Incorporation of 2H from 2H2O into ABA in Tomato Shoots: Evidence for a Large Pool of Precursors

Tomato shoots were supplied simultaneously with S-[¹⁴C]abscisicacid and ²H₂O for 4 h. After a further 6 h incubation no deuteriumhad been incorporated into abscisic acid (ABA) extracted fromwilted or turgid shoots although up to 93% of the ABA had beensynthesized in the presence of ²H₂O as shown by the dilutionof [¹⁴C]ABA. This provides substantial evidence for a largepool of ABA precursors. When tomato shoots were incubated for6d in 80% ²H₂O between 20% and 32% of ABA molecules became labelledwith from 1 to 14 deuterium atoms. From this data the minimumsize of the precursor pool was calculated to be approximately35 times that of ABA. In addition, ABA from wilted plants containedsignificantly less deuterium than that from turgid plants suggestinga second or an enlarged pool of precursor in wilted plants. Key words: Abscisic acid, precursor pool, ²H incorporation     

Protein Turnover in Isolated Barley Leaf Segments and the Effects of Stress

Leaf segments prepared from the first leaves of barley (Hordeumvulgare) exhibit a rapid loss of protein when given a matricstress with polyethylene glycol. Protein synthesis was reducedby the stress but a greater effect of stress was seen on proteindegradation. Growing leaves were exposed to ³H₂O for 4 d ormore to label total protein, and the half-life of protein 2-³H,in the isolated segments prepared from such leaves, was shownto be c. 140 h in the absence of stress. Stress reduced thisto c. 62 h. A short pulse with ³H₂O preferentially labels rapidlyturning-over protein and a 24 h pulse given to isolated leafsegments labelled proteins with a half-life of c. 64 h in thepresence or absence of stress. Degradation of the 24 h pulse-labelledproteins was inhibited by cycloheximide. Proline accumulationoccurred in the stressed segments and was inhibited by cycloheximide.The results are discussed in the light of current views concerningprotein degradation and possible relationships between proteolysisand proline accumulation.     

The Effect of Growth in D2O on the Metabolism of Winter Rye

The metabolism of winter rye seedlings (Secale cereale, L. ev.Winter) cultured in 99.8 per cent D₂O was investigated. Comparedwith water-grown seedlings, the protein content was much lowerin the D₂O-cultured seedlings and the pattern of incorporationof [³H]leucine and [³H]phenylalanine into protein was substantiallydifferent. Seedlings cultured in D₂O incorporated [³H]thymidineinto DNA, but did not take up [³H]uridine. The results suggestthat some of the toxic effects of D₂O culture on higher plantscan be attributed to a partial block of protein synthesis.     

Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water

N-Acetyl-D-[2-³H]glucosamine was synthesized from N-acetyl-D-mannosamineby alkaline 2-epimerization in pyridine containing ³H₂O andnickelous acetate. The reaction involves reversible formationof an enol intermediate and therefore also resulted in incorporationof tritium into N-acetylmannosamine. After completed reaction,the two N-acetylhexosamines were separated from other radioactiveproducts and Morgan-Elson chromogens by chromatography on acolumn of Sephadex G-10, which was eluted with 10% ethanol,and were then separated from each other by chromatography onSephadex G-15 in 0·27 M sodium borate (pH 7·8).The location of the incorporated tritium was established bytreatment of the N-acetylhexosamines with borate under the conditionsof the Morgan-Elson reaction, which converts the sugars to Kuhn'schromogen I with concomitant loss of the C-2 hydrogen. As expected,this treatment resulted in the formation of ³H₂O, indicatingthat the tritium was located at C-2. [2-³H]Glucosamine was preparedby acid hydrolysis of the labelled N-acetylglucosamine and wasconverted to [2-³H]glucosamine 6-phosphate by incubation withhexokinase and ATP. The sugar phosphate was used as a substratefor glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10 [EC] )in a simple ³H₂O release assay. N-acetyl[2-³H]glucosamine N-acetyl[2-³H]mannosamine [2-³H]glucosamine glucosamine 6-phosphate deaminase [2-³H]mannosamine     

Oxalate inhibits renal proximal tubule cell proliferation via oxidative stress, p38 MAPK/JNK, and cPLA2 signaling pathways

Exposure of renal proximal tubule cells to oxalate may play an important role in cell proliferation, but the signaling pathways involved in this effect have not been elucidated. Thus the present study was performed to examine the effect of oxalate on ³H-labeled thymidine incorporation and its related signal pathway in primary cultured rabbit renal proximal tubule cells (PTCs). The effects of oxalate on [³H]thymidine incorporation, lactate dehydrogenase (LDH) release, Trypan blue exclusion, H₂O₂ release, activation of mitogen-activated protein kinases (MAPKs), and ³H-labeled arachidonic acid (AA) release were examined in primary cultured renal PTCs. Oxalate inhibited [³H]thymidine incorporation in a time- and dose-dependent manner. However, its analogs did not affect [³H]thymidine incorporation. Oxalate (1 mM) significantly increased H₂O₂ release, which was blocked by N-acetyl-L-cysteine (NAC) and catalase (antioxidants). Oxalate significantly increased p38 MAPK and stress-activated protein kinase (SAPK)/c-Jun NH₂-terminal kinase (JNK) activity, not p44/42 MAPK. Oxalate stimulated [³H]AA release and translocation of cytosolic phospholipase A₂ (cPLA₂) from the cytosolic fraction to the membrane fraction. Indeed, oxalate significantly increased prostaglandin E₂ (PGE₂) production compared with control. Oxalate-induced inhibition of [³H]thymidine incorporation and increase of [³H]AA release were prevented by antioxidants (NAC), a p38 MAPK inhibitor (SB-203580), a SAPK/JNK inhibitor (SP-600125), or PLA₂ inhibitors [mepacrine and arachidonyl trifluoromethyl ketone (AACOCF₃)], but not by a p44/42 MAPK inhibitor (PD-98059). These findings suggest that oxalate inhibits renal PTC proliferation via oxidative stress, p38 MAPK/JNK, and cPLA₂ signaling pathways. kidney; mitogen-activated protein kinase; phospholipase A₂     

Proton Translocation Associated with Denitrification in a Photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

H⁺ translocation driven by NO₃^–, NO₂^– and N₂O reductionswith endogenous substrates in cells of Rhodopseudomonas sphaeroidesforma sp. denitrificans was investigated by the oxidant pulsemethod. Upon injection of nitrogenous oxides to anaerobic cellsin darkness, an alkaline transient in the external medium wasobserved, followed by acidification. The alkaline transientwas enhanced by carbonyl cyanide m-chlorophenylhydrazone. When a viologen dye was used as an electron donor in the presenceof 1 mM Af-ethylmaleimide and 0.1 mM 2-n-heptyl-4-hydroxyquinoline-N-oxideto preclude respiration-linked H⁺ extrusion, addition of KNO₃,KNO₂ and N₂O caused only a rapid alkalinization. The H⁺ consumptionstoichiometries, H⁺/2e^– ratios for NO₃^– reductionto NO₂^–, NO₂^– reduction to 1/2 N₂O and N₂O reductionto N₂ were –1.90, –3.18 and –2.04, respectively.These values agreed well with the fact that all reductions ofnitrogenous oxides in denitrification occur on the periplasmicside of the cytoplasmic membrane. When corrected for H⁺ consumption in the periplasm, the H⁺ extrusionstoichiometries, H⁺/2e^– ratios with endogenous substratesin the presence of K⁺/valinomycin for NO₃^– reduction toNO₂^–, NO₂^– reduction to 1/2 N₂O and N₂O reductionto N₂ were 4.05, 4.95 and 6.01, respectively. (Received August 4, 1982; Accepted January 13, 1983)     

Oligomycin-insensitive incorporation of 32P into chloroplast protein by illumination

³²P incorporation into the protein fraction of chloroplast fragmentsby short illumination was investigated under various phosphorylatingconditions. ³²P incorporation was generally accompanied by cyclic and non-cyclicphotophosphorylations and also by formation of a high energyintermediate "X_E". However, the addition of a DPIP-ascorbatecouple caused inhibition of ³²P incorporation, while ATP formationproceeded. Effects of inhibitors and uncouplers of photophosphorylationon the formation of protein-bound ³²P were generally similarto those on ATP formation. AT³²P was not utilized for protein-bound ³²P formation in thedark by chloroplast fragments, but its radioactivity was transferredinto the chloroplast protein fraction in the light. Oligomycininhibited ATP formation but did not inhibit protein-bound ³²Pformation. m-Cl-CCP blocked both reactions. This suggests thatprotein-bound ³²P is not an actual intermediate in the phosphorylativeprocess leading to formation of ATP. It is probably formed ona side pathway from an intermediate of ATP formation. Analyses of protein-bound ³²P after digestion with proteaseand lipase showed that the ³²P incorporated was bound to peptidesin chloroplast lamellae. The possible form of this bound ³²Pis discussed. (Received November 22, 1971; )     

Oxygen Diffusion in Lupin Nodules: I.VISUALIZATION OF DIFFUSION BARRIER OPERATION

Root nodules of Lupinus albus (L.) cv. Multolupa were subjectedto short- and medium-term stresses by lowering rhizosphere temperaturefrom 25 to 16°C (2 h), detopping plants (3 h), darkeningplants (21 h) or exposing roots to 20 mol m^–3 KNO₃ for4 d. All experimental treatments produced increases in oxygendiffusion resistance, compared with control plants. These correlatedwith structural changes in the nodule cortex, which is describedin detail for the first time. The most noticeable change isthe occlusion of intercellular spaces by a glycoprotein whichwas identified using the monoclonal antibody MAC236. This glycoproteinwas also found surrounding bacteria in intercellular spacesof the cortex of control nodules. Key words: Oxygen diffusion resistance, glycoprotein, nodules, nitrogen fixation, Lupinus albus     

14C2H4: Distribution of 14C-labeled tissue metabolites in pea seedlings

The ¹⁴C-metabolite distribution pattern following ¹⁴C₂H₄ metabolismin intact pea seedlings (Pisum sativum L.) was determined undervarious conditions. After a 24 hr exposure to ¹⁴C₂H₄, the majorityof ¹⁴C-metabolites were water-soluble (60–70%) with lesseramounts in the protein (10–15%), lipid (1%), and insoluble(1–2%) fractions. Ion exchange chromatography of the water-solublecomponents into basic, neutral, and acidic fractions revealeda 50 : 40 : 10 distribution, respectively. Chromatography ofthe neutral fraction revealed two regions of radioactivity (Rf=0.38)and 0.63 which did not cochromatograph with twenty-two knownsugars or neutral metabolites. Chromatograms of the basic fractioncontained 3 regions of radioactivity. Similar distribution patternswere noted when ¹⁴C₂H₄ exposure was followed by a 6 hr air chaseor when 5% CO₂, an antagonist of ethylene action, was presentduring the exposure. Marked differences in the ¹⁴C-metabolite distribution patternswere obtained when ¹⁴CO₂ was substituted for ¹⁴C₂H₄. These resultsindicate that the metabolic pathway involved in ethylene metabolismis different from that involved in intermediary carbon metabolism. ¹ Contribution No. 2338 from Central Research and DevelopmentDepartment, Experimental Station, E. I. du Pont de Nemours andCompany, Wilmington, Delaware. (Received June 28, 1976; )     

Measurement of mouse vascular smooth muscle and atheroma cell proliferation by 2H2O incorporation into DNA

Vascular smooth muscle cell (VSMC) and leukocyte proliferation are central features of atherosclerosis. Using ²H₂O to label the deoxyribose moiety of newly synthesized DNA in VSMC and atheroma cells from mouse aorta, we developed a method to measure DNA replication and, hence, cell division. Cell turnover/proliferation in aortae from normal and apolipoprotein E (ApoE)-knockout (ApoE^–/–) mice was measured. Mice were injected with ²H₂O to achieve 2% body water enrichments and then maintained on 4% ²H₂O in drinking water for weeks to months. DNA from the intimal-medial layer of the aorta was extracted and hydrolyzed to deoxyribonucleosides. Purified deoxyadenosine was derivatized to pentane tetraacetate for analysis of ²H enrichment by gas chromatography-mass spectrometry. VSMC proliferation was measurable but slow in adult mice (0.12 ± 0.08%/day) and higher in young mice (0.25 ± 0.08%/day). VSMC delabeling revealed that ²H died away slowly in VSMC DNA, confirming the low turnover rate. Atheroma cell proliferation was elevated in ApoE^–/– mice fed low- or high-fat diets for 15 wk, concurrent with histological appearance of atherosclerosis. Validation of the method for VSMC was confirmed by comparison of in vitro rat VSMC proliferation rates using ²H₂O with cell counts and bromodeoxyuridine proliferative index. In summary, proliferation of VSMC and atheroma cells can be quantified reliably and sensitively without radioactivity and may be an informative biomarker in vascular hyperplastic diseases, including atherosclerosis. atherosclerosis; gas chromatography-mass spectrometry; stable isotopes; animal model     

Defoliation-Induced Stress in Nodules of White Clover: I. CHANGES IN PHYSIOLOGICAL PARAMETERS AND PROTEIN SYNTHESIS

Nodule function and protein synthesis were studied in defoliationstressed white clover plants. Uncut control plants (C) werecompared with plants from two defoliation treatments: (1) continuousdefoliation (CD) where all leaves and petioles were removedeach day; and (2) defoliated/recovered (DR) where, after removalof all leaves and petioles, new leaves were then allowed toregrow. After a single defoliation N₂ fixation (acetylene reductionactivity) and nitrogenase-linked respiration declined by morethan 80% within 3 h and by nearly 100% by 24 h. DR plants beganto fix nitrogen again at a very low level 3 d later and thereafterrose to control levels by 15 d. Continuously defoliated plantsnever recovered N₂ fixation capacity. Nodule protein complementwas assessed by polyacrylamide gel electrophoresis. Major changesoccurred in buffer soluble protein band patterns by 6 d in CDplants, but few changes were evident in SDS soluble proteins.By 9 and 14 d significant disruption of all proteins was evident.The prominent host plant protein, leghaemoglobin (Lb) had disappearedby 14 d. In DR plants the intensity of staining was reducedbut no major changes in band patterns were evident and by 21d nodules were rejuvenated. [³⁵S]-labelled methionine was incorporated into nodule proteinsfrom all treatments throughout the experiment. However, continuousdefoliation caused increasing variability between replicatesin the labelled band patterns. By 21 d CD, much of the labelledprotein was present as amorphous low M_r material which suggestseither disruption of the protein synthesizing machinery or rapidhydrolysis by proteolytic enzymes. Surprisingly [³⁵S]-methionine was never found in Lb from nodulesof any treatment. It is possible that white clover Lb does notcontain any methionine residues or that no synthesis of Lb occurred. Key words: Trifolium repens, white clover, defoliation, protein synthesis, nodules     

Glycollate Formation during the Photorespiration of Acetate by Chlorella

WhenChlorella pyrenoidosa photoassimilates ³H-¹⁴C-acetate theglycollic acid formed shows a high ³H/¹⁴C ratio, the only othercompounds showing similar ratios being glycerate and serine.The ³H/¹⁴C ratio of glycollate was unaffected by the TCA cycleinhibitors MFA, diethylmalonate and arsenite showing that ³Hin glycollate does not result from the oxidation of acetatevia the TCA cycle, the resulting NADP³H₂ or NAD³H₂ being usedfor the reduction of the glycollate precursor. Although DCMUdecreased the ³H/¹⁴C ratio, complete inhibition of glycollatelabelling was not observed with 10^–6 M DCMU, at whichconcentration complete inhibition of the Hill reaction is achieved.Although the ³H/¹⁴C ratio was unaltered, total dpm of both ¹⁴Cand ³H in glycollate were increased by INH. The ³H/¹⁴C ratiosof glycerate and serine were decreased by INH, as were the totaldpm of ³H and ¹⁴C incorporated into these compounds. Thus, INHinhibits the further metabolism of glycollate to glycerate andserine. The effect of INH on incorporation of ¹⁴C-I-acetateinto various cell fractions was investigated. The incorporationof ¹⁴C into polysaccharide and lipid was decreased, while theincorporation of ¹⁴C into the water-soluble fraction of cellsand therelease of ¹⁴CO₂ were little affected. Although glycollicacid was an early product of acetate photoassimilation in Chlorellapyrenoidosa, glycollate excretion does not take place undera wide range of environmental conditions shown to favour glycollateexcretion by other algae. However, small amounts of labelledglycollate were detected in the supernatant from the cells duringthe photoassimilation of ³H-¹⁴C-acetate, but this glycollatedid not show the high ³H/¹⁴C ratio of glycollate present withinthe cell. The failure of Chlorella pyrenoidosa to excrete appreciableamounts of glycollate when photoassimilating acetate or carbondioxide was considered to result from the presence of glycollateoxidase (EC 1.1.3.1 [EC] ) which allowed the further metabolism ofglycollate. Besides glycollate oxidase, glyoxylate reductasewas also demonstrated in Chlorella pyrenoidosa so that glycollatecould function in hydrogen transfer during the photoassimilationof acetate.     

孝顺竹愈伤组织增殖培养基优化研究

为筛选适宜孝顺竹愈伤组织继代增殖培养基,控制褐变发生,提高再生体系效率,对培养基组成如5种基本培养基、6种有机添加物、7种糖类和5种大量元素等因子进行试验分析。结果表明：培养20 d,基本培养基以MS效果较好,愈伤组织增殖2.8倍,白至淡黄色,致密;有机添加物以1.0 g·L^-1脯氨酸效果较显著,愈伤组织增殖3.64倍,淡黄色,致密均一;碳源以30 g·L^-1麦芽糖效果较好,愈伤组织增殖2.96倍,白至淡黄色,致密。5种大量元素中NH₄NO₃对孝顺竹愈伤组织增殖的影响达到显著水平,以825 mg·L^-1为较佳浓度,培养29 d愈伤组织增殖可达5倍以上,部分出现根分化;适宜孝顺竹愈伤组织培养的大量元素组合为：KNO₃ 475 mg·L^-1+NH₄NO₃ 825 mg·L^-1+MgSO₄·7H₂O 185 mg·L^-1+KH₂PO₄ 340 mg·L^-1+CaCl₂·7H₂O 440 mg·L^-1。     

Assimilation of 15N2 and 15NO3- by Partially Nitrate-Tolerant Nodulation Mutants of Soybean

Growth-chamber studies were conducted to evaluate nitrogen assimilationby three hypernodulated soybean [Glycine max (L.) Merr.] mutants(NOD1–3, NOD2–4, NOD3–7) and the Williamsparent. Seeds were inoculated at planting and transplanted atday 7 to nutrient solution with 1 mol m^–3 urea (optimizesnodule formation) or 5 mol m^–3 NO₃^– (inhibits noduleformation). At 25 d after planting, separate plants were exposedto ¹⁵NO₂ or ¹⁵NO₃^– for 3 to 48 h to evaluate N₂ fixationand NO₃^– assimilation. Plant growth was less for hypernodulatedmutants than for Williams with both NO₃^– and urea nutrition.The major portion of symbiotically fixed ¹⁵N was rapidly assimilated(30 min) into an ethanol-soluble fraction, but by 24 h aftertreatment the ethanolinsoluble fraction in each plant part wasmost strongly labelled. Distribution patterns of ¹⁵N among organswere very similar among lines for both N growth treatments aftera 24 h ¹⁵N₂ fixation period; approximate distributions were40% in nodules, 12% in roots, 14% in stems, and 34% in leaves.With urea-grown plants the totalmg ¹⁵N fixed plant^–1 24h^–1 was 1·18 (Williams), 1·40 (N0D1-3),107 (NOD2-4), and 0·80 (NOD3-7). The 5 mol m^-3 NO₃^- treatmentresulted in a 95 to 97% decrease in nodule mass and ¹⁵N₂ fixationby Williams, while the three mutants retained 30 to 40% of thenodule mass and 17 to 19% of the ¹⁵N₂ fixation of respectiveurea-grown controls. The hypernodulated mutants, which had restrictedroot growth, absorbed less ¹⁵NO₃^- than Williams, irrespectiveof prior N growthcondition. The ¹⁵N from ¹⁵NO₃^- was primarilyretained in the soluble fraction of all plant parts through24 h. The ¹⁵N incorporation studies confirmed that nodule developmentis less sensitive to external NO₃^- in mutant lines than in theWilliams parent, and provide evidence that subsequent metabolismand distribution within the plant was not different among lines.These results further confirm that the hypernodulated mutantsof Williams are similar in many respects to the hyper- or supernodulatedmutants in the Bragg background, and suggest that a common mutationalevent affectingautoregulatory control of nodulation has beentargeted. Key words: Glycine max (L.) Merr., soybean, N₂fixation, nitrate assimilation, nodulation mutants, ¹⁵N isotope     

Dark Hydrogen Evolution and Adenine Nucleotides of Subtropical Marine Unicellular Green Algae during Anaerobic Incubation

The relation between dark anaerobic hydrogen (H₂) evolutionof marine unicellular green algae and the energetic state ofthe cells, as revealed by adenine nucleotide (AN) levels, wasstudied. One of the 4 investigated strains produced H₂ continuouslyfor 48 h and maintained its adenylate energy charge (EC) atabout 0.6, whereas the H₂ evolution of the other strains stoppedwithin 24 h and their EC decreased to low values. Prolongedaerobic preincubation resulted in reduced H₂ evolution and astronger decrease in EC during subsequent anaerobiosis. Amongcultures of the same strain, H₂ evolution was correlated withthe initial pool levels of total AN and chlorophyll and withthe ATP level and EC during anaerobiosis. These results indicatea dependence between H₂ evolution and capacity for starvation.EC was not affected by complete inhibition of H₂ evolution bycarbon monoxide in one strain but was significantly loweredin another strain. This difference between the two strains isinterpreted as reflecting a different dependence on, or utilizationof, alternative pathways for disposal of electrons during fermentation,although H₂ evolution apparently played a minor and non-essentialrole in the dark anaerobic fermentation. ¹ Present address: Institute of Oceanic Research and Development,Tokai University, Orido, Shimizu, Shizuoka 424, Japan. ² Present address: Institute of Marine Research, Directorateof Fisheries, P.O. Box 1870, N-5011 Bergen-Nordnes, Norway. (Received November 19, 1986; Accepted March 17, 1987)     

A possible role of hydroxyproline-protein in oxygen-sensitive growth

Exposure of Avena coleoptile sections to 8% O₂ brought aboutrespiration decrease, resulting in a decrease of ATP production.The pH at the cell wall surface slightly rose in sections exposedto 8% O₂, while their growth was greatly accelerated. Moreover,this growth acceleration was observed even in sections treatedwith CCCP known to make membranes permeable for protons. Weconcluded that the growth acceleration with reduction of O₂concentration is probably not the result of secretion of H₊ions into cell wall compartments. Results of this study provided evidence to support the hypothesisthat there is an inverse relationship between hydroxyproline-proteinlevel and the ability of a cell to undergo rapid cell elongation.Total labeling of the cell wall fraction with ¹⁴C-proline wasunaffected by 8% O₂ treatment, although the radioactivitiesof hydroxyproline incorporated into this fraction during thetreatments fell to about 45% of the control. Moreover, the radioactivitiesof hydroxyproline incorporated into the SLS-insoluble cell wallfraction of sections exposed to 8% O₂ decreased to about 30%of the control. This decrease of hydroxyproline was also observedin sections treated with cycloheximide, which inhibits the secretionof H₊ ions into the cell wall compartment. Reduction of O₂ concentrationin the surrounding atmosphere affects not only the hydroxylationof peptidyl proline, but also the binding of hydroxyproline-protein(s)to cell wall polysaccharides, and the resulting decrease ofthe protein rigidly bound to them may induce cell elongation. (Received December 5, 1975; )     

Possible physiological mechanisms for production of hydrogen peroxide by the ichthyotoxic flagellate Heterosigma akashiwo

Blooms of the toxic red tide phytoplankton Heterosigma akashiwo(Raphidophyceae) are responsible for substantial losses withinthe aquaculture industry. The toxicological mechanisms of H.akashiwoblooms are complex and to date, heavily debated. One putativetype of ichthyotoxin includes the production of reactive oxygenspecies (ROS) that could alter gill structure and function,resulting in asphyxiation. In this study, we investigated thepotential of H.akashiwo to produce extracellular hydrogen peroxide,and have investigated which cellular processes are responsiblefor this production. Within all experiments, H.akashiwo producedsubstantial amounts of hydrogen peroxide (up to 7.6 pmol min^–110⁴ cells^–1), resulting in extracellular concentrationsof ~0.5 µmol l^–1 H₂O₂. Measured rates of hydrogenperoxide production were directly proportional to cell density,but at higher cell densities, accuracy of H₂O₂ detection wasreduced. Whereas light intensity did not alter H₂O₂ production,rates of production were stimulated when temperature was elevated.Hydrogen peroxide production was not only dependent on growthphase, but also was regulated by the availability of iron inthe medium. Reduction of total iron to 1 nmol l^–1 enhancedthe production of H₂O₂ relative to iron replete conditions (10µmol l^–1 iron). From this, we collectively concludethat production of extracellular H₂O₂ by H.akashiwo occurs througha metabolic pathway that is not directly linked to photosynthesis.     

Estradiol-17beta stimulates proliferation of mouse embryonic stem cells: involvement of MAPKs and CDKs as well as protooncogenes

Although the importance of estradiol-17 (E₂) in many physiological processes has been reported, to date no researchers have investigated the effects of E₂ on embryonic stem (ES) cell proliferation. Therefore, in the present study, we have examined the effect of E₂ on the DNA synthesis of murine ES (ES-E14TG2a) cells and its related signaling pathways. The results of this study show that E₂ (10^–9 M) significantly increased [³H]thymidine incorporation at >4 h and that E₂ (>10^–12 M) induced an increase of [³H]thymidine incorporation after 8-h incubation. Moreover, E₂ (>10^–12 M) also increased 5'-bromo-2'-deoxyuridine (BrdU) incorporation and cell number. Indeed, E₂ stimulated estrogen receptor (ER)- and - protein levels and increased mRNA expression levels of protooncogenes (c-fos, c-jun, and c-myc). Tamoxifen (antiestrogen) completely inhibited E₂-induced increases in [³H]thymidine incorporation. In addition, estradiol-6-O-carboxymethyl oxime-BSA (E₂-BSA; 10^–9 M) increased [³H]thymidine incorporation at >1 h, and E₂-BSA (>10^–12 M) increased [³H]thymidine incorporation after 1-h incubation. E₂-BSA-induced increase in BrdU incorporation also occurred in a dose-dependent manner. Tamoxifen had no effect on E₂-BSA-induced increase of [³H]thymidine incorporation. Also, E₂ and E₂-BSA displayed maximal phosphorylation of p44/42 MAPKs at 10 and 5 min, respectively. E₂ increased cyclins D1 and E as well as cyclin-dependent kinase (CDK)2 and CDK4. In contrast, E₂ decreased the levels of p21^cip1 and p27^kip1 (CDK-inhibitory proteins). Increases of these cell cycle regulators were blocked by 10^–5 M PD-98059 (MEK inhibitor). Moreover, E₂-induced increase of [³H]thymidine incorporation was inhibited by PD-98059 or butyrolactone I (CDK2 inhibitor). In conclusion, estradiol-17 stimulates the proliferation of murine ES cells, and this action is mediated by MAPKs, CDKs, or protooncogenes. cyclin-dependent kinase; mitogen-activated protein kinase     

Contrasting Incorporation of 2H from 2H2O into ABA, Xanthoxin and Carotenoids in Tomato Shoots

Nonhebel, H. M. and Milborrow, B. V. 1987. Contrasting incorporationof ²H from ²H²O into ABA, xanthoxin and carotenoids in tomatoshoots.—J. exp. Bot. 38: 980–991. The incorporation of ²H into abscisie acid, xanthoxin, ß-carotene,lutein, lutein epoxide and violaxanthin in tomato shoots incubatedfor 6 d in 70% ²H₂O was compared to investigate whether thesecompounds are precursors of abscisie acid. On average, 5% ofabscisie acid molecules became labelled with a single ²H atomand 21% with from 3 to 14 atoms of ²H. However, mass spectralanalysis of endogenous xanthoxin extracted from the same plants,in darkness, under nitrogen, and derivatized to the pentafluorobenzyloximeshowed incorporation of only single ²H atoms, ruling out xanthoxinas an abscisie acid precursor. Normal-phase HPLC analysis oftomato shoot extracts showed four major carotenoid peaks; ß-carotene,lutein, lutein epoxide and violaxanthin. Calculations basedon the measured carotenoid pool sizes and on the calculatedminimum pool size of the ABA precursor predicted that at least6·8% of violaxanthin molecules or 7·9% of luteinepoxide molecules should become labelled with from 3 to 14 ²Hatoms if these molecules are precursors of abscisie acid. However,mass spectral analysis of xanthoxin derived from purified violaxanthinand lutein epoxide showed no molecules with more than a single²H atom, with detection limits of less than 1% and 0·2%respectively. Similarly, mass spectra of ß-caroteneand lutein did not show any ²H. We conclude that these carotenoidsare not precursors of abscisie acid. Key words: Abscisic acid, xanthoxin, carotenoids     

Theoretical Basis of Protocols for Seed Storage III. Optimum Moisture Contents for Pea Seeds Stored at Different Temperatures

In previous work, we demonstrated that there was an optimummoisture level for seed storage at a given temperature (Vertucciand Roos, 1990), and suggested, using thermodynamic considerations,that the optimum moisture content increased as the storage temperaturedecreased (Vertucci and Roos, 1993b). In this paper, we presentdata from a two year study of aging rates in pea (Pisum sativum)seeds supporting the hypothesis that the optimum moisture contentfor storage varies with temperature. Seed viability and vigourwere monitored during storage under dark or lighted conditionsat relative humidities between 1 and 90%, and temperatures between-5 and 65°C. The optimum moisture content varied from 0·015g H₂O g^-1 d.wt at 65°C to 0·101 g H₂O g^-1 d.wt at15°C under dark conditions and from 0·057 at 35°Cto 0·092 g H₂O g^-1 d.wt at -5°C under lighted conditions.Our results suggest that optimum moisture contents cannot beconsidered independently of temperature. This conclusion hasimportant implications for 'ultra-dry' and cryopreservationtechnologies.Copyright 1994, 1999 Academic Press Seed storage, seed aging, seed longevity, water content, temperature, glass, desiccation damage, ultradry, Pisum sativum L., pea, cryopreservation