Adenine Supplementation Research Articles

2,8-Dihydroxyadenine-induced nephropathy causes hexosylceramide accumulation with increased mTOR signaling, reduced levels of protective SirT3 expression and impaired renal mitochondrial function

AimChronic kidney disease (CKD) is accompanied by increased cardiovascular risk and heart failure (HF). In rodents, 2,8-dihydroxyadenine (DHA)-induced nephropathy is a frequently used CKD model. Cardiac and kidney tubular cells share high energy demand to guarantee constant contractive force of the heart or reabsorption/secretion of primary filtrated molecules and waste products by the kidney. Here we analyze time-dependent mechanisms of kidney damage and cardiac consequences under consideration of energetic pathways with the focus on mitochondrial function and lipid metabolism in mice. Methods and resultsCKD was induced by alternating dietary adenine supplementation (0.2 % or 0.05 % of adenine) in C57BL/6J mice for 9 weeks. Progressive kidney damage led to reduced creatinine clearance, kidney fibrosis and renal inflammation after 3, 6, and 9 weeks. No difference in cardiac function, mitochondrial respiration nor left ventricular fibrosis was observed at any time point. Investigating mechanisms of renal damage, protective SirT3 was decreased in CKD, which contrasted an increase in protein kinase B (AKT) expression, mechanistic target of rapamycin (mTOR) downstream signaling, induction of oxidative and endoplasmic reticulum (ER) stress. This occurred together with impaired renal mitochondrial function and accumulation of hexosylceramides (HexCer) as an established mediator of inflammation and mitochondrial dysfunction in the kidney. Conclusions2,8-DHA-induced CKD results in renal activation of the mTOR downstream signaling, endoplasmic reticulum stress, tubular injury, fibrosis, inflammation, oxidative stress and impaired kidney mitochondrial function in conjunction with renal hexosylceramide accumulation in C57BL/6J mice.

Soluble guanylate cyclase agonist, isoliquiritigenin attenuates renal damage and aortic calcification in a rat model of chronic kidney failure

AimsChronic kidney disease (CKD) is a growing fatal health problem worldwide associated with vascular calcification. Therapeutic approaches are limited with higher costs and poor outcomes. Adenine supplementation is one of the most relevant CKD models to human. Insufficient Nitric Oxide (NO)/ cyclic Guanosine Monophosphate (cGMP) signaling plays a key role in rapid development of renal fibrosis. Natural products display proven protection against CKD. Current study therefore explored isoliquiritigenin, a bioflavonoid extracted from licorice roots, potential as a natural activator for soluble Guanylate Cyclase (sGC) in a CKD rat model. Materials and methods60 male Wistar rats were grouped into Control group (n = 10) and the remaining rats received adenine (200 mg/kg, p.o) for 2 wk to induce CKD. They were equally sub-grouped into: Adenine untreated group and 4 groups orally treated by isoliquiritigenin low or high dose (20 or 40 mg/kg) with/without a selective sGC inhibitor, ODQ (1-H(1,2,4)oxadiazolo(4,3-a)-quinoxalin-1-one, 2 mg/kg, i.p) for 8 wk. Key findingsLong-term treatment with isoliquiritigenin dose-dependently and effectively amended adenine-induced chronic renal and endothelial dysfunction. It not only alleviated renal fibrosis and apoptosis markers but also aortic calcification. Additionally, this chalcone neutralized renal inflammatory response and oxidative stress. Isoliquiritigenin beneficial effects were associated with up-regulation of serum NO, renal and aortic sGC, cGMP and its dependent protein kinase (PKG). However, co-treatment with ODQ antagonized isoliquiritigenin therapeutic impact. SignificanceIsoliquiritigenin seems to exert protective effects against CKD and vascular calcification by activating sGC, increasing cGMP and its downstream PKG.

Increased PHOSPHO1 expression mediates cortical bone mineral density in renal osteodystrophy.

Patients with advanced chronic kidney disease (CKD) often present with skeletal abnormalities, a condition known as renal osteodystrophy (ROD). While tissue non-specific alkaline phosphatase (TNAP) and PHOSPHO1 are critical for bone mineralization, their role in the etiology of ROD is unclear. To address this, ROD was induced in both WT and Phospho1 knockout (P1KO) mice through dietary adenine supplementation. The mice presented with hyperphosphatemia, hyperparathyroidism, and elevated levels of FGF23 and bone turnover markers. In particular, we noted that in CKD mice, bone mineral density (BMD) was increased in cortical bone (P < 0.05) but decreased in trabecular bone (P < 0.05). These changes were accompanied by decreased TNAP (P < 0.01) and increased PHOSPHO1 (P < 0.001) expression in WT CKD bones. In P1KO CKD mice, the cortical BMD phenotype was rescued, suggesting that the increased cortical BMD of CKD mice was driven by increased PHOSPHO1 expression. Other structural parameters were also improved in P1KO CKD mice. We further investigated the driver of the mineralization defects, by studying the effects of FGF23, PTH, and phosphate administration on PHOSPHO1 and TNAP expression by primary murine osteoblasts. We found both PHOSPHO1 and TNAP expressions to be downregulated in response to phosphate and PTH. The in vitro data suggest that the TNAP reduction in CKD-MBD is driven by the hyperphosphatemia and/or hyperparathyroidism noted in these mice, while the higher PHOSPHO1 expression may be a compensatory mechanism. Increased PHOSPHO1 expression in ROD may contribute to the disordered skeletal mineralization characteristic of this progressive disorder.

MO587: Chronic Kidney Disease Induces Endotoxin-Related Activation of The Innate Immune System

Abstract BACKGROUND AND AIMS Chronic inflammation is common in patients with chronic kidney disease (CKD) and is associated with poor outcomes. Although the etiology is only partly understood, the gut–kidney axis is considered an important contributor [1]. Lipopolysaccharide (LPS), also known as endotoxin, is a well-characterized pyrogen found in the exterior cell membrane of most of the gram-negative bacteria. It plays an important role in promoting intestinal inflammatory responses. When absorbed through the intestinal epithelium, LPS induces inflammation by activating macrophages and monocytes. Due to short half-life and semi-quantitative assay characteristics of the Limulus Amebocyte Lysate (LAL) assay, direct quantification of LPS is not suitable to quantify activation of the innate immune system. Lipoprotein-binding protein (LBP), a more stable biomarker, is an acute phase protein produced mostly by the hepatocytes and intestinal epithelial cells and is an essential component of an effective innate immune response to LPS. Circulating LBP significantly enhances the sensitivity of CD14 + cells (mostly monocytes and macrophages) to stimulation by LPS. LBP has been shown to facilitate binding of LPS to CD14 receptor. Levels of LBP peak in serum shortly after endotoxemia and remain increased up to 72 h later. An increased plasma LBP indicates gut epithelial barrier dysfunction [2]. Whether LBP is altered by CKD is not known. METHOD We analyzed the effects of CKD on LBP plasma concentrations. We used samples from the Leuven mild-to-moderate CKD cohort (NCT00441623). To study causality, we used animal models of experimentally induced CKD. To exclude model-related bias, two different rat models of CKD, i.e. 5/6th nephrectomy and adenine supplementation, were used. LBP was measured using commercially available ELISA kits (Hycultbiotech, The Netherlands). RESULTS In a cohort of 460 patients with CKD, we found a significant increase in LBP levels across the different stages of CKD (ANOVA, P: 0.001). When analyzed as a continuous variable, LBP is significantly inversely correlated with eGFR (P &amp;lt; 0.001; Spearman, r: −0.221), and positively correlated with CRP (P &amp;lt; 0.001; Spearman, r: 0.592). During a median follow-up of 56 (IQR, 53–59) months, 70 patients died, with more deaths observed among patients with LBP in higher tertiles [tertiles 1–3: 11, 22, and 37 events, respectively (Fig. 1)]. In univariate Cox proportional hazard analysis, plasma LBP was significantly associated with mortality [hazard ratio (HR) of 1.032; 95% confidence interval (95% CI): 1.014– 1.046; P &amp;lt; 0.001). This association remained significant in multivariate models with adjustment for age, sex and BMI. In two different rat models, induction of CKD resulted in a significant increase in LBP (P &amp;lt; 0.001). In these animals, we observed a significant inverse association between eGFR (and measured creatinine clearance) and LBP concentrations (P &amp;lt; 0.001; Spearman, r: −0.460). CONCLUSION Patients with CKD have higher levels of LBP, with higher levels of plasma LBP present in patients with more advanced CKD. Plasma LBP is an independent predictor of mortality. Experimentally induced CKD equally results in significantly increased LBP. Our data suggest that CKD leads to increased passage of LPS across the intestinal barrier. These findings strengthen the hypothesis of the gut–kidney axis as a source of chronic inflammation.

Targeting Metabolic Vulnerabilities in Primary Effusion Lymphoma Using the Novel Nucleoside Analog 6-Eti

Although nucleoside analogues have been used effectively in the clinic for the treatment of a wide range of hematological malignancies, lack of response to currently available nucleoside analogues and drug resistance limit their utility. A rare but highly aggressive cancer is primary effusion lymphoma (PEL). Through high throughput screening, we have discovered a novel nucleoside analog, called 6-ethylthioinosine (6-ETI) as a potent and selective inhibitor of PEL, with little activity in other lymphomas tested. PEL is a rare B-cell non-Hodgkin's lymphoma characterized by lymphomatous effusions in body cavities. It is associated with Kaposi's sarcoma herpesvirus (KSHV/HHV-8) infection and occurs mainly in immunocompromised patients. PEL is known to frequently be resistant to conventional chemotherapy (CHOP and EPOCH) resulting in poor prognosis and a rather incurable disease. Our studies demonstrated that 6-ETI is a pro-drug activated by adenosine kinase (ADK), an enzyme that is overexpressed in PEL cell lines and primary PEL specimens, as well as other plasma cell malignancies, including plasmablastic lymphoma (PBL) and multiple myeloma (MM). The latter is also responsive to 6-ETI in vitro and in mouse models. 6-ETI induces S phase arrest and inhibits DNA synthesis. RNA sequencing of in vitro generated PEL resistant clones and CRISPR knock out of ADK (ADK KO), respectively, indicated that mutations or loss of expression of ADK renders cells resistant to treatment. This data demonstrates that ADK expression can be used as a predictive biomarker of response to 6-ETI, which can help identify which patients are more likely to respond to this treatment.We investigated which pathways are differentially regulated in sensitive and resistant cells to better delineate the mechanism of action of 6-ETI and to design effective combinatorial regimens and prevent resistance. We found that drug sensitivity was associated with AMPK activation and inhibition of PI3K/mTOR/p70S6K signaling. Little is known about the function of ADK in plasma cell neoplasms. Knock-out of this protein in PEL, or use of ADK chemical inhibitors, do not affect their viability. Thus, we used ADK KO cell lines to examine the role of ADK in these tumors and to determine if cells undergo adaptations that may contribute to 6-ETI resistance and represent potential vulnerabilities to combat it. We performed metabolic and transcriptomic profiling of wild type (WT) (6-ETI sensitive) and ADK KO (6-ETI resistant) cells to achieve a comprehensive assessment of all the metabolic perturbations and gene expression changes induced by knocking out ADK. We also treated these cells with 6-ETI to examine the effects in sensitive and resistant cells. This integrated analysis revealed that 6-ETI depletes sensitive PEL cells of their nucleotide pools accompanied by the downregulation of several genes in purine and pyrimidine biosynthesis pathways. We found that adenine supplementation rescues sensitive PEL cells from 6-ETI induced cytotoxicity, reverses p70S6K inhibition and restores DNA synthesis suggesting that purine metabolism is a critical mediator of 6-ETI induced cytotoxicity. Using seahorse bioenergetic assay, we show that ADK KO resistant cells have impaired mitochondrial respiration indicating that ADK plays a critical role in mitochondrial bioenergetics. Metabolic profiling of these ADK KO resistant cells showed that these cells have elevated levels of de novo pyrimidine metabolic intermediates. Metabolic flux through de novo pyrimidine is controlled by the rate limiting enzyme CAD. The activity of CAD is regulated by ribosomal protein S6 Kinase 1(S6K1) by phosphorylation at its (Ser1859) site. Using western blotting, we observed a striking increase of phosphorylation of CAD at its S6K1 site (Ser1859) in ADK KO cells compared to WT cells.This is the first to date study that characterizes the role of ADK in lymphomas. Our data indicates that ADK KO cells have undergone metabolic reprogramming to upregulate de novo pyrimidine biosynthesis and p70S6K signaling. Moreover, we found that 6-ETI synergizes with the pan PI3K inhibitor BKM120 highlighting nucleotide metabolism and PI3K/mTOR signaling as key therapeutic vulnerabilities targeted by this novel nucleoside analog. DisclosuresNo relevant conflicts of interest to declare.

Modulation of adenine phosphoribosyltransferase-mediated salvage pathway to accelerate diabetic wound healing.

Adenine phosphoribosyltransferase (APRT) is the key enzyme involved in purine salvage by the incorporation of adenine and phosphoribosyl pyrophosphate to provide adenylate nucleotides. To evaluate the role of APRT in the repair processes of cutaneous wounds in healthy skin and in diabetic patients, a diabetic mouse model (db/db) and age-matched wild-type mice were used. Moreover, the topical application of adenine was assessed. In vitro studies, analytical, histological, and immunohistochemical methods were used. Diabetic mice treated with adenine exhibited elevated ATP levels in organismic skin and accelerated wound healing. In vitro studies showed that APRT utilized adenine to rescue cellular ATP levels and proliferation from hydrogen peroxide-induced oxidative damage. HPLC-ESI-MS/MS-based analysis of total adenylate nucleotides in NIH-3T3 fibroblasts demonstrated that adenine addition enlarged the cellular adenylate pool, reduced the adenylate energy charge, and provided additional AMP for the further generation of ATP. These data indicate an upregulation of APRT in skin wounds, highlighting its role during the healing of diabetic wounds through regulation of the nucleotide pool after injury. Furthermore, topical adenine supplementation resulted in an enlargement of the adenylate pool needed for the generation of ATP, an important molecule for wound repair.

Alteration of serum amino acid profiles by dietary adenine supplementation inhibits fatty liver development in rats

Studies on animal models have demonstrated that feeding a low-arginine diet inhibits triacylglycerol (TAG) secretion from the liver, resulting in marked fatty liver development in rats. Here, we first showed that culturing hepatocytes in the medium mimicking the serum amino acid profile of low-arginine diet-fed rats induced TAG accumulation in the cells, indicating that the specific amino acid profile caused TAG accumulation in hepatocytes. Dietary adenine supplementation completely recovered hepatic TAG secretion and abolished hepatic TAG accumulation in rats. A comprehensive non-linear analysis revealed that inhibition of hepatic TAG accumulation by dietary adenine supplementation could be predicted using only serum amino acid concentration data. Comparison of serum amino acid concentrations indicated that histidine, methionine, and branched-chain amino acid (BCAA) concentrations were altered by adenine supplementation. Furthermore, when the serum amino acid profiles of low-arginine diet-fed rats were altered by modifying methionine or BCAA concentrations in their diets, their hepatic TAG accumulation was abolished. Altogether, these results suggest that an increase in methionine and BCAA levels in the serum in response to dietary arginine deficiency is a key causative factor for hepatic TAG accumulation, and dietary adenine supplementation could disrupt this phenomenon by altering serum amino acid profiles.

590-P: Modulation of Adenine Phosphoribosyltransferase-Mediated Purine Salvage to Accelerate Diabetic Wound Healing

Aim: Adenine phosphoribosyltransferase (APRT) is the key enzyme in purine salvage by the incorporation of adenine and phosphoribosyl pyrophosphate to provide adenylate nucleotide. Our aim was to evaluate the role of APRT in diabetic wound healing. Methods: The molecular mechanism of APRT-mediated cell survival was validated by the haploid HAP1 cell. The adenylate nucleotides in NIH-3T3 fibroblast response to adenine was monitoring by HPLC-MS/MS method. The effects of cell behaviors were evaluated by mice NIH-3T3 fibroblast and human epithelial keratinocytes. Genetically diabetic mice received surgery for full-thickness of the cutaneous wound and the adenine addition as the substrate for APRT on the wound. Results: In the wild type and APRT knockout HAP1 cell, our data revealed that APRT utilized adenine to rescue cellular ATP levels and proliferation against hydrogen peroxide-induced oxidative damage. The time-course of monitoring the total adenylate nucleotides in NIH-3T3 fibroblast showed that adenine addition enlarged the cellular adenylate pool, reduced the adenylate energy charge, and provided more AMP for the generation of ATP in further. In mice NIH-3T3 fibroblasts and human NHEK keratinocytes, adenine elevated cellular ATP levels in a dose-dependent manner and facilitated cell migration. The up-regulated APRT found in wound skin was correlated with the demands of wound repair in diabetic mice. Administration of adenine on the wound of diabetic mice exhibited elevated ATP levels in organismic skin and accelerated wound healing. Conclusions: Our data indicated the role of APRT during diabetic wound healing by regulating the nucleotide pool after injury and demonstrated the improvement by topical adenine, which highlights its value as a promising agent in therapeutic intervention. This study provided an explanation for the up-regulation of APRT in tissue repair and adenine supplement resulted in an enlargement of the adenylate pool for ATP generation. Disclosure G. Young: Employee; Self; Energenesis-Biomedical. J. Lin: Employee; Self; Energenesis-Biomedical. Y. Cheng: Employee; Self; Energenesis-Biomedical. H. Chen: Board Member; Self; Energenesis-Biomedical.

Dual control of NAD+ synthesis by purine metabolites in yeast.

Metabolism is a highly integrated process resulting in energy and biomass production. While individual metabolic routes are well characterized, the mechanisms ensuring crosstalk between pathways are poorly described, although they are crucial for homeostasis. Here, we establish a co-regulation of purine and pyridine metabolism in response to external adenine through two separable mechanisms. First, adenine depletion promotes transcriptional upregulation of the de novo NAD+ biosynthesis genes by a mechanism requiring the key-purine intermediates ZMP/SZMP and the Bas1/Pho2 transcription factors. Second, adenine supplementation favors the pyridine salvage route resulting in an ATP-dependent increase of intracellular NAD+. This control operates at the level of the nicotinic acid mononucleotide adenylyl-transferase Nma1 and can be bypassed by overexpressing this enzyme. Therefore, in yeast, pyridine metabolism is under the dual control of ZMP/SZMP and ATP, revealing a much wider regulatory role for these intermediate metabolites in an integrated biosynthesis network.

Adenine Addition Restores Cell Viability and Butanol Production in Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) Cultivated at 37°C.

We have developed butanol-producing consolidated bioprocessing from cellulosic substrates through coculture of cellulolytic clostridia and butanol-producing Clostridium saccharoperbutylacetonicum strain N1-4. However, the butanol fermentation by strain N1-4 (which has an optimal growth temperature of 30°C) is sensitive to the higher cultivation temperature of 37°C; the nature of this deleterious effect remains unclear. Comparison of the intracellular metabolites of strain N1-4 cultivated at 30°C and 37°C revealed decreased levels of multiple primary metabolites (notably including nucleic acids and cofactors) during growth at the higher temperature. Supplementation of the culture medium with 250 mg/liter adenine enhanced both cell growth (with the optical density at 600 nm increasing from 4.3 to 10.2) and butanol production (increasing from 3.9 g/liter to 9.6 g/liter) at 37°C, compared to those obtained without adenine supplementation, such that the supplemented 37°C culture exhibited growth and butanol production approaching those observed at 30°C in the absence of adenine supplementation. These improved properties were based on the maintenance of cell viability. We further showed that adenine supplementation enhanced cell viability during growth at 37°C by maintaining ATP levels and inhibiting spore formation. This work represents the first demonstration (to our knowledge) of the importance of adenine-related metabolism for clostridial butanol production, suggesting a new means of enhancing target pathways based on metabolite levels.IMPORTANCE Metabolomic analysis revealed decreased levels of multiple primary metabolites during growth at 37°C, compared to 30°C, in C. saccharoperbutylacetonicum strain N1-4. We found that adenine supplementation restored the cell growth and butanol production of strain N1-4 at 37°C. The effects of adenine supplementation reflected the maintenance of cell viability originating from the maintenance of ATP levels and the inhibition of spore formation. Thus, our metabolomic analysis identified the depleted metabolites that were required to maintain cell viability. Our strategy, which is expected to be applicable to a wide range of organisms, permits the identification of the limiting metabolic pathway, which can serve as a new target for molecular breeding. The other novel finding of this work is that adenine supplementation inhibits clostridial spore formation. The mechanism linking spore formation and metabolomic status in butanol-producing clostridia is expected to be the focus of further research.

Disruption of de novo purine biosynthesis in Pseudomonas fluorescens Pf0-1 leads to reduced biofilm formation and a reduction in cell size of surface-attached but not planktonic cells.

Pseudomonas fluorescens Pf0-1 is one of the model organisms for biofilm research. Our previous transposon mutagenesis study suggested a requirement for the de novo purine nucleotide biosynthesis pathway for biofilm formation by this organism. This study was performed to verify that observation and investigate the basis for the defects in biofilm formation shown by purine biosynthesis mutants. Constructing deletion mutations in 8 genes in this pathway, we found that they all showed reductions in biofilm formation that could be partly or completely restored by nucleotide supplementation or genetic complementation. We demonstrated that, despite a reduction in biofilm formation, more viable mutant cells were recovered from the surface-attached population than from the planktonic phase under conditions of purine deprivation. Analyses using scanning electron microscopy revealed that the surface-attached mutant cells were 25 ∼ 30% shorter in length than WT, which partly explains the reduced biomass in the mutant biofilms. The laser diffraction particle analyses confirmed this finding, and further indicated that the WT biofilm cells were smaller than their planktonic counterparts. The defects in biofilm formation and reductions in cell size shown by the mutants were fully recovered upon adenine or hypoxanthine supplementation, indicating that the purine shortages caused reductions in cell size. Our results are consistent with surface attachment serving as a survival strategy during nutrient deprivation, and indicate that changes in the cell size may be a natural response of P. fluorescens to growth on a surface. Finally, cell sizes in WT biofilms became slightly smaller in the presence of exogenous adenine than in its absence. Our findings suggest that purine nucleotides or related metabolites may influence the regulation of cell size in this bacterium.

Activation of AMP-Activated Protein Kinase by Adenine Alleviates TNF-Alpha-Induced Inflammation in Human Umbilical Vein Endothelial Cells

The AMP-activated protein kinase (AMPK) signaling system plays a key role in cellular stress by repressing the inflammatory responses induced by the nuclear factor-kappa B (NF-κB) system. Previous studies suggest that the anti-inflammatory role of AMPK involves activation by adenine, but the mechanism that allows adenine to produce these effects has not yet been elucidated. In human umbilical vein endothelial cells (HUVECs), adenine was observed to induce the phosphorylation of AMPK in both a time- and dose-dependent manner as well as its downstream target acetyl Co-A carboxylase (ACC). Adenine also attenuated NF-κB targeting of gene expression in a dose-dependent manner and decreased monocyte adhesion to HUVECs following tumor necrosis factor (TNF-α) treatment. The short hairpin RNA (shRNA) against AMPK α1 in HUVECs attenuated the adenine-induced inhibition of NF-κB activation in response to TNF-α, thereby suggesting that the anti-inflammatory role of adenine is mediated by AMPK. Following the knockdown of adenosyl phosphoribosyl transferase (APRT) in HUVECs, adenine supplementation failed to induce the phosphorylation of AMPK and ACC. Similarly, the expression of a shRNA against APRT nullified the anti-inflammatory effects of adenine in HUVECs. These results suggested that the role of adenine as an AMPK activator is related to catabolism by APRT, which increases the cellular AMP levels to activate AMPK.

Adenine Supplement Suppresses Diabetic Nephropathy Through AMPK and Sirt-1 Pathways

Adenine Supplement Suppresses Diabetic Nephropathy Through AMPK and Sirt-1 Pathways

Dissection of the PHO pathway in Schizosaccharomyces pombe using epistasis and the alternate repressor adenine.

In Saccharomyces cerevisiae, intracellular phosphate levels are maintained by the PHO pathway, activation of which is assayed by increased phosphatase activity. The PHO pathway of Schizosaccharomyces pombe upregulates phosphatase activity (encoded by pho1 (+)) during low extracellular phosphate levels, but the underlying mechanism is poorly understood. We utilized an alternate repressor of pho1 (+) expression (adenine supplementation) along with epistasis analysis to develop a model of how S. pombe PHO pathway components interact. Analyzing Pho1 activity in S. pombe PHO pathway deletion mutants during adenine starvation, we observed most mutants with a phosphatase defect in phosphate starvation also had a defect in adenine starvation. Pho7, a transcription factor in the PHO pathway, is necessary for an adenine starvation-mediated increase in Pho1 activity. Comparing adenine starvation to phosphate starvation, there are differences in the degree to which individual mutants regulate the two responses. Through epistasis studies, we identified two positive regulatory arms and one repressive arm of the PHO pathway. PKA activation is a positive regulator of Pho1 activity under both environmental conditions and is critical for transducing adenine concentrations in the cell. The synthesis of IP7 also appears critical for the induction of Pho1 activity during adenine starvation, but IP7 is not critical during phosphate starvation, which differs from S. cerevisiae. Finally, Csk1 is critical for repression of pho1 (+) expression during phosphate starvation. We believe all of these regulatory arms converge to increase transcription of pho1 (+) and some of the regulation acts through pho7 (+).

Adenine auxotrophy - be aware: some effects of adenine auxotrophy inSaccharomyces cerevisiaestrain W303-1A

Adenine auxotrophy is a commonly used genetic marker in haploid yeast strains. Strain W303-1A, which carries the ade2-1 mutation, is widely used in physiological and genetic research. Yeast extract-based rich medium contains a low level of adenine, so that adenine is often depleted before glucose. This could affect the cell physiology of adenine auxotrophs grown in rich medium. The aim of our study was to assess the effects of adenine auxotrophy on cell morphology and stress physiology. Our results show that adenine depletion halts cell division, but that culture optical density continues to increase due to cell swelling. Accumulation of trehalose and a coincident 10-fold increase in desiccation stress tolerance is observed in adenine auxotrophs after adenine depletion, when compared to prototrophs. Under adenine starvation, long-term survival of W303-1A is lower than during carbon starvation, but higher than during leucine starvation. We observed drastic adenine-dependent changes in cell stress physiology, suggesting that results may be biased when adenine auxotrophs are grown in rich media without adenine supplementation.

Vascular function in rats with adenine-induced chronic renal failure

The aim of the present study was to characterize the function of resistance arteries, and the aorta, in rats with adenine-induced chronic renal failure (A-CRF). Sprague-Dawley rats were randomized to chow with or without adenine supplementation. After 6-10 wk, mesenteric arteries and thoracic aortas were analyzed ex vivo by wire myography. Plasma creatinine concentrations were elevated twofold at 2 wk, and eight-fold at the time of death in A-CRF animals. Ambulatory systolic and diastolic blood pressures measured by radiotelemetry were significantly elevated in A-CRF animals from week 3 and onward. At death, A-CRF animals had anemia, hyperphosphatemia, hyperparathyroidism, and elevated plasma levels of asymmetric dimethylarginine and oxidative stress markers. There were no significant differences between groups in the sensitivity, or maximal response, to ACh, sodium nitroprusside (SNP), norepinephrine, or phenylephrine in either mesenteric arteries or aortas. However, in A-CRF animals, the rate of aortic relaxation was significantly reduced following washout of KCl (both in intact and endothelium-denuded aorta) and in response to ACh and SNP. Also the rate of contraction in response to KCl was significantly reduced in A-CRF animals both in mesenteric arteries and aortas. The media of A-CRF aortas was thickened and showed focal areas of fragmented elastic lamellae and disorganized smooth muscle cells. No vascular calcifications could be detected. These results indicate that severe renal failure for a duration of less than 10 wk in this model primarily affects the aorta and mainly slows the rate of relaxation.

Striatal neurons expressing full-length mutant huntingtin exhibit decreased N-cadherin and altered neuritogenesis

The expanded CAG repeat that causes striatal cell vulnerability in Huntington's disease (HD) encodes a polyglutamine tract in full-length huntingtin that is correlated with cellular [ATP] and [ATP/ADP]. Since striatal neurons are vulnerable to energy deficit, we have investigated, in Hdh CAG knock-in mice and striatal cells, the hypothesis that decreased energetics may affect neuronal (N)-cadherin, a candidate energy-sensitive adhesion protein that may contribute to HD striatal cell sensitivity. In vivo, N-cadherin was sensitive to ischemia and to the effects of full-length mutant huntingtin, progressively decreasing in Hdh(Q111) striatum with age. In cultured striatal cells, N-cadherin was decreased by ATP depletion and STHdh(Q111) striatal cells exhibited dramatically decreased N-cadherin, due to decreased Cdh2 mRNA and enhanced N-cadherin turnover, which was partially normalized by adenine supplementation to increase [ATP] and [ATP/ADP]. Consistent with decreased N-cadherin function, STHdh(Q111) striatal cells displayed profound deficits in calcium-dependent N-cadherin-mediated cell clustering and cell-substratum adhesion, and primary Hdh(Q111) striatal neuronal cells exhibited decreased N-cadherin and an abundance of immature neurites, featuring diffuse, rather than clustered, staining for N-cadherin and synaptic vesicle markers, which was partially rescued by adenine treatment. Thus, mutant full-length huntingtin, via energetic deficit, contributes to decreased N-cadherin levels in striatal neurons, with detrimental effects on neurite maturation, strongly suggesting that N-cadherin-mediated signaling merits investigation early in the HD pathogenic disease process.

Evaluation of heavy metal toxicity in eukaryotes using a simple functional assay

Although carcinogenesis caused by metals has been intensively investigated, the mechanisms of action, especially at the molecular level, are still unclear. This work aimed to investigate Cd(2+), Cu(2+), Ni(2+), Cr(3+), and Zn(2+) mutagenicity and its relationship with oxidative stress. We have applied the Functional Assay for the Separation of Alleles in Yeast (FASAY) with only minor modifications to detect p53 defects caused by metals. In this method, human p53-coding gene (TP53) expressed in Saccharomyces cerevisiae activates transcription of the ADE2 reporter gene. Yeast cells, expressing p53, were exposed to increased concentrations of metals and, then, plated on media supplemented or not with adenine. Yeast colonies containing functional p53 grow independently of adenine supplementation and colonies containing nonfunctional p53 are dependent on this nutrient. Mutations in the TP53 are implicated in the pathogenesis of half of all human tumors. According to our results, Cd(2+) was found to be the most toxic metal and produced the highest oxidative damage to lipids and proteins. At low concentrations (40 μM), this metal decreased viability and completely inhibited cell growth, while higher concentrations were necessary to produce the same toxic effect by Cu(2+), Cr(3+), and Ni(2+). Zn(2+) showed no significant toxicity. Cd(2+) strongly induced damages and altered the function of p53, while Cu(2+), followed by Cr(3+), showed lower percentages of p53-mutant colonies. Our results point towards a relationship between the loss of functional p53 protein and oxidative stress, a mechanism that can be associated with tumor formation induced by heavy metals in mammalian cells. By this adaptation of FASAY developed by us it is possible to easily and rapidly detect mutations caused by metals or other stresses.

Continuous culture and proteomic analysis of Escherichia coli DH5α and its acetate-tolerant mutant DA19 under conditions of nitrogen source limitation

Escherichia coli DH5α grows poorly in a chemically defined medium, but its acetate-tolerant mutant, DA19, grows fast and produces less acetate. To investigate the differences in metabolism between the two strains, the samples of continuous cultures at steady state were analyzed by two-dimensional SDS-PAGE (2-D SDS-PAGE) and peptide mass fingerprinting (PMF). The results indicated that in DA19, expression of gnd, yddS, and purH (except for adenine supplement) was up-regulated, and expression of atpA, glnK, and ompR, which encode membrane proteins, was significantly up-regulated. Expression of ackA and yahK in DA19 was down-regulated. In the presence of acetate, hchA and ydcW in DH5α were significantly down-regulated. This study shed light on the differences in metabolic properties between DH5α and DA19, and provided some ideas for improving strains through metabolic engineering.

Intracellular Water Lifetime Depends on Cellular Energetic State

Intra- and extracellular water undergoes equilibrium exchange via mechanisms that include passive diffusion across the membrane and movement through channels, like aquaporin. Longitudinal 1H2O NMR relaxography can employ extracellular relaxation agent GdDTPA2- to distinguish intra- and extracellular 1H2O signals by creating a difference in their relaxation time constant (T1) values. Inverse Laplace transform of relaxation decay produces the relaxogram, the T1 distribution, which reports apparent water populations, pi′ and pe′. The true water fractions, pi and pe - measures of the volume fractions vi and ve, are determined if the kinetics of transcytolemmal water exchange (the mean intracellular water life time τi) are quantified using two-site-exchange analysis. The “well-mixed” kinetic expression is: τi-1 = PW(A/V), where PW is the water permeability coefficient; A and V are the individual cell surface area and volume, respectively. Furthermore: vi/τi = PWS, where S is the total cell surface area.We determined the relationship between τi and cellular energetics in suspensions of Saccharomyces cerevisiae. Under aerobic conditions τi was 333 (± 4) ms; under anaerobic conditions τi was 670 (± 62) ms. Changing from aerobic to anaerobic conditions also resulted in a decrease of ATP content. In the yeast strain MR6, which requires adenine supplementation to grow, ATP content increased with medium adenine concentration. As ATP was thus increased, the τi was found to inversely correlate with ATP content (R2 = 0.96). Therefore, τi is a sensitive indicator of the cellular energetic state and energetics affects the kinetics of transcytolemmal water exchange. The aerobic PWS was greater than the anaerobic PWS. The underlying mechanism(s) are not yet clear. Experiments with baker's yeast stored in an anaerobic state found that inhibition of protein synthesis prevented the anaerobic-to-aerobic τi decrease and that aquaporin is not responsible.1