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At least 1,693 records · Page 94

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DOE OSTI2016

Transcriptional regulation of the human Liver X Receptor α gene by Hepatocyte Nuclear Factor 4α

Liver X Receptors (LXRs) are sterol-activated transcription factors that play major roles in cellular cholesterol homeostasis, HDL biogenesis and reverse cholesterol transport. The aim of the present study was to investigate the mechanisms that control the expression of the human LXRα gene in hepatic cells. A series of reporter plasmids containing consecutive 5′ deletions of the hLXRα promoter upstream of the luciferase gene were constructed and the activity of each construct was measured in HepG2 cells. This analysis showed that the activity of the human LXRα promoter was significantly reduced by deleting the −111 to −42 region suggesting the presence of positive regulatory elements in this short proximal fragment. Bioinformatics data including motif search and ChIP-Seq revealed the presence of a potential binding motif for Hepatocyte Nuclear Factor 4 α (HNF-4α) in this area. Overexpression of HNF-4α in HEK 293T cells increased the expression of all LXRα promoter constructs except −42/+384. In line, silencing the expression of endogenous HNF-4α in HepG2 cells was associated with reduced LXRα protein levels and reduced activity of the −111/+384 LXRα promoter but not of the −42/+384 promoter. Using ChiP assays in HepG2 cells combined with DNAP assays we mapped the novel HNF-4α specific binding motif (H4-SBM) in the −50 to −40 region of the human LXRα promoter. A triple mutation in this H4-SBM abolished HNF-4α binding and reduced the activity of the promoter to 65% relative to the wild type. Furthermore, the mutant promoter could not be transactivated by HNF-4α. In conclusion, our data indicate that HNF-4α may have a wider role in cell and plasma cholesterol homeostasis by controlling the expression of LXRα in hepatic cells. - Highlights: • The human LXRα promoter contains a HNF-4α specific binding motif in the proximal −50/−40 region. • Mutations in this motif abolished HNF4α binding and transactivation of the hLXRα promoter. • Silencing of HNF-4α was associated with reduced LXRα protein levels and promoter activity. • The HNF-4α/LXRα axis may have a wide role in cell and plasma cholesterol homeostasis.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

The dual role of cGMP in oocyte maturation of zebrafish

Highlights: • Activation of the NO/sGC/cGMP pathway could lead to oocyte maturation (OM). • Inhibition of the NO/sGC/cGMP pathway could lead to OM. • NO level increased in follicular cells but remained constant in oocytes during OM. • cGMP level increased in the follicular cells but decreased in oocytes during OM. The roles of cyclic guanosine monophosphate (cGMP) signaling in oocyte maturation attracts much attention in mammals, but its roles in fish are still largely unknown. Using zebrafish as a model, we demonstrated for the first time in fish that cGMP is involved in oocyte maturation, and its functional model in oocyte maturation is different from that of mammals. The intracellular cGMP could be regulated by nitric oxide (NO), we found that all three NO synthase enzymes and four soluble guanylyl cyclases (sGC) are expressed in the zebrafish ovary. Intriguingly, either the activation or inhibition of the NO/sGC/cGMP pathway in fully grown follicles could lead to oocyte maturation. During oocyte maturation, cGMP levels increased in the follicular cell layer but decreased in oocytes, while NO levels increased in follicular cells but remained constant in oocytes. Based on these findings in zebrafish, we propose a hypothetical model on the dual role of cGMP in oocyte maturation: in follicular cells the LH signal could increase the level of NO and cGMP which induces oocyte maturation, while in the oocyte the decreased cGMP level can also induce oocyte maturation. These findings help us to understand the molecular mechanism of fish oocyte maturation.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2013

A Prospective, Multicenter Study of Complementary/Alternative Medicine (CAM) Utilization During Definitive Radiation for Breast Cancer

Purpose: Although complementary and alternative medicine (CAM) utilization in breast cancer patients is reported to be high, there are few data on CAM practices in breast patients specifically during radiation. This prospective, multi-institutional study was conducted to define CAM utilization in breast cancer during definitive radiation. Materials/Methods: A validated CAM instrument with a self-skin assessment was administered to 360 Stage 0-III breast cancer patients from 5 centers during the last week of radiation. All data were analyzed to detect significant differences between users/nonusers. Results: CAM usage was reported in 54% of the study cohort (n=194/360). Of CAM users, 71% reported activity-based CAM (eg, Reiki, meditation), 26% topical CAM, and 45% oral CAM. Only 16% received advice/counseling from naturopathic/homeopathic/medical professionals before initiating CAM. CAM use significantly correlated with higher education level (P<.001), inversely correlated with concomitant hormone/radiation therapy use (P=.010), with a trend toward greater use in younger patients (P=.066). On multivariate analysis, level of education (OR: 6.821, 95% CI: 2.307-20.168, P<.001) and hormones/radiation therapy (OR: 0.573, 95% CI: 0.347-0.949, P=.031) independently predicted for CAM use. Significantly lower skin toxicity scores were reported in CAM users vs nonusers, respectively (mild: 34% vs 25%, severe: 17% vs 29%, P=.017). Conclusion: This is the first prospective study to assess CAM practices in breast patients during radiation, with definition of these practices as the first step for future investigation of CAM/radiation interactions. These results should alert radiation oncologists that a large percentage of breast cancer patients use CAM during radiation without disclosure or consideration for potential interactions, and should encourage increased awareness, communication, and documentation of CAM practices in patients undergoing radiation treatment for breast cancer.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

DOE OSTI2014

Modeling coupled sorption and transformation of 17β-estradiol–17-sulfate in soil–water systems

Animal manure is the primary source of exogenous free estrogens in the environment, which are known endocrine-disrupting chemicals to disorder the reproduction system of organisms. Conjugated estrogens can act as precursors to free estrogens, which may increase the total estrogenicity in the environment. In this study, a comprehensive model was used to simultaneously simulate the coupled sorption and transformation of a sulfate estrogen conjugate, 17 beta-estradiol-17-sulfate (E2-17S), in various soil-water systems (non-sterile/sterile; topsoil/subsoil). The simulated processes included multiple transformation pathways (i.e. hydroxylation, hydrolysis, and oxidation) and mass transfer between the aqueous, reversibly sorbed, and irreversibly sorbed phases of all soils for E2-17S and its metabolites. The conceptual model was conceived based on a series of linear sorption and first-order transformation expressions. The model was inversely solved Using finite difference to estimate process parameters. A global optimization method was applied for the inverse analysis along with variable model restrictions to estimate 36 parameters. The model provided a satisfactory simultaneous fit (R-adj(2) = 0.93 and d = 0.87) of all the experimental data and reliable parameter estimates. This modeling study improved the understanding on fate and transport of estrogen conjugates under various soil-water conditions.

17β-Estradiol–17-sulfate↗

DOE OSTI2012

Effect of mono-(2-ethylhexyl) phthalate on human and mouse fetal testis: In vitro and in vivo approaches

The present study was conducted to determine whether exposure to the mono-(2-ethylhexyl) phthalate (MEHP) represents a genuine threat to male human reproductive function. To this aim, we investigated the effects on human male fetal germ cells of a 10{sup −5} M exposure. This dose is slightly above the mean concentrations found in human fetal cord blood samples by biomonitoring studies. The in vitro experimental approach was further validated for phthalate toxicity assessment by comparing the effects of in vitro and in vivo exposure in mouse testes. Human fetal testes were recovered during the first trimester (7–12 weeks) of gestation and cultured in the presence or not of 10{sup −5} M MEHP for three days. Apoptosis was quantified by measuring the percentage of Caspase-3 positive germ cells. The concentration of phthalate reaching the fetal gonads was determined by radioactivity measurements, after incubations with {sup 14}C-MEHP. A 10{sup −5} M exposure significantly increased the rate of apoptosis in human male fetal germ cells. The intratesticular MEHP concentration measured corresponded to the concentration added in vitro to the culture medium. Furthermore, a comparable effect on germ cell apoptosis in mouse fetal testes was induced both in vitro and in vivo. This study suggests that this 10{sup −5} M exposure is sufficient to induce changes to the in vivo development of the human fetal male germ cells. -- Highlights: ► 10{sup −5} M of MEHP impairs germ cell development in the human fetal testis. ► Organotypic culture is a suitable approach to investigate phthalate effects in human. ► MEHP is not metabolized in the human fetal testis. ► In mice, MEHP triggers similar effects both in vivo and in vitro.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2012

Opposing regulation of cytochrome P450 expression by CAR and PXR in hypothyroid mice

Clinical hypothyroidism affects various metabolic processes including drug metabolism. CYP2B and CYP3A are important cytochrome P450 drug metabolizing enzymes that are regulated by the xenobiotic receptors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2). We evaluated the regulation of the hepatic expression of CYPs by CAR and PXR in the hypothyroid state induced by a low-iodine diet containing 0.15% propylthiouracil. Expression of Cyp3a11 was suppressed in hypothyroid C57BL/6 wild type (WT) mice and a further decrement was observed in hypothyroid CAR{sup −/−} mice, but not in hypothyroid PXR{sup −/−} mice. In contrast, expression of Cyp2b10 was induced in both WT and PXR{sup −/−} hypothyroid mice, and this induction was abolished in CAR{sup −/−} mice and in and CAR{sup −/−} PXR{sup −/−} double knockouts. CAR mRNA expression was increased by hypothyroidism, while PXR expression remained unchanged. Carbamazepine (CBZ) is a commonly used antiepileptic that is metabolized by CYP3A isoforms. After CBZ treatment of normal chow fed mice, serum CBZ levels were highest in CAR{sup −/−} mice and lowest in WT and PXR{sup −/−} mice. Hypothyroid WT or PXR{sup −/−} mice survived chronic CBZ treatment, but all hypothyroid CAR{sup −/−} and CAR{sup −/−} PXR{sup −/−} mice died, with CAR{sup −/−}PXR{sup −/−} mice surviving longer than CAR{sup −/−} mice (12.3 ± 3.3 days vs. 6.3 ± 2.1 days, p = 0.04). All these findings suggest that hypothyroid status affects xenobiotic metabolism, with opposing responses of CAR and PXR and their CYP targets that can cancel each other out, decreasing serious metabolic derangement in response to a xenobiotic challenge. -- Highlights: ► Hypothyroid status activates CAR in mice and induces Cyp2b10 expression. ► Hypothyroid status suppresses PXR activity in mice and represses Cyp3a11 expression. ► These responses balance each other out in normal mice. ► Hypothyroidism sensitizes CAR null mice to toxic effects of carbamazepine.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2014

Chrysin alleviates testicular dysfunction in adjuvant arthritic rats via suppression of inflammation and apoptosis: Comparison with celecoxib

Long standing rheumatoid arthritis (RA) is associated with testicular dysfunction and subfertility. Few studies have addressed the pathogenesis of testicular injury in RA and its modulation by effective agents. Thus, the current study aimed at evaluating the effects of two testosterone boosting agents; chrysin, a natural flavone and celecoxib, a selective COX-2 inhibitor, in testicular impairment in rats with adjuvant arthritis, an experimental model of RA. Chrysin (25 and 50 mg/kg) and celecoxib (5 mg/kg) were orally administered to Wistar rats once daily for 21 days starting 1 h before arthritis induction. Chrysin suppressed paw edema with comparable efficacy to celecoxib. More important, chrysin, dose-dependently and celecoxib attenuated the testicular injury via reversing lowered gonadosomatic index and histopathologic alterations with preservation of spermatogenesis. Both agents upregulated steroidogenic acute regulatory (StAR) mRNA expression and serum testosterone with concomitant restoration of LH and FSH. Furthermore, they suppressed inflammation via abrogation of myeloperoxidase, TNF-α and protein expression of COX-2 and iNOS besides elevation of IL-10. Alleviation of the testicular impairment was accompanied with suppression of oxidative stress via lowering testicular lipid peroxides and nitric oxide. With respect to apoptosis, both agents downregulated FasL mRNA expression and caspase-3 activity in favor of cell survival. For the first time, these findings highlight the protective effects of chrysin and celecoxib against testicular dysfunction in experimental RA which were mediated via boosting testosterone in addition to attenuation of testicular inflammation, oxidative stress and apoptosis. Generally, the 50 mg/kg dose of chrysin exerted comparable protective actions to celecoxib. - Highlights: • Chrysin and celecoxib alleviated testicular suppression in adjuvant arthritis. • They attenuated histopathological damage and preserved spermatogenesis. • They upregulated StAR expression and testosterone with restoration of LH and FSH. • They abrogated myeloperoxidase, TNF-α and COX-2 and iNOS expression. • They suppressed oxidative stress and FasL-associated apoptosis.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2016

Activation of the Constitutive Androstane Receptor induces hepatic lipogenesis and regulates Pnpla3 gene expression in a LXR-independent way

The Constitutive Androstane Receptor (CAR, NR1I3) has been newly described as a regulator of energy metabolism. A relevant number of studies using animal models of obesity suggest that CAR activation could be beneficial on the metabolic balance. However, this remains controversial and the underlying mechanisms are still unknown. This work aimed to investigate the effect of CAR activation on hepatic energy metabolism during physiological conditions, i.e. in mouse models not subjected to metabolic/nutritional stress. Gene expression profiling in the liver of CAR knockout and control mice on chow diet and treated with a CAR agonist highlighted CAR-mediated up-regulations of lipogenic genes, concomitant with neutral lipid accumulation. A strong CAR-mediated up-regulation of the patatin-like phospholipase domain-containing protein 3 (Pnpla3) was demonstrated. Pnpla3 is a gene whose polymorphism is associated with the pathogenesis of nonalcoholic fatty liver disease (NAFLD) development. This observation was confirmed in human hepatocytes treated with the antiepileptic drug and CAR activator, phenobarbital and in immortalized human hepatocytes treated with CITCO. Studying the molecular mechanisms controlling Pnpla3 gene expression, we demonstrated that CAR does not act by a direct regulation of Pnpla3 transcription or via the Liver X Receptor but may rather involve the transcription factor Carbohydrate Responsive Element-binding protein. These data provide new insights into the regulation by CAR of glycolytic and lipogenic genes and on pathogenesis of steatosis. This also raises the question concerning the impact of drugs and environmental contaminants in lipid-associated metabolic diseases. - Highlights: • Induction of hepatic glycolytic and lipogenic genes upon CAR activation by TCPOBOP. • These effects are not mediated by the nuclear receptor LXR. • CAR activation resulted in hepatic lipid accumulation. • Pnpla3 expression is regulated by CAR in mouse liver and human hepatocytes.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Naloxegol, an opioid antagonist with reduced CNS penetration: Mode-of-action and human relevance for rat testicular tumours

Naloxegol is an opioid antagonist which has been developed for the treatment of patients with opioid induced constipation. In the nonclinical safety program naloxegol was shown to have a very benign toxicity profile. In the rat, but not the mouse, 2-year carcinogenicity study a change in tumour pattern with an increase in testicular Leydig cell tumours (LCT) was observed after dosing at high (supra-pharmacological) concentrations. To establish the basis of the increase in LCT and to assess its potential relevance to humans, studies to exclude and potentially identify mode-of-action (MoA) were performed. A genotoxic mechanism was ruled out following negative results in the Ames, mouse lymphoma, and micronucleus assays. An effect on androgen metabolism was excluded since the treatment of rats with naloxegol for 14 days did not result in any induction of CYP protein levels. It was demonstrated that administration of centrally restricted opioid antagonists naloxegol or methylnaltrexone at high doses induced an increase in LH release with no clear increase in testosterone, in contrast to the centrally acting opioid antagonist naloxone, which showed marked increases in both LH and testosterone. LCT due to increased LH stimulation is common in rats but not documented in humans. Collectively, the lack of genotoxicity signal, the lack of androgen effect, the increase in LH secretion in rats, which is no considered to be relevant for LCT formation in humans, and high margins to clinical exposures, the observed increase in LCT in the rat is not expected to be clinically relevant. - Highlights: • Naloxegol elicited neuroendocrine responses in form of increased LH release. • In contrast to naloxone, naloxegol did not markedly increase testosterone. • Naloxegol treatment showed increases in rat testicular Leydig cell tumours. • A reduction in pituitary tumours was consistent with opioid antagonism. • Increase in LH mediated rat Leydig cell tumours is not relevant for patients.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Mineral nutrient signaling controls photosynthesis: focus on iron deficiency-induced chlorosis

Photosynthetic organisms convert light energy into chemical energy stored in carbohydrates. To perform this process, an adequate supply of essential mineral elements, such as iron, is required in the chloroplast. Because iron plays a crucial role during electron transport and chlorophyll formation, iron deficiency alters photosynthesis and promotes chlorosis, or the yellowing of leaves. Intriguingly, iron deficiency-induced chlorosis can be reverted by the depletion of other micronutrients [i.e., manganese (Mn)] or macronutrients [i.e., sulfur (S) or phosphorus (P)], raising the question of how plants integrate nutrient status to control photosynthesis. Here, in this work, we review how improving our understanding of the complex relationship between nutrient homeostasis and photosynthesis has great potential for crop improvement.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2010

Nuclear translocation of the 1,25D{sub 3}-MARRS (membrane associated rapid response to steroids) receptor protein and NF{kappa}B in differentiating NB4 leukemia cells

1,25 Dihydroxyvitamin D{sub 3} (1,25D{sub 3}) primes NB4 promyelocytic leukemia cells to differentiate along the monocyte/macrophage lineage through a non-genomic mechanism. Here we show that NB4 cells express high levels of the recently identified membrane receptor for 1,25D{sub 3}, which is a distinct gene product from the classical nuclear vitamin D receptor. This 57 kDa protein, named 1,25D{sub 3}-MARRS (Membrane Activated Rapid Response to Steroids)/ERp57/PIA3 appears to associate in a complex with the transcription factor, nuclear factor kappa B (NF{kappa}B). In unstimulated cells, 1,25D{sub 3}-MARRS can be co-immunoprecipitated with antibodies directed at NF{kappa}B, and NF{kappa}B is co-precipitated when antibodies against 1,25D{sub 3}-MARRS or ERp57 are used. Confocal microscopy and subcellular fractionation studies demonstrate that both 1,25D{sub 3}-MARRS and NF{kappa}B begin translocating to the nucleus within minutes of co-stimulation with 1,25D{sub 3} and phorbol ester. The predominant nuclear localization of both proteins precedes the expression of the monocyte/macrophage phenotype and suggests that this event may be critical to the differentiation pathway. This suggests a role for 1,25D{sub 3}-MARRS in the nucleus as a regulator of gene expression. Here it may also regulate the activity of NF{kappa}B and other factors with which it may be interacting.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2015

JAZF1 promotes proliferation of C2C12 cells, but retards their myogenic differentiation through transcriptional repression of MEF2C and MRF4—Implications for the role of Jazf1 variants in oncogenesis and type 2 diabetes

Single-nucleotide polymorphisms associated with type 2 diabetes (T2D) have been identified in Jazf1, which is also involved in the oncogenesis of endometrial stromal tumors. To understand how Jazf1 variants confer a risk of tumorigenesis and T2D, we explored the functional roles of JAZF1 and searched for JAZF1 target genes in myogenic C2C12 cells. Consistent with an increase of Jazf1 transcripts during myoblast proliferation and their decrease during myogenic differentiation in regenerating skeletal muscle, JAZF1 overexpression promoted cell proliferation, whereas it retarded myogenic differentiation. Examination of myogenic genes revealed that JAZF1 overexpression transcriptionally repressed MEF2C and MRF4 and their downstream genes. AMP deaminase1 (AMPD1) was identified as a candidate for JAZF1 target by gene array analysis. However, promoter assays of Ampd1 demonstrated that mutation of the putative binding site for the TR4/JAZF1 complex did not alleviate the repressive effects of JAZF1 on promoter activity. Instead, JAZF1-mediated repression of Ampd1 occurred through the MEF2-binding site and E-box within the Ampd1 proximal regulatory elements. Consistently, MEF2C and MRF4 expression enhanced Ampd1 promoter activity. AMPD1 overexpression and JAZF1 downregulation impaired AMPK phosphorylation, while JAZF1 overexpression also reduced it. Collectively, these results suggest that aberrant JAZF1 expression contributes to the oncogenesis and T2D pathogenesis. - Highlights: • JAZF1 promotes cell cycle progression and proliferation of myoblasts. • JAZF1 retards myogenic differentiation and hypertrophy of myotubes. • JAZF1 transcriptionally represses Mef2C and Mrf4 expression. • JAZF1 has an impact on the phosphorylation of AMPK.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2019

Mevalonate Pathway Promiscuity Enables Noncanonical Terpene Production

The lepidoptera (butterflies and moths) make the six-carbon compounds homoisopentenyl pyrophosphate (HIPP) and homodimethylallyl pyrophosphate (HDMAPP) that are incorporated into 16, 17, and 18 carbon farnesyl pyrophosphate (FPP) analogues. In this work we heterologously expressed the lepidopteran modified mevalonate pathway, a propionyl-CoA ligase, and terpene cyclases in E. coli to produce several novel terpenes containing 16 carbons. Changing the terpene cyclase generated different novel terpene product profiles. To further validate the new compounds we confirmed 13 C propionate was incorporated, and that the masses and fragmentation patterns were consistent with novel 16 carbon terpenes by GC-QTOF. On the basis of the available farnesyl pyrophosphate analogues lepidoptera produce, this approach should greatly expand the reachable biochemical space with applications in areas where terpenes have traditionally found uses.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Structural and functional analyses explain Pea KAI2 receptor diversity and reveal stereoselective catalysis during signal perception

KAI2 proteins are plant α/β hydrolase receptors which perceive smoke-derived butenolide signals and endogenous, yet unidentified KAI2-ligands (KLs). The number of functional KAI2 receptors varies among species and KAI2 gene duplication and sub-functionalization likely plays an adaptative role by altering specificity towards different KLs. Legumes represent one of the largest families of flowering plants and contain many agronomic crops. Prior to their diversification, KAI2 underwent duplication resulting in KAI2A and KAI2B. Here we demonstrate that Pisum sativum KAI2A and KAI2B are active receptors and enzymes with divergent ligand stereoselectivity. KAI2B has a higher affinity for and hydrolyses a broader range of substrates including strigolactone-like stereoisomers. We determine the crystal structures of PsKAI2B in apo and butenolide-bound states. The biochemical, structural, and mass spectra analyses of KAI2s reveal a transient intermediate on the catalytic serine and a stable adduct on the catalytic histidine, confirming its role as a bona fide enzyme. Our work uncovers the stereoselectivity of ligand perception and catalysis by diverged KAI2 receptors and proposes adaptive sensitivity to KAR/KL and strigolactones by KAI2B.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2015

Defining a Two-pronged Structural Model for PB1 (Phox/Bem1p) Domain Interaction in Plant Auxin Responses

Phox/Bem1p (PB1) domains are universal structural modules that use surfaces of different charge for protein-protein association. In plants, PB1-mediated interactions of auxin response factors (ARF) and auxin/indole 3-acetic acid inducible proteins regulate transcriptional events modulated by the phytohormone auxin. Here we investigate the thermodynamic and structural basis for Arabidopsis thaliana ARF7 PB1 domain self-interaction. Isothermal titration calorimetry and NMR experiments indicate that key residues on both the basic and acidic faces of the PB1 domain contribute to and organize coordinately to stabilize protein-protein interactions. Calorimetric analysis of ARF7PB1 site-directed mutants defines a two-pronged electrostatic interaction. Furthermore, the canonical PB1 interaction between a lysine and a cluster of acidic residues provides one prong with an arginine and a second cluster of acidic residues defining the other prong. Evolutionary conservation of this core recognition feature and other co-varying interface sequences allows for versatile PB1-mediated interactions in auxin signaling.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia

Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17α-hydroxyprogesterone (17α-OH-progesterone) to 11-deoxycortisol. More than 100 CYP21A2 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure of WT human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17α-OH-progesterone). We found that the 17α-OH-progesterone- and progesterone-bound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either salt-wasting or nonclassical forms of CAH were expressed, purified, and analyzed. The catalytic activities of these 12 variants ranged from 0.00009% to 30% of WT P450 21A2 and the extent of heme incorporation from 10% to 95% of the WT. Substrate dissociation constants (K s ) for four variants were 37–13,000-fold higher than for WT P450 21A2. Cytochrome b 5 , which augments several P450 activities, inhibited P450 21A2 activity. Similar to the WT enzyme, high noncompetitive intermolecular kinetic deuterium isotope effects (≥ 5.5) were observed for all six P450 21A2 variants examined for 21-hydroxylation of 21-d3-progesterone, indicating that C–H bond breaking is a rate-limiting step over a 104-fold range of catalytic efficiency. Using UV-visible and CD spectroscopy, we found that P450 21A2 thermal stability assessed in bacterial cells and with purified enzymes differed among salt-wasting- and nonclassical-associated variants, but these differences did not correlate with catalytic activity. Our in-depth investigation of CAH-associated P450 21A2 variants reveals critical insight into the effects of disease-causing mutations on this important enzyme.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Systemic stomatal responses in plants: Coordinating development, stress, and pathogen defense under a changing climate

To successfully survive, develop, grow and reproduce, multicellular organisms must coordinate their molecular, physiological, developmental and metabolic responses among their different cells and tissues. This process is mediated by cell-to-cell, vascular and/or volatile communication, and involves electric, chemical and/or hydraulic signals. Within this context, stomata serve a dual role by coordinating their responses to the environment with their neighbouring cells at the epidermis, but also with other stomata present on other parts of the plant. As stomata represent one of the most important conduits between the plant and its above-ground environment, as well as directly affect photosynthesis, respiration and the hydraulic status of the plant by controlling its gas and vapour exchange with the atmosphere, coordinating the overall response of stomata within and between different leaves and tissues plays a cardinal role in plant growth, development and reproduction. Here, we discuss different examples of local and systemic stomatal coordination, the different signalling pathways that mediate them, and the importance of systemic stomatal coordination to our food supply, ecosystems and weather patterns, under our changing climate. Importantly, we further discuss the potential biotechnological implications of regulating systemic stomatal responses for enhancing agricultural productivity in a warmer and CO 2 -rich environment.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

A Molecular View of Plant Local Adaptation: Incorporating Stress-Response Networks

Ecological specialization in plants occurs primarily through local adaptation to different environments. Local adaptation is widely thought to result in costly fitness trade-offs that result in maladaptation to alternative environments. However, recent studies suggest that such trade-offs are not universal. Additionally, there is currently a limited understanding of the molecular mechanisms responsible for fitness trade-offs associated with adaptation. Here, we review the literature on stress responses in plants to identify potential mechanisms underlying local adaptation and ecological specialization. We focus on drought, high and low temperature, flooding, herbivore, and pathogen stresses. We then synthesize our findings with recent advances in the local adaptation and plant molecular biology literature. In the process, we identify mechanisms that could cause fitness trade-offs and outline scenarios where trade-offs are not a necessary consequence of adaptation. Lastly, future studies should aim to explicitly integrate molecular mechanisms into studies of local adaptation.

54 ENVIRONMENTAL SCIENCES↗