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.     

Risk of all-cause mortality in HIV infected patients is associated with clinical, immunologic predictors and the CCR5 Δ32 deletion

Objective

Investigation of the interplay between the CCR5 Δ32/wt genotype and demographic, epidemiological, clinical and immunological factors associated with mortality in the cART era.

Design

Longitudinal data from 507 HIV-infected patients following the Δ32 allele detection were analyzed.

Methods

Cumulative 15 years mortality was calculated using Kaplan-Meyer methodology. Hazard ratios were estimated using univariate Cox models. Basing on Akakie information criteria and statistical significance multivariate Cox model was constructed and effect plots presenting adjusted hazard ratio time-dependency were drawn. Analysis of the association of all-cause mortality and CCR5 Δ32/wt genotype prior to the antiretroviral treatment (cART) initiation (n = 507) and on the therapy (n = 422) was also performed.

Results

A mortality rate of 2.66 (CI 2.57–3.19) per 100 person-years was observed. Univariate analysis factors modifying the risk of death included the CCR5 genotype, gender, history of cART, AIDS diagnosis and also CD4 lymphocyte nadir, zenith, the latest CD4 count and stable levels >500 cells/µl. For multivariate analysis the following predictors were selected: CCR5 genotype (HR for wt/wt 2.53, CI 1.16–5.53, p = 0.02), gender (HR for males 1.91, 95%CI 1.1–3.36, p = 0.023), introduction of combined antiretroviral treatment (HR 4.85, CI 3.0–7.89, if untreated or treated <1 month, p<0.0001) CD4 count of 500 cells/µl for six months or more (HR 4.16, CI 1.95–8.88 if not achieved, p = 0.028), the latest CD4 count (HR 5.44, CI 3.39–8.74 for <100 cells/µl, p<0.0001) and history of AIDS (HR 1.69, CI 1.03–2.79, p = 0.039). Among untreated individuals the Δ32/wt genotype was associated with notably better survival (p = 0.026), while among cART treated individuals the Δ32 mutation did not correlate significantly with higher survival rates (p = 0.23).

Conclusions

The Δ32 CCR5 allele is associated with a reduction of the risk of all-cause mortality in HIV (+) patients alongside clinical and immunologic predictors such as AIDS, history of cART, lymphocyte CD4 cell count and gender.     

The Fbx4 tumor suppressor regulates cyclin D1 accumulation and prevents neoplastic transformation

Skp1-Cul1-F-box (SCF) E3 ubiquitin ligase complexes modulate the accumulation of key cell cycle regulatory proteins. Following the G(1)/S transition, SCF(Fbx4) targets cyclin D1 for proteasomal degradation, a critical event necessary for DNA replication fidelity. Deregulated cyclin D1 drives tumorigenesis, and inactivating mutations in Fbx4 have been identified in human cancer, suggesting that Fbx4 may function as a tumor suppressor. Fbx4(+/-) and Fbx4(-/-) mice succumb to multiple tumor phenotypes, including lymphomas, histiocytic sarcomas and, less frequently, mammary and hepatocellular carcinomas. Tumors and premalignant tissue from Fbx4(+/-) and Fbx4(-/-) mice exhibit elevated cyclin D1, an observation consistent with cyclin D1 as a target of Fbx4. Molecular dissection of the Fbx4 regulatory network in murine embryonic fibroblasts (MEFs) revealed that loss of Fbx4 results in cyclin D1 stabilization and nuclear accumulation throughout cell division. Increased proliferation in early passage primary MEFs is antagonized by DNA damage checkpoint activation, consistent with nuclear cyclin D1-driven genomic instability. Furthermore, Fbx4(-/-) MEFs exhibited increased susceptibility to Ras-dependent transformation in vitro, analogous to tumorigenesis observed in mice. Collectively, these data reveal a requisite role for the SCF(Fbx4) E3 ubiquitin ligase in regulating cyclin D1 accumulation, consistent with tumor suppressive function in vivo.     

A biotrophic fungal infection of the great burnet Sanguisorba officinalis indirectly affects caterpillar performance of the endangered scarce large blue butterfly Phengaris teleius

Interactions between ecological communities of herbivores and microbes are commonly mediated by a shared plant. A tripartite interaction between a pathogenic fungus-host plant-herbivorous insect is an example of such mutual influences. In such a system a fungal pathogen commonly has a negative influence on the morphology and biochemistry of the host plant, with consequences for insect herbivore performance. Here we studied whether the biotrophic fbngus Podosphaera ferruginea, attacking the great burnet Sanguisorba officinalis, affects caterpillar performance of the endangered scarce large blue butterfly Phengaris teleius. Our results showed that the pathogenic ftmgus affected the number and size of inflorescences produced by food-plants and, more importantly, had in direct, plant-mediated effects on the abun dance, body mass and immune response of caterpillars. Specifically, we found the relationship between caterpillar abundance and variability in inflorescence size on a plant to be positive among healthy food-plants, and negative among infected food-plants. Caterpillars that fed on healthy food-plants were smaller than those that fed on infected food-plants in one studied season, while there was no such difference in the other season. We observed the relationship between caterpillar immune response and the proportion of infected great burnets within a habitat patch to be positive when caterpillars fed on healthy food-plants, and negative when caterpillars fed on infected food-plants. Our results suggest that this biotrophic fungal infection of the great burnet may impose a significant indirect influence on P. teleius caterpillar performance with potential consequences for the population dynamics and structure of this endangered butterfly.     

Denervation of the porcine ovaries performed during the early luteal phase influenced morphology and function of the gonad

We studied both morphology and steroidogenic activity of ovaries in gilts after bilateral surgical denervation performed on day 3 of the estrous cycle. Blood samples were collected from day 4 of the first estrous cycle to day 11 of the subsequent cycle. Denervation resulted in a dramatic reduction in the number or in the disappearance of tyrosine hydroxylase/dopamine-beta-hydroxylase- and/or neuropeptide Y-immunoreactive nerve fibres. On day 11 of the second cycle, the number of follicles (3-6 mm in diameter) was lower (p<0.001) in the denervated ovaries, while corpora lutea were not present. Neurectomy also led to a decrease in the concentrations of progesterone, androstendione and 17beta-estradiol in the follicular fluid originated from small (1-3 mm in diameter) as well as medium-sized follicles (3-6 mm in diameter). Similar to follicular fluid, concentration of androstendione in the follicular wall of medium-sized follicles decreased in experimental gilts in comparison to that of control animals. In addition, plasma concentrations of LH and steroid hormones were lower in the control than in the experimental group. Our results show that denervation of ovaries during the early luteal phase of the estrous cycle in gilts resulted in the changes in both morphology and steroidogenic activity. These results confirm the important role of the peripheral nerves in the function of ovaries.     

Does Long-Term High Fat Diet Always Lead to Smaller Hippocampi Volumes,Metabolite Concentrations,and Worse Learning and Memory? A Magnetic Resonance and Behavioral Study in Wistar Rats

BackgroundObesity is a worldwide epidemic with more than 600 million affected individuals. Human studies have demonstrated some alterations in brains of otherwise healthy obese individuals and elevated risk of neurodegenerative disease of old age; these studies have also pointed to slightly diminished memory and executive functions among healthy obese individuals. Similar findings were obtained in animal models of obesity induced by high fat diet. On the other hand, low carbohydrate high fat diets are currently promoted for losing weight (e.g., Atkin’s style diets). However, the long-term effects of such diets are not known. Additionally, high fat diets leading to (mild) ketonemia were shown to improve brain function in elderly humans and in some animal models.AimTo evaluate the hypothesis that long-term use of a high fat diet was associated with decreases in spatial memory, smaller hippocampi and hippocampi metabolite concentrations in Wistar rats.MethodsTwenty five male Wistar rats were put on high fat diet (HFD; 60% calories from fat, 30% from carbohydrates) on their 55^th day of life, while 25 control male rats (CONs) remained on chow. Adequate levels of essential nutrients were provided. Both groups underwent memory tests in 8-arm radial maze at 3^rd, 6^th, 9^th, and 12^th month. ¹H magnetic resonance spectroscopy was employed to measure concentrations of tNAA (marker of neuronal integrity) at one month and one year, whereas MRI was used to evaluate hippocampal volumes.ResultsObese rats (OBRs) consumed similar amount of calories as CONs, but less proteins. However, their protein intake was within recommended amounts. Throughout the experiment OBRs had statistically higher concentrations of blood ketone bodies than CONs, but still within normal values. At post-mortem assessment, OBRs had 38% larger fat deposits than CONs (p<0.05), as evaluated by volume of epididymis fat, an acknowledged marker of fat deposits in rats. Contrary to our expectations, OBRs had better scores of memory behavioral tasks than CONs throughout the experiment. At one year, their hippocampi were by 2.6% larger than in CONs (p = 0.05), whereas concentration of tNAA was 9.8% higher (p = 0.014).ConclusionLong-term HFD in our study resulted in better memory, larger hippocampal volumes, as well as higher hippocampal metabolite concentrations, possibly due to increased levels of blood ketone bodies. The results should be interpreted with caution, as results from animal models do not necessarily directly translate in human condition.     

Functional alterations in endothelial NO, PGI₂ and EDHF pathways in aorta in ApoE/LDLR⁻/⁻ mice

Adequate endothelial production of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin (PGI?) is critical to the maintenance of vascular homeostasis. However, it is not clear whether alterations in each of these vasodilatory pathways contribute to the impaired endothelial function in murine atherosclerosis. In the present study, we analyze the alterations in NO-, EDHF- and PGI?-dependent endothelial function in the thoracic aorta in relation to the development of atherosclerotic plaques in apoE/LDLR?/? mice. We found that in the aorta of 2-month-old apoE/LDLR?/? mice there was no lipid deposition, subendothelial macrophage accumulation; and matrix metalloproteinase (MMP) activity was low, consistent with the absence of atherosclerotic plaques. Interestingly, at this stage the endothelium was already activated and hypertrophic as evidenced by electron microscopy, while acetylcholine-induced NO-dependent relaxation in the thoracic aorta was impaired, with concomitant upregulation of cyclooxygenase-2 (COX-2)/PGI? and EDHF (epoxyeicosatrienoic acids, EETs) pathways. In the aorta of 3-6-month-old apoE/LDLR?/? mice, lipid deposition, macrophage accumulation and MMP activity in the intima were gradually increased, while impairment of NO-dependent function and compensatory upregulation of COX-2/PGI? and EDHF pathways were more accentuated. These results suggest that impairment of NO-dependent relaxation precedes the development of atherosclerosis in the aorta and early upregulation of COX-2/PGI? and EDHF pathways may compensate for the loss of the biological activity of NO.     

Basal activation of the P2X7 ATP receptor elevates mitochondrial calcium and potential, increases cellular ATP levels, and promotes serum-independent growth

P2X7 is a bifunctional receptor (P2X7R) for extracellular ATP that, depending on the level of activation, forms a cation-selective channel or a large conductance nonselective pore. The P2X7R has a strong proapoptotic activity but can also support growth. Here, we describe the mechanism involved in growth stimulation. Transfection of P2X7R increases resting mitochondrial potential (delta psi(mt)), basal mitochondrial Ca2+ ([Ca2+]mt), intracellular ATP content, and confers ability to grow in the absence of serum. These changes require a full pore-forming function, because they are abolished in cells transfected with a mutated P2X7R that retains channel activity but cannot form the nonselective pore, and depend on an autocrine/paracrine tonic stimulation by secreted ATP. On the other hand, sustained stimulation of P2X7R causes a delta psi(mt) drop, a large increase in [Ca2+]mt, mitochondrial fragmentation, and cell death. These findings reveal a hitherto undescribed mechanism for growth stimulation by a plasma membrane pore.     

Further evidence for the absence of polyproline II stretch in the XAO peptide

It has been suggested that the alanine-based peptide with sequence Ac-XX-[A](7)-OO-NH(2), termed XAO where X denotes diaminobutyric acid and O denotes ornithine, exists in a predominantly polyproline-helix (P(II)) conformation in aqueous solution. In our recent work, we demonstrated that this "polyproline conformation" should be regarded as a set of local conformational states rather than as the overall conformation of the molecule. In this work, we present further evidence to support this statement. Differential scanning calorimetry measurements showed only a very small peak in the heat capacity of an aqueous solution of XAO at 57 degrees C, whereas the suggested transition to the P(II) structure should occur at approximately 30 degrees C. We also demonstrate that the temperature dependence of the (3)J(HNHalpha) coupling constants of the alanine residues can be explained qualitatively in terms of Boltzmann averaging over all local conformational states; therefore, this temperature dependence proves that a conformational transition does not occur. Canonical MD simulations with the solvent represented by the generalized Born model, and with time-averaged NMR-derived restraints, demonstrate the presence of an ensemble of structures with a substantial amount of local P(II) conformational states but not with an overall P(II) conformation.     

Induction of the murine “W phenotype” in long-term cultures of human cord blood cells by c-kit antisense oligomers

The murine white (W) spotting locus is the site of the c-kit gene and encodes a tyrosine kinase receptor while the complementary Steel (Sl) iocus encodes its ligand. Mutations at either locus have profound effects on hematopoiesis, particularly erythroid and mast cell proliferation. We added c-kit antisense oligonucleotides to long-term suspension cultures of enriched human umbilical cord progenitor cells. This resulted in the suppression of c-kit gene expression and the preferential suppression of the generation of erythroid burst-forming cells (BFU-E) which extended over the life of the culture (3 weeks). The results provide an in vitro model of the “W phenotype” in human hematopoiesis and confirm the importance of c-kit gene function in early erythropoiesis. Because the generation of BFU-E was suppressed even after c-kit gene expression had recovered, this gene product may be critical to the erythroid commitment process. © 1993 Wiley-Liss, Inc.