Compartmentation Analysis of Paraquat Fluxes in Maize Roots as a Means of Estimating the Rate of Vacuolar Accumulation and Translocation to Shoots

Efflux analysis conducted after five loading periods of various lengths (2, 6, 12, 18, or 24 h) was used to investigate uptake, compartmentation, and translocation of [14C]paraquat in maize (Zea mays L.) seedlings. The time course for net paraquat uptake (paraquat concentration in uptake solution = 25[mu]M) into maize roots was linear (56.7 nmol g-1 root fresh weight h-1) for 24 h. Estimates of changes in paraquat content in the vacuole, cytoplasm, and cell wall after 2-, 6-, 12-, 18-, and 24-h loading periods indicated that the cell wall saturated rapidly, whereas accumulation of paraquat into the vacuole increased linearly (12.4 nmol g-1 root fresh weight h-1) over 24 h. In contrast to vacuolar accumulation, cytoplasmic paraquat content appeared to approach saturation. The half-time for paraquat efflux from the cell wall (16.6 min [plus or minus] 1.2 SD) and cytoplasm (58.8 min [plus or minus] 8.9 SD remained relatively constant regardless of the length of the loading period, whereas the half-time for efflux from the vacuole was considerably longer and increased linearly with increased loading time (6.1-18.7 h). The time course for paraquat translocation to the shoot was linear within a 24-h exposure to radiolabeled herbicide, but translocation did not begin until 5 h after initiation of treatment. The experimental approach used in these experiments provides a valuable method for examining the movement of paraquat in maize seedlings. Results indicate that the herbicide slowly accumulates in the vacuole of root cells but is also translocated to the shoot.     

Exposure to nitric oxide protects against oxidative damage but increases the labile iron pool in sorghum embryonic axes

Sodium nitroprusside (SNP) and diethylenetriamine NONOate (DETA NONOate), were used as the source of exogenous NO to study the effect of NO upon germination of sorghum (Sorghum bicolor (L.) Moench) seeds through its possible interaction with iron. Modulation of cellular Fe status could be an important factor for the establishment of oxidative stress and the regulation of plant physiology. Fresh and dry weights of the embryonic axes were significantly increased in the presence of 0.1 mM SNP, as compared to control. Spin trapping EPR was used to assess the NO content in axes from control seeds after 24 h of imbibition (2.4+/-0.2 nmol NO g(-1) FW) and seeds exposed to 0.01, 0.1, and 1 mM SNP (3.1+/-0.3, 4.6+/-0.2, and 6.0+/-0.9 nmol NO g(-1) FW, respectively) and 1 mM DETA NONOate (6.2+/-0.6 nmol NO g(-1) FW). Incubation of seeds with 1 mM SNP protected against oxidative damage to lipids and maintained membrane integrity. The content of the deferoxamine-Fe (III) complex significantly increased in homogenates of axes excised from seeds incubated in the presence of 1 mM SNP or 1 mM DETA NONOate as compared to the control (19+/-2 nmol Fe g(-1) FW, 15.2+/-0.5 nmol Fe g(-1) FW, and 8+/-1 nmol Fe g(-1) FW, respectively), whereas total Fe content in the axes was not affected by the NO donor exposure. Data presented here provide experimental evidence to support the hypothesis that increased availability of NO drives not only protective effects to biomacromolecules, but to increasing the Fe availability for promoting cellular development as well.     

Acetylene Reduction by Symbiosomes and Free Bacteroids from Broad Bean (Vicia faba L.) Nodules (Role of Oxalate)

We report the presence of oxalate in the organic acid fraction of broad bean (Vicia faba L.) nodule cytosol. Using both high-performance liquid chromatography and enzymic assays, high levels of oxalate were detected (70.4 [plus or minus] 2.4 mM). To study the potential role of oxalate as an energy-yielding substrate for nitrogenase activity, free bacteroids were isolated from nodules and found to oxidize oxalate in support of C2H2 reduction under O2 tensions that were lower than those required to oxidize succinate, another dicarboxylate commonly detected in legume nodules. Symbiosomes of broad bean, isolated for the first time from amide-producing nodules, were provided with [14C]oxalate and found to have uptake kinetics with a lower affinity [Km(oxalate) = 330 [mu]M] than that for free bacteroids [Km(oxalate) = 130 [mu]M]. In anaerobic preparations of symbiosomes supplied with purified oxyleghemoglobin, O2 consumption was stimulated by oxalate from 20.2 [plus or minus] 0.8 nmol O2 min-1mg-1 protein to 24.5 [plus or minus] 1.1 nmol O2 min-1 mg-1 protein but always remained lower than the rate of O2 consumption in free bacteroids (32.2 [plus or minus] 1.4 nmol O2 min-1 mg-1 protein). Under these conditions, C2H2 reduction activity was 9.7 [plus or minus] 0.8 and 15.1 [plus or minus] 0.9 nmol C2H4 min-1 mg-1 protein for symbiosomes and bacteroids, respectively. These data support the suggestion that oxalate may play a role as a carbon substrate in support of N2 fixation in broad bean nodules.     

Involvement of Abscisic Acid in Ethylene-Induced Cotyledon Abscission in Cotton Seedlings

Cotton (Gossypium hirsutum L. cv LG102) seedlings raised from seeds exposed to 100 [mu]M norflurazon (NFZ) during imbibition contained reduced levels of free abscisic acid (ABA) and were visibly achlorophyllous. Exposure of untreated cotton seedlings to ethylene concentrations >1 [mu]L/L for 24 h resulted in cotyledon abscission. In contrast, exposure of NFZ-treated seedlings to concentrations of ethylene [less than or equal to]50 [mu]L/L elicited no cotyledon abscission. Application of ABA, an ABA analog, or jasmonic acid to NFZ-treated seedlings restored ethylene-induced abscission. Isolated cotyledonary node explants prepared from NFZ-treated seedlings exhibited an altered dose-response pattern of ethylene-induced petiole abscission. Endogenous levels of free IAA were unaltered in NFZ-treated seedlings. Ethylene treatment (50 [mu]L/L, 24 h) had no effect on free indoleacetic acid (IAA) levels in either control or NFZ-treated seedlings. Levels of conjugated (ester plus amide) IAA were substantially increased in NFZ-treated seedlings regardless of ethylene treatment. These results indicate that endogenous ABA plays an essential, but physiologically undefined, role in ethylene-induced cotyledon abscission in cotton.     

Prevention of imbibitional injury in low vigor soybean embryonic axes by osmotic control of water uptake

Deterioration of soybean [ Glycine max (L.) Merr. cv. Essex] seeds during accelerated aging at 41°C and 100% relative humidity predisposes the embryonic axis to injury during the initial period of imbibition. This injury was prevented or greatly reduced in severity when excised axes were imbibed on blotters containing 30% polyethylene glycol which slowed the rate of water uptake and when axes were pre-equilibrated to a high moisture level. Rates of water uptake by "high"(no treatment) and "low vigor"(accelerated aged) excised axes were identical. However, high vigor axes tolerated rapid water uptake during early imbition, whereas low vigor axes did not. Leakage of electrolytes during early imbibition was nearly six times greater in low than in high vigor axes. Polyethylene glycol significantly reduced the leakage of electrolytes from both low and high vigor axes. The data are in agreement with the hypothesis that seed deterioration in soybeans involves membrane changes which may predispose embryonic tissues to injury during imbibition. Reduction of the rate of water uptake during the initial period of imbibition would allow extra time for membrane repair or rearrangement, thus permitting the tissues to develop in a more orderly manner. The data indicate that deterioration in soybean seeds involves, at least in part, a decrease in ability of seed axes to tolerate rapid water uptake at the start of imbibition and that this weakness may be compensated by osmotic control of water uptake.     

Metabolic Changes in Axes of Germinating Vigna unguiculata Seeds as Related to Effects of Removal of Cotyledons

[¹⁴C]-Labeled amino acids and sucrose were fed to Vigna unguiculataseeds through cut-ends of cotyledons, and incorporations ofradioactivity into trichloroacetic acid- and 80% ethanol-insolublefractions of axes, respectively, were followed during 48 h ofthe post-imbibition development. The results of these studies,together with determinations of changes in dry weight and proteincontents after the onset of imbibition, indicated that the reservematerials stored in cotyledons were available for active growthof axes only after 12 h of post-imbibition. However, pulse-labelingexperiments, where [³H]-labeled leucine and uridine were feddirectly to axes attached to or detached from cotyledons, indicatedthat synthesis of protein and RNA in both axes was very pronouncedeven at earlier stages (2–8 h) of post-imbibition. Albuminand globulin proteins of axes disappeared most rapidly duringthe 6–12 h period of post-imbibition. Cycloheximide, -amanitinand cordycepin added to imbibing axes inhibited the degradationof major globulin proteins, whereas the inhibitors had littleeffect on the degradation of major albumin proteins. Both proteolyticand amylolytic activities were found to occur in embryonic axesof ‘dry’ seeds, and increased to higher levels asthe germination proceeded. Axes at early stages of germinationmay degrade the self-sustained reserve proteins and utilizethem for the synthesis of new proteins. (Received June 11, 1983; Accepted August 16, 1983)     

Regulation of Purine Metabolism in Intact Leaves of Coffea arabica

The capacity of Coffea arabica leaves (5- x 5-mm pieces) to synthesize de novo and catabolize purine nucleotides to provide precursors for caffeine (1,3,7-trimethylxanthine) was investigated. Consistent with de novo synthesis, glycine, bicarbonate, and formate were incorporated into the purine ring of inosine 5[prime]-monophosphate (IMP) and adenine nucleotides ([sigma]Ade); azaserine, a known inhibitor of purine de novo synthesis, inhibited incorporation. Activity of the de novo pathway in C. arabica per g fresh weight of leaf tissue during a 3-h incubation period was 8 [plus or minus] 4 nmol of formate incorporated into IMP, 61 [plus or minus] 7 nmol into [sigma]Ade, and 150 nmol into caffeine (the latter during a 7-h incubation). Coffee leaves exhibited classical purine catabolism. Radiolabeled formate, inosine, adenosine, and adenine were incorporated into hypoxanthine and xanthine, which were catabolized to allantoin and urea. Urease activity was demonstrated. Per g fresh weight, coffee leaf squares incorporated 90 [plus or minus] 22 nmol of xanthine into caffeine in 7 h but degraded 102 [plus or minus] 1 nmol of xanthine to allantoin in 3 h. Feedback control of de novo purine biosynthesis was contrasted in C. arabica and Cucurbita pepo, a species that does not synthesize purine alkaloids. End-product inhibition was demonstrated to occur in both species but at different enzyme reactions.     

Content of low-molecular-weight thiols during the imbibition of Pea seeds

The metabolism of low-molecular-weight thiols was investigated in seeds of Pisum sativum L. cv. Kleine Rheinländerin during imbibition in water for 14 h. The amount of oxidized glutathione (GSSG) decreased from 319 nmol (g dry weight)⁻¹ in dry seeds to 38 nmol (g dry weight)⁻¹ within the first 14 h of imbibition. The decrease may have been due to the reduction of GSSG to reduced glutathione (GSH), catalyzed by the enzyme glutathione reductase (GR; EC 1.6.4.2). The enzyme activity was high in dry seeds [25 nkat (g dry weight)⁻¹] and decreased to 20 nkat (g dry weight)⁻¹ within 14 h of imbibition. The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) decreased from 100 nkat (g dry weight)⁻¹ in dry seeds to 67 nkat (g dry weight)⁻¹ after 14 h of imbibition. Within 14 h the amount of γ-glutamyl-cysteine (γ-GC) decreased from 135 to 38 nmol (g dry weight)⁻¹, whereas the cysteine content rose from 81 nmol (g dry weight)⁻¹ in dry seeds to a maximum of 170 nmol (g dry weight)⁻¹ after 12 h of imbibition, which may be due to the degradation of γ-GC into cysteine.     

Osmoregulation by Oat Coleoptile Protoplasts (Effect of Auxin)

The effect of auxin on the physiology of protoplasts from growing oat (Avena sativa L.) coleoptiles was investigated. Protoplasts, isolated iso-osmotically from peeled oat coleoptile segments, were found to swell steadily over many hours. Incubated in 1 mM CaCl2, 10 mM KCl, 10 mM 2-(morpholino)ethanesulfonic acid/1,3-bis-[tris(hydroxymethyl)methylamino]propane, pH 6.5, and mannitol to 300 milliosmolal, protoplasts swelled 28.9% [plus or minus] 2.0 (standard error) after 6 h. Addition of 10 [mu]M indoleacetic acid (IAA) increased swelling to 41.1% [plus or minus] 2.1 (standard error) after 6 h. Swelling (in the absence of IAA) was partially dependent on K+ in the bath medium, whereas auxin-induced swelling was entirely dependent on K+. Replacement of mannitol in the bath by Glc increased swelling (in the absence of IAA) and eliminated auxin-induced swelling. Swelling with or without IAA was inhibited by osmotic shock and was completely reversed by 0.1 mM NaN3. Sodium orthovanadate, applied at 0.5 mM, only gradually inhibited swelling under various conditions but was most effective with protoplasts prepared from tissue preincubated in vanadate. Our data are interpreted to suggest that IAA increases the conductance of the plasma membrane to K+.     

Transport and Metabolism of Dihydrozeatin Riboside in Germinating Lupin Seeds

[³H]-dihydrozeatin riboside was applied selectively to the embryonicaxes or to the cotyledons of germinating lupin (Lupinus luteusL. cv. Weiko III) seeds 6 h following the start of imbibition.There was little transport of dihydrozeatin riboside from embryoto cotyledons up to 6 h after the application, but a substantialamount of radioactivity had moved into the cotyledons at theend of the 10 h incubation period. However, there was no detectablemovement of [³H]-dihydrozeatin riboside from the cotyledonsto the embryonic axis. This indicated a highly polarized movementof cytokinins during the early stages of seed germination. Exogenouslyapplied [³H]-dihydrozeatin riboside was found to be very stable,both when applied to the embryonic axes and cotyledons of intactseed, or following excision, and there was little metabolismwith only small amounts of radioactivity found associated withdegradative metabolites. The embryonic axis of this specieshas recently been found to synthesize cytokinins within 12 hfrom the start of imbibition, and the results of this studyindicate that the embryo-derived cytokinin is probably transportedto the cotyledons where it accumulates and subsequently participatesin the control of cotyledon function. Key words: Lupinus luteus, cytokinin transport and metabolism, dihydrozeatin riboside, seed germination     

Cyclic AMP and free fatty acids in the longer-term regulation of pyruvate dehydrogenase kinase in rat soleus muscle.

Starvation increased pyruvate dehydrogenase (PDH) kinase activity in extracts of freshly excised rat soleus 2.2-fold (from 0.6 min-1 in fed rats to 1.31 min-1 in 48-h-starved rats). In fed rats, activities were unchanged following 24 h of culture in medium 199, but increased 2.1-fold on 24 h of culture with 50 microM dibutyryl cAMP plus 1 mM n-octanoate and 1.6-1.7-fold with either agent alone. Approx. 70% of the increase in PDH kinase induced by starvation was lost following 24 h of culture in medium 199; the loss was prevented by 50 microM dibutyryl cAMP plus 1 mM n-octanoate. cAMP concentrations in fresh soleus muscle were 1 nmol/g (fed rats) and 1.6 nmol/g (starved rats). After 20-60 min of culture the fed-starved difference disappeared and [cAMP] fell to 0.4 nmol/g. Calcitonin-gene-related peptide (CGRP) increased cAMP 3-fold; the increase was maintained throughout 24 h of culture, but was readily reversed at 30 min or 24 h of culture by 60-min incubation with CGRP-free medium. Starvation of the rat (48 h) had no effect on the sensitivity of soleus towards the [cAMP]-increasing effect of CGRP. It is concluded that culture may reverse effects of starvation on PDH kinase activity by lowering cAMP and by removal from the in vivo effects of circulating free fatty acids; and that starvation and CGRP had no detectable long-term effects on the cAMP system in soleus muscle.     

萌发中花生胚轴的耐干性与热稳定蛋白

成熟花生种子吸胀１８ ｈ 发芽率达１００ ％ 。在这１８ ｈ 的范围内,胚轴即使经干燥处理,萌发生长率仍保持１００ ％ ,而热稳定蛋白含量变化很小。吸胀２４ ｈ 后,经干燥的花生胚完全丧失萌发生长能力。ＳＤＳＰＡＧＥ和双向电泳表明,花生胚轴的热稳定蛋白主要是贮藏蛋白,该蛋白中的花生球蛋白大亚基,伴花生球蛋白Ｉ和２Ｓ 蛋白的降解与胚轴的耐干性丧失有关。     

2,4-Dichlorophenoxyacetic Acid and Related Chlorinated Compounds Inhibit Two Auxin-Regulated Type-III Tobacco Glutathione S-Transferases

Two auxin-inducible glutathione S-transferase (GST, EC 2.5.1.18) isozymes from tobacco (Nicotiana tabacum, White Burley) were partially characterized. GST1-1 and GST2-1 are members of a recently identified new type of plant GST isozymes that we will here refer to as type III. Both enzymes were active, with 1-chloro-2,4-dinitrobenzene as a substrate, when expressed in bacteria as fusion proteins. The apparent Km for 1-chloro-2,4-dinitrobenzene was found to be 0.85 [plus or minus] 0.25 mM for GST1-1 and 0.20 [plus or minus] 0.15 mM for GST2-1. The apparent Km for glutathione was similar for both enzymes, 0.40 [plus or minus] 0.15 mM. The in vitro activity of both enzymes could be inhibited by the synthetic auxin 2,4-dichlorophenoxyacetic acid, with an apparent Ki of 80 [plus or minus] 40 [mu]M for GST1-1 and 200 [plus or minus] 100 [mu]M for GST2-1. The GST1-1 was also inhibited by structurally related substances, such as 2,4-dichlorobenzoic acid, with a roughly similar Ki. The nonchlorinated structures benzoic acid and phenoxyacetic acid did not inhibit. p-Chloroisobutyric acid, or clofibric acid, an auxin-transport inhibitor, was found to be an active inhibitor as well. The strongest inhibitor identified, however, was a phenylacetic acid derivative, ethacrynic acid, which showed an apparent Ki of 5 [plus or minus] 5 [mu]M for both enzymes. This substance is a known inducer as well as a substrate of specific mammalian GSTs. The results presented here indicate that the type III plant GSTs might be involved in the metabolism or transport of chlorinated substances that are structurally related to auxins. The possibility that auxins are endogenous ligands or substrates for GSTs is discussed.     

Correction

Phosphoribosylpyrophosphate synthetase (PRS; EC 2.7.6.1) from Hevea brasiliensis Mull. Arg. latex was located in the cytosol. After purification, its apparent molecular weight under nondenaturing conditions was estimated at 200,000 [plus or minus] 10,000; a single band at 57,000 [plus or minus] 3,000 was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme seemed to be a homotetramer. Its affinity constants were estimated at 200 [plus or minus] 30 [mu]M for adenosine triphosphate and 40 [plus or minus] 2 [mu]M for ribose-5-phosphate. The purified enzyme proved to be functional in a paraphysiological medium (cytosol deproteinized by ultrafiltration). Optimum pH was 7.5 in buffer and 6.5 in a paraphysiological medium. No PRS activity was detected in the absence of the Mg2+ ion. Of the numerous compounds tested, only Mn2+, phosphoribosylpyrophosphate and inorganic phosphate affected the enzymatic reaction. Mn2+ (inhibitor constant = 20 [mu]M) and phosphoribosylpyrophosphate (inhibitor constant = 30 [mu]M) were inhibitors. PRS responded allosterically (Hill's coefficient = 2.3) to ribose-5-phosphate in the presence of a physiological concentration of inorganic phosphate (10 mM). These results are set in the physiological context of laticifers.     

Purification and Characterization of Phosphoribosylpyrophosphate Synthetase from Rubber Tree Latex

Phosphoribosylpyrophosphate synthetase (PRS; EC 2.7.6.1) from Hevea brasiliensis Mull. Arg. latex was located in the cytosol. After purification, its apparent molecular weight under nondenaturing conditions was estimated at 200,000 [plus or minus] 10,000; a single band at 57,000 [plus or minus] 3,000 was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme seemed to be a homotetramer. Its affinity constants were estimated at 200 [plus or minus] 30 [mu]M for adenosine triphosphate and 40 [plus or minus] 2 [mu]M for ribose-5-phosphate. The purified enzyme proved to be functional in a paraphysiological medium (cytosol deproteinized by ultrafiltration). Optimum pH was 7.5 in buffer and 6.5 in a paraphysiological medium. No PRS activity was detected in the absence of the Mg2+ ion. Of the numerous compounds tested, only Mn2+, phosphoribosylpyrophosphate, and inorganic phosphate affected the enzymatic reaction. Mn2+ (inhibitor constant = 20 [mu]M) and phosphoribosylpyrophosphate (inhibitor constant = 30 [mu]M) were inhibitors. PRS responded allosterically (Hill's coefficient = 2.3) to ribulose-5-phosphate in the presence of a physiological concentration of inorganic phosphate (10 mM). These results are set in the physiological context of laticifers.     

Reactive oxygen species release, vitamin E, fatty acid and phytosterol contents of artificially aged radish (Raphanus sativus L.) seeds during germination

Seeds of Raphanus sativus L. subjected to accelerated ageing were investigated for reactive oxygen species (ROS) release and for content of vitamin E (tocopherol, TOC, and tocotrienol, TOC-3), fatty acids and phytosterols in seed coats, cotyledons and embryonic axes during germination. In unaged seeds, ROS release occurred mainly in seed coats of non-imbibed seeds and in seedlings (48?h of imbibition). TOC and TOC-3 were mainly represented by the ??-isoform, abundant in embryonic axes. Fatty acids were mainly found in cotyledons. In seed coat and embryonic axis, phytosterols consisted mainly of sitosterols. The effects of ageing were mainly visible in embryonic axes at 48?h of imbibition. Deterioration was associated with a decrease in fresh weight increase percentage, germination percentage, ??-TOC and total fatty acid content. An increase in ROS release from seed coats and in ??-TOC, ??-TOC, ??-TOC-3 content in embryonic axis was also observed. The use of ??-TOC and total fatty acids in embryonic axis as parameters of seed quality evaluation during storage was suggested.     

Changes in trigonelline (N-methylnicotinic acid) content and nicotinic acid metabolism during germination of mungbean (Phaseolus aureus) seeds

Changes in trigonelline content and in biosynthetic activity were determined in the cotyledons and embryonic axes of etiolated mungbean (Phaseolus aureus) seedlings during germination. Accumulation of trigonelline (c. 240 nmol per pair of cotyledons) was observed in the cotyledons of dry seeds; trigonelline content decreased 2 d after imbibition. Trigonelline content in the embryonic axes increased with seedling growth and reached a peak (c. 380 nmol per embryonic axis) at day 5. Trigonelline content did not change significantly during the differentiation of hypocotyls, and the concentration was greatest in the apical 5 mm. Nicotinic acid and nicotinamide were better precursors for pyridine nucleotide synthesis than quinolinic acid, but no great differences were found in the synthesis of trigonelline from these three precursors. Trigonelline synthesis was always higher in embryonic axes than in cotyledons. Activity of quinolinate phosphoribosyltransferase (EC 2.4.2.19), nicotinate phosphoribosyltransferase (EC 2.4.2.11), and nicotinamidase (EC 3.5.1.19) was found in cotyledons and embryonic axes, but no nicotinamide phosphoribosyltransferase (EC 2.4.2.12) activity was detected. It follows that quinolinic acid and nicotinic acid were directly converted to nicotinic acid mononucleotide by the respective phosphoribosyltransferases, but nicotinamide appeared to be converted to nicotinic acid mononucleotide after conversion to nicotinic acid. Trigonelline synthase (nicotinate N-methyltransferase, EC 2.1.1.7) activity increased in the embryonic axes, but decreased in cotyledons during germination. [14C]Nicotinic acid and trigonelline absorbed by the cotyledons were transported to the embryonic axes during germination. Trigonelline had no effect on the growth of seedlings, but nicotinic acid and nicotinamide significantly inhibited the growth of roots. Based on these findings, the role of trigonelline synthesis in mungbean seedlings is discussed.     

The amounts,subunit structures,and template-engaged activities of RNA polymerases in germinating soybean axes

During the first 24 hr of soybean axis imbibition and growth, there is about a 25-fold increase in RNA polymerase activity associated with isolated nuclei. Within this same period, there is no increase in the amount of RNA polymerase I or II protein in soybean axes. There is no alteration in subunit structure of RNA polymerase II during germination and growth, with the possible exception of conversion of the 215,000 subunit to a 180,000 polypeptide, and no alteration in phosphorylation pattern of RNA polymerase II subunits. These results suggest that the rates of RNA synthesis during imbibition, germination, and growth of soybean axes are not regulated by altering the amount or subunit structure or by posttranslational modification of RNA polymerase II subunits.     

Dissociation between release and gene expression of gonadotropin alpha-subunit in gonadotropin-releasing hormone-stimulated alpha T3-1 cell line.

The alpha T3-1 cell line which was derived by targeted tumorigenesis in transgenic mice [Windle et al. (1990) Mol. Endocrinol. 4, 597-603] possesses high-affinity binding sites for GnRH analogs coupled to enhanced phosphoinositide turnover and phospholipase D activity. Incubation of alpha T3-1 cells with [D-Trp6]-GnRH analog (GnRH-A) resulted in a rapid increase in gonadotropin alpha-subunit mRNA levels which was detected already at 30 min of incubation (0.1 nM GnRH-A, 3-fold, p < 0.01). The effect diminished with time to reach basal levels at about 12 h of incubation, with a secondary rise in alpha mRNA levels between 12 and 24 h of incubation. Addition of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA, 100 ng/mL) or the Ca2+ ionophore ionomycin (1 microM) to alpha T3-1 cells also resulted in a rapid increase in alpha-subunit mRNA levels. Surprisingly, GnRH-induced alpha-subunit release was detected only after a lag of 4 h of incubation. Thus, dissociation between exocytosis and gene expression can be demonstrated in GnRH-stimulated alpha T3-1 cell line.     

Dibutyryl cyclic AMP decreases glutamine synthetase in cultured 3T3-L1 adipocytes

Glutamine synthetase specific activity increases greater than 100-fold during the insulin-mediated differentiation of confluent 3T3-L1 cells into adipocytes. Incubation of the adipocytes for 22 h with 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline, 0.2 mM 8-bromo-cyclic AMP, 10 micro M epinephrine, or 1 microgram of alpha 1-24 adrenocorticotropic hormone/ml decreased glutamine synthetase by greater than 60%. During the same incubation period, there was no effect of these compounds on protein or on the specific activities of glucose-6-P dehydrogenase or hexokinase. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase activity was half-maximal at 50 micro M dibutyryl cyclic AMP. Furthermore, between 10 micro M and 5 mM dibutyryl cyclic AMP, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase was similar in the absence or presence of 1 microgram of insulin/ml. Immunotitration of glutamine synthetase activity from 3T3 adipocytes indicates that the dibutyryl cyclic AMP-mediated decrease in the activity is due to a decrease in the cellular content of glutamine synthetase molecules. We studied the effects of dibutyryl cyclic AMP on the synthesis and degradation of glutamine synthetase. Synthesis rate was estimated from the incorporation of L-[35S]methionine into glutamine synthetase during a 60-min incubation period. Degradation rate was estimated from the first order disappearance of radioactivity from glutamine synthetase in 3T3 adipocytes previously incubated with L-[35S]methionine. Glutamine synthetase was isolated by immunoprecipitation followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Incubation of 3T3 adipocytes with dibutyrl cyclic AMP resulted in a rapid decline in the apparent synthesis rate of glutamine synthetase. In addition, dibutyryl cyclic AMP treatment increased the initial rate of glutamine synthetase degradation. The half-life of glutamine synthetase was 24.5 h in control cultures and 16 h in dibutyryl cyclic AMP-treated cultures. In contrast, dibutyryl cyclic AMP had little effect on the synthesis or degradation of soluble protein. Our data indicate that the dibutyryl cyclic AMP-mediated decrease in 3T3 adipocyte glutamine synthetase activity results from a decrease in the synthesis rate and an increase in the initial degradation rate of the enzyme.