), G. arenariaand G. latrobeana. A second group had higher DNA contents rangingfrom 5.27 to 6.59 pg, and consisted of G. curvata, G. cyrtoloba(CC), and G. falcata (FF). The polyploid species, G. tabacina2n=80 (AABB, BBB¹B¹), G. tomentella 2n=78 and 2n=80 (AAEE andDDEE, respectively) contained amounts approximating to the sumsof the respective parental diploid species thought to have givenrise to these allotetraploids. Intraspecific variation was detectedin the DNA content of G. canescens. Within the overall distributionof DNA amounts found in A genome species, each genome containeda range of DNA contents specific to that species. This phenomenonwas also detected amongst B genome species.     

Purification and Immunochemical Characterization of NADP-Dependent Glyceraldehyde 3-Phosphate Dehydrogenase of Euglena gracilis

NADP-dependent glyceraldehyde 3-phosphate dehydrogenase fromEuglena gracilis (EC 1.2.1.13 [EC] ) was purified about 170-fold bya two-step procedure involving DEAE-SH cellulose chromatographyand affinity chromatography on ADP-Sepharose. The homogeneousenzyme from mildly sonicated cells contained equal amounts oftwo types of subunits with mol wts of 34,000 (A) and 38,000(B). The active enzyme had a mol wt 144,000 and is thereforean A₂B₂ tetramer. Enzyme from strongly sonicated Euglena cellscontained, in addition, a second allomer with a probable A₄structure. NADdependent glyceraldehyde 3-phosphate dehydrogenase,a tetramer with 36,000 mol wt subunits, was unrelated immunologicallyto the NADP-dependent enzyme although the latter also showedminor NAD-dependent activity. Both isoenzymes of the NADPlinkedglyceraldehyde 3-phosphate dehydrogenase, however, were immunologicallyidentical. ¹Dedicated, to Prof. Dr. O. H. Volk on his 80th birthday. (Received October 13, 1982; Accepted March 21, 1984)     

Sample preconditioning for measurement of fluorescence induction of chlorophyll a in marine phytoplankton

Conditions for measuring fluorescence induction curves (time-scalems) of in vivo chlorophyll ^a were studied using cultures ofDunaliella tertiolecta Butcher (Chlorophyceae) and of Thalassiosirapseudonana Hustedt (3H) (Bacillariophyceae), and samples ofnatural phytoplankton populations from the Grand Banks. Thearea above the fluorescence induction curve (A_DCMU) and themaximum fluorescence intensity (F_max) measured in the presenceof 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) were computedby microcomputer. Cells must be ‘conditioned’ or‘adapted’ prior to obtaining a fluorescence inductioncurve; dark-adaptation resulted in a lower A_DCMU and F_max thandid adaptation in far-red (720 nm) light, and was the conditioningmethod chosen. A_DCMU and F_max increased linearly with increasingirradiance up to 32.8 W m^–2 the highest actinic irradianceavailable. Information on the light history of D. tertiolectawas obtained by following the time-course of change in A_DCMUand in F_max for cells exposed for 10 min to far-red or to bluelight. The rise-time of the fluorescence induction curve andvalues of F_max were greater for samples of D. tertiolecta concentratedonto glass-fiber filters than for liquid samples, however, valuesof A_DCMU for filtered and liquid samples were not significantlydifferent. Samples of Grand Banks phytoplankton collected ontoglass-fiber filters and frozen for 28 d exhibited a significantdecrease in F_max and in A_DCMU relative to the same freshly-filteredsamples. Filtration and freezing of samples is not recommended. ^*This paper is the result of a study made at the Group for AquaticPrimary Productivity (GAP). Second International Workshop heldat the National Oceanographic Institute. Haifa. Israel in April–May1984.     

Activity of Gibberellins A1 to A0 in the Avena First-leaf Bioassay, and Location after Chromatography

Activity curves are determined for gibberellins A₁ to A₀ bythe Avena first-leaf bioassay method. Gibberellins A¹, A₄ andA₅ can be detected at 10^-11 or 10^-10 g/ml and give optimum activityof approximately 230 per cent elongation (water controls = 100per cent). Gibberellins A²A³, and A⁹ can be detected at 10^-3g/mland give optimum activity of approximately 200 per cent. GibberellinsA₆ and A₇ can be detected at 10^-5g/ml; GA₇ gives optimum activityof around 190 per cent. All the gibberellins except GA₈ canbe detected by this bioassay method after chromatography inn-butanol: 1.5 N ammonia (3: 1) and benzene: acetic acid: water(4: 2: 1) when applied to the paper at concentrations from O.Ito µg. The sensitivity of the method is compared withthat of other gibberellin bioassay methods.     

Relationship between T-wave amplitude and oxygen pulse in guinea pigs in hyperbaric helium and hydrogen

Diving isknown to induce a change in the amplitude of the T wave(A_Tw) ofelectrocardiograms, but it is unknown whether this is linked to achange in cardiovascular performance. We analyzed A_Tw in guinea pigs at 10-60atm and 25-36°C, breathing 2%O₂ in either helium (heliox;n = 10) or hydrogen (hydrox;n = 9) for 1 h at each pressure. Coretemperature and electrocardiograms were detected by using implantedradiotelemeters. O₂ consumption rate was measured by using gas chromatography. In a previous study (S. R. Kayar and E. C. Parker. J. Appl.Physiol. 82: 988-997, 1997), we analyzed theO₂ pulse, i.e., theO₂ consumption rate per heartbeat, in the same animals. By multivariate regression analysis, weidentified variables that were significant toO₂ pulse: body surface area,chamber temperature, core temperature, and pressure. In this study,inclusion of A_Tw made asignificantly better model with fewer variables. After normalizing forchamber temperature and pressure, theO₂ pulse increased with increasing A_Tw in heliox(P = 0.001) but with decreasingA_Tw in hydrox(P < 0.001). ThusA_Tw is associated with thedifferences in O₂ pulse foranimals breathing heliox vs. hydrox.

     

Identification and Semi-Quantification of Gibberellins from the Pollen and Anthers of Zea mays by Immunoassay and GC/MS

Radioimmunoassays and enzyme-linked immunosorbent assays formethyl esters of gibberellins A₁, A₃, A₄, and A₇ were establishedusing an antiserum specific for GA₁-Me. The antiserum was characterizedby high titer and specificity for such C₁₉-GAs with 3ß-hydroxylgroup as GA₁, GA₃, GA₄ and GA₇. Combination of this antiserumand HPLC enabled us to identify and quantify GA, and GA₄ fromthe pollen of Zea mays with a high degree of reliability. Similarly,identification and quantification of GA₉ and GA₂₀ were alsomade possible by use of an antiserum specific for GA₂₀-Me. Combineduse of immunoassays and GC/MS enabled us to identify nine GAsfrom the pollen and four from the anthers of Zea mays. The identificationof non-13-hydroxylated GAs, such as GA₄ and GA₉, in additionto 13-hydroxylated GAs from the pollen and the anthers suggeststhat the early-non-hydroxylation pathway, as well as the early-13-hydrox-ylationpathway, operates in the male reproductive organs of Zea mays,and that the organ-specific biosynthesis and/or localizationof GAs in Zea mays is similar to that in Oryza saliva. (Received May 7, 1990; Accepted August 20, 1990)     

Identification and absolute configuration of dihydroxy-arachidonic acids formed by oxygenation of 5S-HETE by native and aspirin-acetylated COX-2

Biosynthesis of the prostaglandin endoperoxide by the cyclooxygenase (COX) enzymes is accompanied by formation of a small amount of 11R-hydroxyeicosatetraenoic acid (HETE), 15R-HETE, and 15S-HETE as by-products. Acetylation of COX-2 by aspirin abrogates prostaglandin synthesis and triggers formation of 15R-HETE as the sole product of oxygenation of arachidonic acid. Here, we investigated the formation of by-products of the transformation of 5S-HETE by native COX-2 and by aspirin-acetylated COX-2 using HPLC-ultraviolet, GC-MS, and LC-MS analysis. 5S,15S- dihydroxy (di)HETE, 5S,15R-diHETE, and 5S,11R-diHETE were identified as by-products of native COX-2, in addition to the previously described di-endoperoxide (5S,15S-dihydroxy-9S,11R,8S,12S-diperoxy-6E,13E-eicosadienoic acid) as the major oxygenation product. 5S,15R-diHETE was the only product formed by aspirin-acetylated COX-2. Both 5,15-diHETE and 5,11-diHETE were detected in CT26 mouse colon carcinoma cells as well as in lipopolysaccharide-activated RAW264.7 cells incubated with 5S-HETE, and their formation was attenuated in the presence of the COX-2 specific inhibitor, NS-398. Aspirin-treated CT26 cells gave 5,15-diHETE as the most prominent product formed from 5S-HETE. 5S,15S-diHETE has been described as a product of the cross-over of 5-lipoxygenase (5-LOX) and 15-LOX activities in elicited rat mononuclear cells and human leukocytes, and our studies implicate cross-over of the 5-LOX and COX-2 pathways as an additional biosynthetic route.     

Interactions of the Growth Retardants Daminozide and Piproctanyl Bromide, and Gibberellins A1, A3, A4+7, A5 and A13 in Stem Extension and Inflorescence Development in Chrysanthemum morifolium Ramat

Interactions between the growth retardants daminozide (a substitutedsuccinamic acid) or piproctanyl bromide (a quaternary ammonium,piperidinium compound), and a subsequent application of a singledose (40 µg) of either gibberellin A₁, A₃, A₄₊₇ or A₁₂,showed that, in Chrysanthemum morifolium Ramat. cv. Bright GoldenAnne, a strong interdependence exists between elongation ofthe lateral shoot and the rate of development of its terminalinflorescence. A₁, A₃, and A₄₊₇ were highly active in overcoming the restrictionson both internode extension and the rate of flower-bud developmentimposed by either retardant, suggesting that these two retardanteffects are caused by a deficiency of active gibberellins (GN).In the absence of retardant, A₁, A₃, and A₄₊₇ markedly increasedstem elongation, and flowering occurred earlier than in plantsreceiving neither retardant nor GN. A₁₃ the only 20-carbon GNtested, was much less active, while A₅ had a relatively greatereffect on the time of flowering than on shoot elongation. Thus,it is not necessarily the rate of stem extension which determinesthe rate of inflorescence development. The response to different amounts of A₁, A₃ or A₁₃ (1, 5, 10,20, or 50 µg per shoot) neither suggest that different‘threshold’ levels of a particular GN are requiredto induce increases in either stem elongation or in the rateat which inflorescences develop, nor did a change in the dosegiven lead to any consistent differential effect on these twoprocesses. Chrysanthemum morifolium Ramat., stem extension, inflorescence development, growth retardants, gibberellins     

Limitation to Photosynthesis in Water-stressed Leaves: Stomata vs. Metabolism and the Role of ATP

Decreasing relative water content (RWC) of leaves progressivelydecreases stomatal conductance (g_s), slowing CO₂ assimilation(A) which eventually stops, after which CO₂ is evolved. In somestudies, photosynthetic potential (A_pot), measured under saturatingCO₂, is unaffected by a small loss of RWC but becomes progressivelymore inhibited, and less stimulated by elevated CO₂, below athreshold RWC (Type 1 response). In other studies, A_pot andthe stimulation of A by elevated CO₂ decreases progressivelyas RWC falls (Type 2 response). Decreased A_pot is caused byimpaired metabolism. Consequently, as RWC declines, the relativelimitation of A by g_s decreases, and metabolic limitation increases.Causes of decreased A_pot are considered. Limitation of ribulosebisphosphate (RuBP) synthesis is the likely cause of decreasedA_pot at low RWC, not inhibition or loss of photosynthetic carbonreduction cycle enzymes, including RuBP carboxylase/oxygenase(Rubisco). Limitation of RuBP synthesis is probably caused byinhibition of ATP synthesis, due to progressive inactivationor loss of Coupling Factor resulting from increasing ionic (Mg²⁺)concentration, not to reduced capacity for electron or protontransport, or inadequate trans-thylakoid proton gradient (pH).Inhibition of A_pot by accumulation of assimilates or inadequateinorganic phosphate is not considered significant. DecreasedATP content and imbalance with reductant status affect cellmetabolism substantially: possible consequences are discussedwith reference to accumulation of amino acids and alterationsin protein complement under water stress.