Intercellular communication between follicular angiotensin receptors and Xenopus laevis oocytes: medication by an inositol 1,4,5- trisphosphate-dependent mechanism

In Xenopus laevis oocytes, activation of angiotensin II (AII) receptors on the surrounding follicular cells sends a signal through gap junctions to elevate cytoplasmic calcium concentration ([Ca2+]i) within the oocyte. The two major candidates for signal transfer through gap junctions into the oocyte during AII receptor stimulation are Ins(1,4,5)P3 and Ca2+. In [3H]inositol-injected follicular oocytes, AII stimulated two- to fourfold increases in phosphoinositide hydrolysis and production of inositol phosphates. Injection of the glycosaminoglycan, heparin, which selectively blocks Ins(1,4,5)P3 receptors, prevented both AII-stimulated and Ins(1,4,5)P3-induced Ca2+ mobilization in Xenopus follicular oocytes but did not affect mobilization of Ca2+ by ionomycin or GTP. These results indicate that the AII-regulated process of gap junction communication between follicular cells and the oocyte operates through an Ins(1,4,5)P3-dependent mechanism rather than through transfer of Ca2+ into the ooplasm and subsequent Ca(2+)-induced Ca2+ release.     

Metabolism of Indole-3-Acetic Acid in Arabidopsis

The metabolism of indole-3-acetic acid (IAA) was investigated in 14-d-old Arabidopsis plants grown in liquid culture. After ruling out metabolites formed as an effect of nonsterile conditions, high-level feeding, and spontaneous interconversions, a simple metabolic pattern emerged. Oxindole-3-acetic acid (OxIAA), OxIAA conjugated to a hexose moiety via the carboxyl group, and the conjugates indole-3-acetyl aspartic acid (IAAsp) and indole-3-acetyl glutamate (IAGlu) were identified by mass spectrometry as primary products of IAA fed to the plants. Refeeding experiments demonstrated that none of these conjugates could be hydrolyzed back to IAA to any measurable extent at this developmental stage. IAAsp was further oxidized, especially when high levels of IAA were fed into the system, yielding OxIAAsp and OH-IAAsp. This contrasted with the metabolic fate of IAGlu, since that conjugate was not further metabolized. At IAA concentrations below 0.5 μm, most of the supplied IAA was metabolized via the OxIAA pathway, whereas only a minor portion was conjugated. However, increasing the IAA concentrations to 5 μm drastically altered the metabolic pattern, with marked induction of conjugation to IAAsp and IAGlu. This investigation used concentrations for feeding experiments that were near endogenous levels, showing that the metabolic pathways controlling the IAA pool size in Arabidopsis are limited and, therefore, make good targets for mutant screens provided that precautions are taken to avoid inducing artificial metabolism.The plant hormone IAA is an important signal molecule in the regulation of plant development. Its central role as a growth regulator makes it necessary for the plant to have mechanisms that strictly control its concentration. The hormone is believed to be active primarily as the free acid, and endogenous levels are controlled in vivo by processes such as synthesis, oxidation, and conjugation. IAA has been shown to form conjugates with sugars, amino acids, and small peptides. Conjugates are believed to be involved in IAA transport, in the storage of IAA for subsequent use, in the homeostatic control of the pool of the free hormone, and as a first step in the catabolic pathways (Cohen and Bandurski, 1978; Nowacki and Bandurski, 1980; Tuominen et al., 1994; Östin et al., 1995; Normanly, 1997). It is generally accepted that in some species conjugated IAA is the major source of free IAA during the initial stages of seed germination (Ueda and Bandurski, 1969; Sandberg et al., 1987; Bialek and Cohen, 1989), and there is also evidence that in some plants (but not all; see Bialek et al., 1992), the young seedling is entirely dependent on the release of free IAA from conjugated pools until the plant itself is capable of de novo synthesis (Epstein et al., 1980; Sandberg et al., 1987).The function of conjugated IAA during vegetative growth is somewhat less clear. It has been shown that conjugated IAA constitutes as much as 90% of the total IAA in the plant during vegetative growth (Normanly, 1997). However, the role of the IAA conjugates at this stage of the plant''s life cycle remains unknown. Analysis of endogenous IAA conjugates in vegetative tissues has revealed the presence of a variety of different compounds, including indole-3-acetyl-inositol, indole-3-acetyl-Ala, IAAsp, and IAGlu (Anderson and Sandberg, 1982; Cohen and Baldi, 1983; Chisnell, 1984; Cohen and Ernstsen, 1991; Östin et al., 1992). Studies of vegetative tissues have indicated that IAAsp, one of the major conjugates in many plants, is the first intermediate in an irreversible deactivation pathway (Tsurumi and Wada, 1986; Tuominen et al., 1994; Östin, 1995). Another mechanism that is believed to be involved in the homeostatic control of the IAA pool is catabolism by direct oxidation of IAA to OxIAA, which has been shown to occur in several plant species (Reinecke and Bandurski, 1983; Ernstsen et al., 1987).One area in the study of IAA metabolism in which our knowledge is increasing is the analysis of the homeostatic controls of IAA levels in plants. It has been possible, for instance, to increase the levels of IAA in transgenic plants expressing iaaM and iaaH genes from Agrobacterium tumefaciens. Analysis of these transgenic plants has indicated that plants have several pathways that can compensate for the increased production of IAA (Klee et al., 1987; Sitbon, 1992). It is expected that future studies using now-available genes will provide further insight into IAA metabolism. For example, a gene in maize encoding IAA-Glc synthetase has been identified, and several genes (including ILR1, which may be involved in hydrolysis of the indole-3-acetyl-Leu conjugate) have been cloned from Arabidopsis (Szerszen et al., 1994; Bartel and Fink, 1995). Furthermore, Chou et al. (1996) identified a gene that hydrolyzes the conjugate IAAsp to free IAA in the bacterium Enterobacter aggloremans.Because of its small genome size, rapid life cycle, and the ease of obtaining mutants, Arabidopsis is increasingly used as a genetic model system to investigate various aspects of plant growth and development. IAA signal transduction is also being investigated intensively in Arabidopsis in many laboratories (Leyser, 1997). Mutants with altered responses to externally added auxins or IAA conjugates have been identified in Arabidopsis. The identified mutants are either signal transduction mutants such as axr1-4 (Lincoln et al., 1990), or have mutations in genes involved in auxin uptake or transport, such as aux1 and pin1 (Okada et al., 1991; Bennett et al., 1996). A few mutants that are unable to regulate IAA levels or are unable to hydrolyze IAA conjugates, sur1-2 and ilr1, respectively, have also been identified (Bartel and Fink, 1995; Boerjan et al., 1995). To our knowledge, no mutant that is auxotrophic for IAA has been identified to date, which may reflect the redundancy in IAA biosynthetic pathways or the lethality of such mutants.In spite of the work reported thus far, many aspects of the metabolism of IAA in Arabidopsis require further investigation, because few details of the processes involved in IAA regulation are known. This lack of knowledge puts severe constraints on genetic analysis of IAA metabolism in Arabidopsis. For example, it is essential to have prior knowledge of IAA metabolism to devise novel and relevant screens with which to identify mutants of IAA metabolism. We have sought to address this issue by identifying the metabolic pathways involved in catabolism and conjugation under conditions that minimally perturb physiological processes. In this investigation we studied the conjugation and catabolic pattern of IAA by supplying relatively low levels of labeled IAA and identifying the catabolites and conjugates by MS. Different feeding systems were tested to optimize the application of IAA and to avoid irregularities in metabolism attributable to culturing, feeding conditions, or microbial activity. It is well documented that IAA metabolism is altered according to the amount of exogenous auxin applied; therefore, we placed special emphasis on distinguishing between catabolic routes that occur at near-physiological concentrations and those that occur at the high auxin concentrations commonly used in mutant screens.     

Structural and Functional Characterization of an Anesthetic Binding Site in the Second Cysteine-Rich Domain of Protein Kinase Cδ?

Elucidating the principles governing anesthetic-protein interactions requires structural determinations at high resolutions not yet achieved with ion channels. Protein kinase C (PKC) activity is modulated by general anesthetics. We solved the structure of the phorbol-binding domain (C1B) of PKCδ complexed with an ether (methoxymethylcycloprane) and with an alcohol (cyclopropylmethanol) at 1.36-Å resolution. The cyclopropane rings of both agents displace a single water molecule in a surface pocket adjacent to the phorbol-binding site, making van der Waals contacts with the backbone and/or side chains of residues Asn-237 to Ser-240. Surprisingly, two water molecules anchored in a hydrogen-bonded chain between Thr-242 and Lys-260 impart elasticity to one side of the binding pocket. The cyclopropane ring takes part in π-acceptor hydrogen bonds with the amide of Met-239. There is a crucial hydrogen bond between the oxygen atoms of the anesthetics and the hydroxyl of Tyr-236. A Tyr-236-Phe mutation results in loss of binding. Thus, both van der Waals interactions and hydrogen-bonding are essential for binding to occur. Ethanol failed to bind because it is too short to benefit from both interactions. Cyclopropylmethanol inhibited phorbol-ester-induced PKCδ activity, but failed to do so in PKCδ containing the Tyr-236-Phe mutation.     

Cortisol stress response and histopathological alteration index in Clarias gariepinus exposed to sublethal concentrations of Qua Iboe crude oil and rig wash

The histological effect on and stress response of post juvenile Clarias gariepinus exposed to Qua Iboe crude oil and rig wash were investigated. Fish weighing 60–90 g and measuring 16–18 cm were exposed for 7–28 days to 8.00 ml^?1 Qua Iboe crude oil and 0.0018 ml^–1 rig wash, both being 0.1 of the 96 hr LC₅₀. Blood samples of C. gariepinus were collected every seven days and evaluated for stress by measuring cortisol concentration. The gills and liver were studied and scored for Gill Alteration Index (GAI) and Hepatic Alteration Index (HAI), respectively. There was an increase in cortisol level up to the 7th and 14th day among the group exposed to Qua Iboe crude oil, with a decrease on the 21st and 28th day. The rig wash group increased in cortisol level up to the 7th day and decreased slightly on the 14th day, after which the trend became irregular. The toxic effects of the Qua Iboe crude oil and rig wash were time dependent, as shown by the histopathological alteration index (HAI) of gill and liver. After 28 days of exposure, the gills had irreparable damage due to high frequency of cellular necrosis and degeneration, whereas the liver had from moderate to severe damage due to the high frequency of cellular degeneration and inflammation. Qua Iboe crude oil and rig wash are both toxic to C. gariepinus, therefore their indiscriminate discharge to the environment must be discouraged.     

Expression of two heterologous promoters, Agrobacterium rhizogenes rolC and cauliflower mosaic virus 35S, in the stem of transgenic hybrid aspen plants during the annual cycle of growth and dormancy

We monitored, for the first time, the activity of two model heterologous promoters, the Agrobacterium rhizogenes rolC and the cauliflower mosaic virus (CaMV) 35S, throughout the annual cycle of growth and dormancy in a perennial species, hybrid aspen. Each promoter was fused to the uidA -glucuronidase (GUS) reporter gene and the constructs were introduced into the hybrid aspen genome by Agrobacterium-mediated transformation. Both wildtype and transgenic plants were cultivated under different regimes of photoperiod and temperature to induce passage through one growth-dormancy-reactivation cycle, and at intervals GUS staining was assessed in stem sections. In rolC::uidA transformants, GUS activity in rapidly growing current-year shoots was not only tissue-specific, being localized to the phloem, but also cell-specific at the shoot base, where it was present only in the companion cells. However, during the onset of dormancy induced by short photoperiod, GUS activity shifted laterally from the phloem to include the cortex and pith. After subsequent exposure to chilling temperatures to induce the transition between the dormancy stages of rest and quiescence, GUS activity almost disappeared from all stem tissues, but regained its original phloem specificity and intensity after the shoots were reactivated by exposing them to long photoperiod and high temperatures. In contrast, GUS activity in the stem of 35S::uidA transformants was strong in all tissues except for the vascular cambium and xylem, and did not vary in intensity during the growth-dormancy-reactivation cycle. The lateral shift and increased intensity of GUS activity in the stem of rolC::uidA transformants during dormancy induction was shown to be associated with the accumulation of starch, and to be mimicked by incubating stem sections in sucrose, as well as glucose and fructose, but not sorbitol, prior to the GUS assay. Our results demonstrate that the activities of the rolC and 35S promoters varied in very different, unpredictable ways during the annual cycle of growth and dormancy in a perennial species, and indicate that the spatial and temporal variation in rolC promoter activity that we observed in the stem of transgenic hybrid aspen plants is attributable to cellular and seasonal changes in sucrose content.     

Origin of the S‐Shaped JV Curve and the Light‐Soaking Issue in Inverted Organic Solar Cells

Inverted organic solar cells generally exhibit a strong s‐shaped kink in the current–voltage characteristics (JV curve) that may be removed by exposure to UV light (light‐soaking) leading to a drastically improved performance. Using in‐device characterization methods the origin of the light‐soaking issue in inverted solar cells employing titanium dioxide (TiO₂) as an electron selective layer is clarified. An injected hole reservoir accumulated at the TiO₂/organic interface of the pristine device is observed from extraction current transients; the hole reservoir increases the recombination and results in an s‐shape in the JV curve of pristine devices. The hole reservoir and the s‐shape is a result of the energetics at the selective contact in the pristine device; the effect of UV exposure is to decrease the work function of the indium tin oxide/TiO₂‐contact, increasing the built‐in potential. This hinders the build‐up of the hole reservoir and the s‐shape is removed. The proposed model is in excellent agreement with drift‐diffusion simulations.     

Dynamics of indole-3-acetic acid and indole-3-ethanol during development and germination of Pinus sylvestris seeds

Indole-3-acetic acid (IAA) and indole-3-ethanol (IEt) were identified in immature seeds of Pinus sylvestris L. by combined gas chromatography-mass spectrometry. Indole-3-methanol was tentatively identified using multiple ion monitoring. Anatomical investigations of seeds, as well as measurements of free and alkali-hydrolysable IAA and IEt, were made during seed development and germination. Levels of free IAA and IEt decreased during seed development. In the later stages of seed maturation most IAA and IEt were present in alkali-hydrolysable forms. Bound IAA and bound IEt rapidly decreased during germination, while levels of free IAA and IEt increased dramatically for a short period.     

Auxin-Cytokinin Interactions in Wild-Type and Transgenic Tobacco

Cytokinins and auxins are important regulators of plant growthand development, but there is incomplete and conflicting evidencethat auxins affect cytokinin metabolism and vice versa. We haveinvestigated these interactions in Nicotiana tabacum L. by separatein planta manipulation of levels of the hormones followed byanalysis of the induced changes in the metabolism of the otherhormone. Cytokinin-overproducing plants (expressing the Agrobacte-riumtumefaciens ipt gene) had lower than wild-type levels of freeIAA, and reduced rates of IAA synthesis and turnover, but therewere no differences in the profiles of metabolites they producedfrom fed IAA. Similarly, auxin-overproducing plants (expressingthe A.tumefaciens iaaM and iaaH genes), had lower levels ofthe major cytokinins than wild-type plants and lower cytokininoxidase activity, but there were no differences in the profilesof metabolites they produced from fed cytokinins. The data demonstratethat cytokinin or auxin overproduction decreases the contentof the other hormone, apparently by decreasing its rate of synthesisand/or transport, rather than by increasing rates of turnoveror conjugation. Implications for the importance of cytokinin: auxin ratios in plant development are considered. (Received September 24, 1996; Accepted December 4, 1996)