AP Accumulation Research Articles

Allelic Variation and Transcriptional Isoforms of Wheat TaMYC1 Gene Regulating Anthocyanin Synthesis in Pericarp

Recently the TaMYC1 gene encoding bHLH transcription factor has been isolated from the bread wheat (Triticum aestivum L.) genome and shown to co-locate with the Pp3 gene conferring purple pericarp color. As a functional evidence of TaMYC1 and Pp3 being the same, higher transcriptional activity of the TaMYC1 gene in colored pericarp compared to uncolored one has been demonstrated. In the current study, we present additional strong evidences of TaMYC1 to be a synonym of Pp3. Furthermore, we have found differences between dominant and recessive Pp3(TaMyc1) alleles. Light enhancement of TaMYC1 transcription was paralleled with increased AP accumulation only in purple-grain wheat. Coexpression of TaMYC1 and the maize MYB TF gene ZmC1 induced AP accumulation in the coleoptile of white-grain wheat. Suppression of TaMYC1 significantly reduced AP content in purple grains. Two distinct TaMYC1 alleles (TaMYC1p and TaMYC1w) were isolated from purple- and white-grained wheat, respectively. A unique, compound cis-acting regulatory element had six copies in the promoter of TaMYC1p, but was present only once in TaMYC1w. Analysis of recombinant inbred lines showed that TaMYC1p was necessary but not sufficient for AP accumulation in the pericarp tissues. Examination of larger sets of germplasm lines indicated that the evolution of purple pericarp in tetraploid wheat was accompanied by the presence of TaMYC1p. Our findings may promote more systematic basic and applied studies of anthocyanins in common wheat and related Triticeae crops.

5-Formyl- and 5-Carboxydeoxycytidines Do Not Cause Accumulation of Harmful Repair Intermediates in Stem Cells.

5-Formyl-dC (fdC) and 5-carboxy-dC (cadC) are newly discovered bases in the mammalian genome that are supposed to be substrates for base excision repair (BER) in the framework of active demethylation. The bases are recognized by the monofunctional thymine DNA glycosylase (Tdg), which cleaves the glycosidic bond of the bases to give potentially harmful abasic sites (AP-sites). Because of the turnover of fdC and cadC during cell state transitions, it is an open question to what extent such harmful AP-sites may accumulate during these processes. Here, we report the development of a new reagent that in combination with mass spectrometry (MS) allows us to quantify the levels of AP-sites. This combination also allowed the quantification of β-elimination (βE) products, which are repair intermediates of bifunctional DNA glycosylases. In combination with feeding of isotopically labeled nucleosides, we were able to trace the intermediates back to their original nucleobases. We show that, while the steady-state levels of fdC and cadC are substantially increased in Tdg-deficient cells, those of both AP- and βE-sites are unaltered. The levels of the detected BER intermediates are 1 and 2 orders of magnitude lower than those of cadC and fdC, respectively. Thus, neither the presence of fdC nor that of cadC in stem cells leads to the accumulation of harmful AP- and βE-site intermediates.

CULTIVATION OF MEDICINAL AND AROMATIC PLANTS IN INDIA - A COMMERCIAL APPROACH

CULTIVATION OF MEDICINAL AND AROMATIC PLANTS IN INDIA - A COMMERCIAL APPROACH

Oscillatory Combustion of Fine-AP/HTPB Propellants: Disproportionate Pyrolysis Response

The combustion of hydroxyl-terminated polybutadiene (HTPB)propellants containing e neammonium perchlorate (AP) was investigated using laser-excited, combustion recoil at 1 ‐5 atm. At 1 atm, binder-rich (70‐75% AP), monomodal e ne-AP (2‐50 µm) propellants exhibit a prominent non-quasi-steady gas and surface reaction zone, homogeneous propellant, one-dimensional e ame response peak at 100 ‐300 Hz, with frequency varying inversely with AP size. Adding coarse AP (resulting in a wide, bimodal AP distribution ) causes this response to disappear at 1 atm. Raising the pressure to 2 atm causes the response to reappear and increase in frequency to 600 ‐800 Hz (2-µm AP). This oscillatory combustion behavior is attributed to time-varying selective or disproportionate pyrolysis of AP and HTPB (unsteady accumulation and depletion of AP at the propellant surface ) and the associated compositional (stoichiometric ) e uctuations that occur in the fuel-rich, premixed gas-phase reaction zone adjacent to the e ne-AP/HTPB solid region. A low Peclet number appears to be a requirement of achieving this condition. Combustion recoil and thermocouple measurements at 1 atm without laser excitation exhibited spontaneous oscillations in the monomodal e ne-AP propellants and corroboratethe disproportionate pyrolysis interpretation of the laser-excitedresonant response. This e nding of a strong disproportionationresponsein compositepropellants with e ne AP or binder-rich, e ne-AP matrix regions has important implications for pressure-coupled response and solid rocket motor stability in that the same response mechanism could operate under oscillatory pressure conditions and at elevated pressures.

Relationship between inositol 1,4,5-trisphosphate mass level and [ 14C]aminopyrine uptake in gastrin-stimulated parietal cells

The relationship between gastrin-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3) content and [ 14C]aminopyrine ([ 14C]AP) uptake (an index of in vitro acid secretion) was investigated in a population of highly enriched rabbit parietal cells (90 ± 5%). Gastrin induced a rapid rise in Ins(1,4,5)P 3 content which was maximal within 15 s of stimulation (2- to 2.5-fold basal level) followed by a rapid decrease within 30 s; a high Ins(1,4,5)P 3 level could also be observed after a longer time of hormone stimulation (180 s). Gastrin dose-dependently induced Ins(1,4,5)P 3 accumulation and [ 14C]AP uptake; both dose-response curves were similar (EC 50 ≈ 0.1 nM). Furthermore, L-365, 260 (3-(acylamino)benzodiazepine), a selective gastrin/ CCK-B receptor antagonist, dose-dependently inhibited Ins(1,4,5)P 3 production and [ 14C]AP accumulation induced by 10 nM gastrin with a similar potency (IC 50 ≈ 1–2 nM). These results led us to conclude that Ins(1,4,5)P 3 is involved in gastrin-stimulated acid secretory activity of gastric parietal cells.

Characterization of a gastrin-type receptor on rabbit gastric parietal cells using L365,260 and L364,718

Previous studies have demonstrated that gastrin and the COOH-terminal octapeptide of cholecystokinin (CCK-8) stimulated in vitro acid secretion from isolated rabbit gastric parietal cells. Both peptides bind to receptor sites located on these cells and induce an increase in phosphoinositide turnover and an uptake of [14C]aminopyrine ([14C]AP) with the same efficacy and potency. In the present study, we used the 3-(benzoylamino)-benzodiazepine analogue L365,260 and the 3-(acylamino)-benzodiazepine analogue L364,718 to investigate what type of receptor (gastrin type or CCK-A type) is involved in the regulation of the H+ secretory activity of the rabbit parietal cell. Neither L365,260 nor L364,718 alone caused stimulation of [3H]inositol phosphates ([3H]InsP) production. Each analogue inhibited 125I-labeled gastrin or 125I-CCK-8 binding to parietal cells and gastrin- or CCK-8-induced [3H]InsP production and [14C]AP accumulation. In all cases, L365,260 was approximately 70-100 times more potent than L364,718 (IC50 approximately 2-4 nM for L365,260 and approximately 0.2-0.4 microM for L364,718). Nevertheless, each antagonist displayed the same potency to inhibit the effects of gastrin or CCK-8. These results demonstrate that gastrin and CCK-8 interact with the same "gastrin-type" receptor on parietal cells. Moreover, L365,260 behaves as a competitive antagonist of the action of gastrin on parietal cells. Gastrin induces a rise in the levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] within the first seconds after parietal cell stimulation. The fact that L365,260 (10 nM) totally suppressed the gastrin-induced formation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 suggests the involvement of these isomers in the mediation of acid secretion through gastrin receptor activation.

H 2-receptor antagonism by a novel compound IT-066 assessed from [ 14C]aminopyrine accumulation in rabbit parietal cells

The H2-receptor antagonistic activities and properties of IT-066 were investigated in rabbit isolated gastric mucosal cell and were compared with those of famotidine and cimetidine. IT-066 inhibited dose dependently the histamine-stimulated [14C]aminopyrine ([14C]AP) accumulation in parietal cells. The antagonism was unsurmountable. The inhibitory action of IT-066 was enhanced by lengthening of its incubation time with the cells. IT-066 following 30 min preincubation with the parietal cells showed 32 and 1560 times the activity of famotidine and cimetidine, respectively, as an inhibitor of [14C]AP accumulation. The inhibitory activity of IT-066 on the histamine effect remained after repeated washing of the cells while with famotidine and cimetidine the response to histamine was restored after washout of the drugs from the cells. These data indicate that the antagonism of IT-066 on histamine H2-receptors is high and is unsurmountable in the isolated parietal cell model.

Cyclic fluctuations in arylphorin, the principal serum storage protein of Lymantria dispar, indicate multiple roles in development

Arylphorin (Ap) was isolated and partially characterized from larval serum of the gypsy moth, Lymantria dispar (Lepidoptera: Lymantriidae). Non-denaturing and SDS-polyacrylamide gel electrophoresis suggest a 440,000 Da hexamer composed of nonidentical subunits of M r 73,000 and 80,000. Tyrosine and phenylalanine comprise 15.6% of the total protein amino acid. L. dispar Ap is immunologically related to Manduca sexta Ap. Hemolymph Ap concentrations throughout the larval stadia were determined using quantitative immunoelectrophoresis. Cyclic fluctuations in hemolymph concentrations are correlated with each molting cycle. A general increase in Ap concentration during each intermolt is followed by a sharp decline between apolysis and ecdysis. The last days of each instar were found to be the best time to sample protein titer with minimum variance. Hemolymph space, estimated by measuring the dilution of an injected foreign protein, is a constant 28% (v/w) of body weight. Total serum Ap per animal was calculated. A divergent allometric relationship between Ap accumulation and weight gain throughout the larval period results in the ultimate domination of the serum protein profile by Ap in the ultimate larval instar of the female in particular. An additional instar of Ap accumulation in the female gypsy moth is suggested to compensate for the lack of a predominantly female-specific storage protein in this species.

Characteristics of the spontaneous gastric endocrine tumor of mastomys

Mastomys is a rodent which has been reported to develop spontaneous antral endocrine tumors with acid hypersecretion and duodenal ulceration. This study documents the establishment of a breeding colony and the characterization of the tumors and their possible secretagogues. Parietal cell secretory characteristics were studied using isolated gastric glands (IGG) of both normal ( n = 5) and tumor-bearing animals. Tumors ( n = 6) and control gastric tissue samples were examined by light transmission microscopy and immunohistochemistry. Gastrin was measured by radioimmunoassay in both plasma and tissue. IGG were prepared by collagenase dispersion and acid sequestration assessed by [ 14C]AP accumulation. Secretory mechanisms of this species were identified by establishment of a histamine dose-response curve and use of 8-bromo-cAMP. Receptor and proton pump inhibitions were assessed using cimetidine (10 −5 M) and the H/K ATPase inhibitor omeprazole (10 −5). Both reduced [ 14C]AP accumulation significantly ( P < 0.05). 8-Bromo-cAMP and histamine significantly stimulated [ 14C]AP accumulation ( P < 0.05). Although parietal cells were substantially increased in tumor animals as compared to controls, the physiological parameters of acid secretion appeared normal in both and were comparable to other species which have been studied. Tumors were Grimelius positive and contained diffuse electron-dense granules. Immunohistochemistry was negative for gastrin, bombesin, serotonin, neuron-specific enolase, calcitonin, and pancreatic polypeptide. Tumor histamine-like immunoreactivity was, however, positive. Normal stomach contained 1001 ± 185 compared to <0.5 pmole/g gastrin in tumors. Plasma gastrin was normal in both groups (29 ± 5) as compared to 26 ± 8 pmole/liter. This study characterizes a spontaneous gastric endocrine tumor which is associated with apparent parietal cell hyperplasia and reports of increased acid secretion and duodenal ulceration. The observations are consistent with the elaboration by the tumor of a nongastrin acid-trophic secretagogue.

Time‐weighted accumulations ap(τ) and Kp(τ)

The planetary geomagnetic indices Kp and ap are widely used in space geophysics. They provide an estimate of maximum magnetic perturbation within a 3‐hour period. Many geophysical properties are clearly related to the indices, through energy transfer from a common disturbance source, but direct correlation is often lacking because of poor matching between the frequency of sampling and the physical response functions. The index ap(τ) is a simple accumulation of the linear ap calculated with an attenuation factor τ included to take account of natural temporal relaxation. The case for ap(τ) and the related Kp(τ) is made using applications to the variability of the plasma environment in the ionosphere and inner magnetosphere. These examples of improved correlation suggest that time‐weighted integration might profitably be applied to other indices.

Inhibition of acid formation by epidermal growth factor in the isolated rabbit gastric glands.

The effects of epidermal growth factor (EGF) on basal and stimulated (with histamine, dibutyryl cyclic AMP, and high concentrations of K+) acid formation have been studied in isolated glands from the rabbit gastric mucosa. The changes in the accumulation of [14C]aminopyrine [14C]AP have been used as an indirect measurement of acid production in the glands. Unstimulated gastric glands accumulated [14C]AP indicating the existence of basal acid production in these glands, and EGF caused a small but significant reduction in basal [14C]AP uptake. A similar reduction of basal [14C]AP uptake was observed after exposure to omeprazole but not after ranitidine or prostaglandin E2 (PGE2). Histamine, dibutyryl cyclic AMP and K+ caused a strong and dose-dependent stimulation of acid formation by the glands. EGF, like omeprazole, reduced dose-dependently the [14C]AP accumulation stimulated by both histamine and dibutyryl cyclic AMP, while ranitidine and PGE2 reduced histamine- but not dibutyryl-cyclic-AMP-stimulated accumulation of [14C]AP. In the absence of other external stimuli, an increased K+ concentration enhanced [14C]AP accumulation to levels similar to those produced by histamine and this effect was not changed by EGF, ranitidine or PGE2 but was inhibited by omeprazole. We conclude that EGF interferes with the final steps of acid production between cyclic nucleotides and proton pump of the parietal cells.

Cyclic GMP and acid production in isolated gastric cells of the rat

Just as cAMP is regarded as an intracellular mediator of histamine, so has cGMP been connected with cholinergic stimulation of gastric acid secretion. The object of the present investigation was to study the possible role of cellular cGMP on 14C-aminopyrine uptake, an indirect measure of parietal cell H+-production, by using mixtures of isolated rat gastric cells and fractions with different parietal cell content. Cellular cAMP and cGMP. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) enhanced the cAMP and cGMP of gastric cells in a time- and concentration-dependent manner, by 98 and 124% (1 mmol/l) and was included in all further studies. In parietal cell enriched fractions, histamine elevated cAMP by 109% (100 mumol/l) without changing cGMP while carbachol did not influence either nucleotide. Various thiols and nitrogen compounds strongly enhanced cellular cGMP, e.g. hydroxylamine and L-cysteine (1 mmol/l) by 527 and 656%, whereas changes in cAMP were minimal. The hydroxylamine response occurred in parietal cell depleted and enriched fractions. 14C-aminopyrine (AP) uptake. IBMX alone reduced the basal AP uptake, potentiated the effect of histamine, and inhibited the effect of carbachol, which alone stimulated basal accumulation by 302%. The most efficacious stimulant of parietal cell H+-production was dibutyryl cAMP (582%, 100 mumol/l), whereas dibutyryl cGMP was without effect. However, this latter compound (1 mmol/l) reduced AP accumulation due to dibutyryl cAMP almost completely. Thiols and nitrogen compounds all more or less reduced AP uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

The mammalian gastric parietal cell in vitro.

To summarize the metabolic status of the parietal cell: There does not seem to be a close relationship between cellular ATP levels and acid secretion. Acid secretion is absolutely dependent on oxygen, and oxygen consumption will increase in direct proportion to the rate of acid secretion. However, the absolute rate of respiration is not closely related to the formation of acid in the subtissue systems. Acid formation can be driven directly by addition of ATP in permeabilized glands, even under apparent anoxic conditions. This correlates well with the presence of the gastric (H+, K+)-ATPase in the parietal cell. If ATP is the main source of energy for the acid secretion, it is quite possible that the relevant ATP pool is compartmentalized and that the content in this pool has a high turnover rate, whereas the ATP used for other cellular functions would be spared. A pure redox mechanism in the gastric mucosa is not possible. However, it remains to be shown that a redox component is not involved in the secretory process. The acid formation measured by AP accumulation in the gastric glands is not an indication of secretory rate. Thus even though ATP appears to restore acid formation in permeabilized glands, this effect has been mainly studied in nonstimulated systems. A detailed study over the energy requirement in the permeabilized resting cell remains to be done. In the mammals we only have information so far about the piglet and the rabbit in terms of substrate preference. The differences between the two could either be due to species or age difference. In both mammals and amphibia, there is no evidence to suggest that acid secretion results in an increase in oxygen consumption purely due to a state IV to III transition of mitochondrial respiration. Rather, increased Krebs-cycle activity would appear to be the major metabolic result of stimulation.

Cytochalasin inhibition of isolated rat gastric parietal cell function

Submicrogram concentrations (0.04-0.29 microM) of the microfilament disrupting agents cytochalasins D, E, and B (CD, CE, CB) were shown to inhibit secretagogue-stimulated 14C-aminopyrine accumulation (AP) in isolated rat gastric mucosal parietal cells. The microtubule disrupting agent colchicine had little influence on AP accumulation. Histamine- and dibutyryl cyclic AMP (DbcAMP)-stimulated AP accumulation was inhibited with an order of potency CD greater than CE approximately equal to CB. CB inhibition of these secretagogue actions was, however, only approximately 65-70% of the maximal stimulated response, whereas CD and CE caused 100% inhibition. On the other hand, carbamylcholine-stimulated AP accumulation was inhibited 100% by all cytochalasins tested with an order of potency CD approximately equal to CE greater than CB. These data are discussed in relation to acid secretagogue-induced morphological changes involving actin filament organization in parietal cells.

Growth Substances in the Roots ofVicia fabaII. THE EFFECTS OF AGEING AND EXCISION OF THE MAIN TAP-ROOT MERISTEM

SUMMARY A quantitative chromatographic study has been made of the changes in the activity and distribution of the main ether-soluble acid auxins of Vicia faba seedling root systems during development and resulting from excision of the main tap-root meristem. An IAA-like auxin (AP (ii)) is apparently synthesized predominantly in apical and at a lower rate in lateral meristems. Production seems to stop when meri stematic growth stops. Its concentration in mature extended cells is much lower and may fall to zero in old cells, suggesting active degradation by an auxin-oxidase. Excision of the main tap-root tip gradually results in a greatly augmented produc tion of AP (ii) in lateral meristems, conceivably the result of correlative growth promotion. A second auxin and root-growth inhibitor (AP (Hi)) is present at higher activity levels than AP (ii) in tap-root meristems and at the same level in lateral meristems. In mature cells its activity is much lower than that of AP (ii). In contrast to AP (ii) it accumulates in both tap-root meristems and mature tissue as the root system ages. It could also be produced during meristematic growth but is not subse quently degraded. As with AP (ii), excision of the tap-root tip brings about a great increase in its concentration in lateral tips. A third auxin and root-growth accelerator (AP (?)) (accelerator a ?) is present in lower concentrations in both meristem and mature tissue. Its concentration tends to decrease with ageing and, in lateral meristems, is not affected by tap-root tip excision. It is suggested that AP (ii) produced by the meristem is normally at suboptimal levels in the extending cells and may be the principal hormone controlling exten sion growth. AP (iii) accumulation may account for growth deceleration on ageing. The role of AP (f) remains obscure. It is unlikely that correlative effects of the tap root tip on lateral root growth are exercised directly via these auxins.