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Optimal nutrition is the foundation for the development and maintenance of a healthy immune system. An optimal supply of nutrients is required for biosynthesis of immune factors and immune cell proliferation. Nutrient deficiency/inadequacy and hidden hunger, which manifests as depleted nutrients reserves, increase the risk of infectious diseases and aggravate disease severity. Therefore, an adequate and balanced diet containing an abundant diversity of foods, nutrients, and non-nutrient chemicals is paramount for an optimal immune defense against infectious diseases, including cold/flu and non-communicable diseases. Some nutrients and foods play a larger role than others in the support of the immune system. Oats are a nutritious whole grain and contain several immunomodulating nutrients. In this narrative review, we discuss the contribution of oat nutrients, including dietary fiber (β-glucans), copper, iron, selenium, and zinc, polyphenolics (ferulic acid and avenanthramides), and proteins (glutamine) in optimizing the innate and adaptive immune system’s response to infections directly by modulating the innate and adaptive immunity and indirectly by eliciting changes in the gut microbiota and related metabolites.

The mitogen-activated protein kinase ERK2 is able to elicit a wide range of context-specific responses to distinct stimuli, but the mechanisms underlying this versatility remain in question. Some cellular functions of ERK2 are mediated through regulation of gene expression. In addition to phosphorylating numerous transcriptional regulators, ERK2 is known to associate with chromatin and has been shown to bind oligonucleotides directly. ERK2 is activated by the upstream kinases MEK1/2, which phosphorylate both tyrosine 185 and threonine 183. ERK2 requires phosphorylation on both sites to be fully active. Some additional ERK2 phosphorylation sites have also been reported, including threonine 188. It has been suggested that this phospho form has distinct properties. We detected some ERK2 phosphorylated on T188 in bacterial preparations of ERK2 by mass spectrometry and further demonstrate that phosphomimetic substitution of this ERK2 residue impairs its kinase activity toward well-defined substrates and also affects its DNA binding. We used electrophoretic mobility shift assays with oligonucleotides derived from the insulin gene promoter and other regions to examine effects of phosphorylation and mutations on the binding of ERK2 to DNA. We show that ERK2 can bind oligonucleotides directly. Phosphorylation and mutations alter DNA binding and support the idea that signaling functions may be influenced through an alternate phosphorylation site. Fil: McReynolds, Andrea C.. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Karra, Aroon S.. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Li, Yan. The University of Texas Southwestern Medical Center; Estados Unidos. National Institute of Neurological Disorders and Stroke; Estados Unidos Fil: Lopez, Elias Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires; Argentina Fil: Turjanski, Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires; Argentina Fil: Dioum, Elhadji. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Lorenz, Kristina. Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V; Alemania Fil: Zaganjor, Elma. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Stippec, Steve. The University of Texas Southwestern Medical Center; Estados Unidos Fil: McGlynn, Kathleen. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Earnest, Svetlana. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Cobb, Melanie H.. The University of Texas Southwestern Medical Center; Estados Unidos

Pancreatic &beta, cell dysfunction and death contribute to the onset of diabetes, and novel strategies of &beta, cell function and survival under diabetogenic conditions need to be explored. We previously demonstrated that Isx9, a small molecule based on the isoxazole scaffold, drives neuroendocrine phenotypes by increasing the expression of genes required for &beta, cell function and improves glycemia in a model of &beta, cell regeneration. We further investigated the role of Isx9 in &beta, cell survival. We find that Isx9 drives the expression of Calbindin-D28K (D28K), a key regulator of calcium homeostasis, and plays a cytoprotective role through its calcium buffering capacity in &beta, cells. Isx9 increased the activity of the calcineurin (CN)/cytoplasmic nuclear factor of the activated T-cells (NFAT) transcription factor, a key regulator of D28K, and improved the recruitment of NFATc1, cAMP response element-binding protein (CREB), and p300 to the D28K promoter. We found that nutrient stimulation increased D28K plasma membrane enrichment and modulated calcium channel activity in order to regulate glucose-induced insulin secretion. Isx9-mediated expression of D28K protected &beta, cells against chronic stress induced by serum withdrawal or chronic inflammation by reducing caspase 3 activity. Consequently, Isx9 improved human islet function after transplantation in NOD-SCID mice in a streptozotocin-induced diabetes model. In summary, Isx9 significantly regulates expression of genes relevant to &beta, cell survival and function, and may be an attractive therapy to treat diabetes and improve islet function post-transplantation.

In the adult and developing brain, G protein-regulated inducer of neurite outgrowth 1 (GPRIN1) is a downstream effector for Gαo/i/z proteins that promotes neurite outgrowth. However, so far, the physiological role of GPRIN1 in brain health has not been reported. We generated a viable GPRIN1 whole-body knockout mouse to assess its physiological role in synaptic function both ex vivo and in vivo. In adult neurons, GPRIN1 is highly localized to the plasma membrane and synapses where it regulates neuronal signal transduction and Ca2+ homeostasis. Our results reveal that GPRIN1 might be a novel protein involved in agonist-stimulated cytoskeletal reorganization, which is crucial for early neuronal network development and in functionally mature neurons. Finally, we show that loss of GPRIN1 leads to a learning deficit in vivo and sensitizes neurons to stress, suggesting a modulatory role in brain health and disease.

Novel strategies are needed to modulate β-cell differentiation and function as potential β-cell replacement or restorative therapies for diabetes. We previously demonstrated that small molecules based on the isoxazole scaffold drive neuroendocrine phenotypes. The nature of the effects of isoxazole compounds on β-cells was incompletely defined. We find that isoxazole induces genes that support neuroendocrine and β-cell phenotypes and suppresses genes important for proliferation. Isoxazole alters β-cell metabolites and protects glucose-responsive signaling pathways under lipotoxic conditions. Finally, we show that isoxazole improves glycemia in a mouse model of β-cell regeneration. Isoxazole is a prime candidate to alter cell fate in different contexts.

Calbindin-D28k, a key regulator of calcium homeostasis plays a cytoprotective role in various tissues. We used serum free (SFM) and charcoal stripped serum (csFBS) culture media as models of cellular stress to modulate calbindin D28k expression and identify regulatory cis-elements and trans-acting factors in kidney and beta cells. The murine calbindin-D28k promoter activity was significantly upregulated under SFM or csFBS condition. Promoter analysis revealed evolutionary conserved regulatory cis-elements and deletion of 23 nt from +117/+139 as critical for basal transcription. Bioinformatics analysis of the promoter revealed conserved NFAT and TFII regulators elements. Forced expression of NFAT stimulated promoter activity. Inhibition of NFAT transcriptional activity by FK506 attenuated calbindin-D28k expression. TFII-I was shown to be necessary for basal promoter activity and to act cooperatively with NFAT. Using chromatin immunoprecipitation (ChIP) assays, NFAT was shown to bind to both proximal and distal promoter regions. ChIP assays also revealed recruitment of TFII to the -36/+139 region. Knockdown of TFII-I decreased promoter activity. In summary, calbindin-D28k expression during serum deprivation is partly regulated by NFAT and TF-II. This regulation may be important in vivo during ischemia and growth factor withdrawal to regulate cellular function and maintenance.

Hemoglobins are ubiquitous in Eukarya and Bacteria but, until now, have not been found in Archaea . A phylogenetic analysis of the recently revealed microbial family of globin-coupled heme-based sensors suggests that these sensors descended from an ancient globin-only progenitor, or a protoglobin (Pgb). Here, we report the discovery and characterization of two Pgbs from the Archaea : Ap Pgb from the obligately aerobic hyperthermophile Aeropyrum pernix , and Ma Pgb from the strictly anaerobic methanogen Methanosarcina acetivorans . Both Ap Pgb and Ma Pgb bind molecular oxygen, nitric oxide, and carbon monoxide by means of a heme moiety that is coordinated to the protein through the F8 histidine (histidine 120). We postulate that these archaeal globins are the ancestors of contemporary hemoglobins.

Understanding how signaling pathways are interconnected is vital for characterizing mechanisms of normal development and disease pathogenesis. In this issue, Van Wageningen et al. (2010) examine phosphorylation networks in Sacharromyces cerevisiae with genome-wide expression profiling to identify recurring themes in signaling redundancy.

Background: Nutrients stimulate calcium dependent activation of energy metabolism, in pancreatic beta cells. Results: Glucose-induced ATP synthase-dependent respiration is strictly calcium-dependent, with little or no effect of calcium on the NAD(P)H response. Conclusion: Calcium coordinates oxidative metabolism and respiration in pancreatic beta cells. Significance: Calcium has novel mitochondrial targets downstream of mitochondrial dehydrogenases., Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio.

New drugs for preserving and restoring pancreatic β-cell function are critically needed for the worldwide epidemic of type 2 diabetes and the cure for type 1 diabetes. We previously identified a family of neurogenic 3,5-disubstituted isoxazoles (Isx) that increased expression of neurogenic differentiation 1 (NeuroD1, also known as BETA2); this transcription factor functions in neuronal and pancreatic β-cell differentiation and is essential for insulin gene transcription. Here, we probed effects of Isx on human cadaveric islets and MIN6 pancreatic β cells. Isx increased the expression and secretion of insulin in islets that made little insulin after prolonged ex vivo culture and increased expression of neurogenic differentiation 1 and other regulators of islet differentiation and insulin gene transcription. Within the first few hours of exposure, Isx caused biphasic activation of ERK1/2 and increased bulk histone acetylation. Although there was little effect on histone deacetylase activity, Isx increased histone acetyl transferase activity in nuclear extracts. Reconstitution assays indicated that Isx increased the activity of the histone acetyl transferase p300 through an ERK1/2-dependent mechanism. In summary, we have identified a small molecule with antidiabetic activity, providing a tool for exploring islet function and a possible lead for therapeutic intervention in diabetes.

Hypoxia-inducible factors (HIFs) are stress-responsive transcriptional regulators of cellular and physiological processes involved in oxygen metabolism. Although much is understood about the molecular machinery that confers HIF responsiveness to oxygen, far less is known about HIF isoform-specific mechanisms of regulation, despite the fact that HIF-1 and HIF-2 exhibit distinct biological roles. We recently determined that the stress-responsive genetic regulator sirtuin 1 (Sirt1) selectively augments HIF-2 signaling during hypoxia. However, the mechanism by which Sirt1 maintains activity during hypoxia is unknown. In this report, we demonstrate that Sirt1 gene expression increases in a HIF-dependent manner during hypoxia in Hep3B and in HT1080 cells. Impairment of HIF signaling affects Sirt1 deacetylase activity as decreased HIF-1 signaling results in the appearance of acetylated HIF-2α, which is detected without pharmacological inhibition of Sirt1. We also find that Sirt1 augments HIF-2 mediated, but not HIF-1 mediated, transcriptional activation of the isolated Sirt1 promoter. These data in summary reveal a bidirectional link of HIF and Sirt1 signaling during hypoxia.

Coordinating Response to Stress Sirtuin 1 (Sirt1) (a protein deacetylase implicated in aging), senses the metabolic state of the cell and modulates the activity of substrate proteins that in turn regulate cellular transcriptional responses. In response to hypoxia, cells activate the transcription factor hypoxia-inducible factor 2 alpha (HIF-2α), which promotes adaptive responses. Dioum et al. (p. 1289 ; see the Perspective by Guarente ) discovered a link between these two important cellular stress response systems—HIF-2α is a substrate of Sirt1. Direct interaction between Sirt1 and HIF-2α results in deacetylation of HIF-2α and enhances its transcriptional activity. In mice lacking Sirt1, the ability of HIF-2α to promote synthesis of the growth factor erythropoietin is diminished. Thus, the regulation of HIF-2α helps to coordinate responses of cells to various stresses.

Mice lacking Epas1, encoding the transcription factor Hypoxia-inducible Factor 2alpha (HIF-2alpha), exhibit an apparent mitochondrial disease state. Similarities between knock-outs of Epas1 and of Sod2, encoding the mitochondrial antioxidant enzyme manganese superoxide dismutase, led to the identification of Sod2 as a HIF-2alpha target gene. However, Sod2 levels in Epas1(-)(/)(-) liver are intermediate between that of Sod(+)(/)(-) and Sod2(-)(/)(-) mice, which have subtle or severe phenotypes, respectively. This suggests that additional HIF-2alpha target genes besides Sod2 contribute to the Epas1(-)(/)(-) mitochondrial disease state. To define the nature of the mitochondrial defect in Epas1(-)(/)(-) liver, we performed biophysical, biochemical, and molecular studies. In the setting of decreased Sod2 levels and increased oxidative stress, we found reduced respiration, sensitized mitochondrial permeability transition pore opening, intact electron transport chain activities, and impaired mitochondrial aconitase activity. Mitochondrial aconitase protein levels were preserved, whereas mRNA and protein levels for frataxin, the oxidative stress-regulated mitochondrial aconitase chaperone protein, were markedly reduced in Epas1(-)(/)(-) livers. The mouse Fxn promoter was preferentially activated by HIF-2alpha through a consensus HIF-responsive enhancer element. In summary, the studies reveal that Fxn, like Sod2, is a nuclear-encoded, mitochondrial-localized HIF-2alpha target gene required for optimal mitochondrial homeostasis. These findings expand upon the previously defined role of HIF-2alpha in the cellular response to oxidative stress and identify a novel link of HIF-2alpha with mitochondrial homeostasis.

Neuronal PAS domain protein 2 (NPAS2) is a mammalian transcription factor that binds DNA as an obligate dimeric partner of BMAL1 and is implicated in the regulation of circadian rhythm. Here we show that both PAS domains of NPAS2 bind heme as a prosthetic group and that the heme status controls DNA binding in vitro. NPAS2-BMAL1 heterodimers, existing in either the apo (heme-free) or holo (heme-loaded) state, bound DNA avidly under favorably reducing ratios of the reduced and oxidized forms of nicotinamide adenine dinucleotide phosphate. Low micromolar concentrations of carbon monoxide inhibited the DNA binding activity of holo-NPAS2 but not that of apo-NPAS2. Upon exposure to carbon monoxide, inactive BMAL1 homodimers were formed at the expense of NPAS2-BMAL1 heterodimers. These results indicate that the heterodimerization of NPAS2, and presumably the expression of its target genes, are regulated by a gas through the heme-based sensor described here.

Phosphorylation of the transcription factor RmFixJ is the key step in the hypoxic induction of Sinorhizobium meliloti nitrogen fixation genes. Oxygen regulates this process by binding reversibly to RmFixL, a heme protein kinase whose deoxy form catalyzes the phosphoryl transfer from ATP to RmFixJ. Here we present the first quantitative measure of the extent by which various heme ligands inhibit the turnover of RmFixJ to phospho-RmFixJ. We also quantitate the inhibition by ligands of the reaction of RmFixL with ATP, in the absence of RmFixJ, to form phospho-RmFixL, i.e., the "autophosphorylation". Phospho-RmFixL formed from autophosphorylation will transfer its phosphoryl group to RmFixJ in an oxygen-independent "phosphotransfer." Here we show that the mode of substrate presentation, i.e., simultaneous versus sequential, influences the regulation of phosphoryl transfer by heme status. Inhibition factors for O(2), CO, NO, CN(-), and imidazole in the presence of RmFixJ are drastically different from the inhibition of autophosphorylation by the same ligands. Oxidation of the heme iron in unliganded RmFixL is known to have no effect on either of the sequential reactions; yet oxidation causes a 100-fold decrease in RmFixJ turnover when ATP and RmFixJ are presented simultaneously. The profound difference between the regulation of isolated RmFixL versus the complex of RmFixL with RmFixJ shows that interaction of a response regulator with its histidine-kinase partner need not be limited to the enzymatic regions of the histidine kinase, but can extend also to its sensory domain.

Protein-DNA interactions (PDIs) mediate a broad range of functions essential for cellular differentiation, function, and survival. However, it is still a daunting task to comprehensively identify and profile sequence-specific PDIs in complex genomes. Here, we have used a combined bioinformatics and protein microarray-based strategy to systematically characterize the human protein-DNA interactome. We identified 17,718 PDIs between 460 DNA motifs predicted to regulate transcription and 4,191 human proteins of various functional classes. Among them, we recovered many known PDIs for transcription factors (TFs). We also identified a large number of new PDIs for known TFs, as well as for previously uncharacterized TFs. Remarkably, we found that over three hundred proteins not previously annotated as TFs also showed sequence-specific PDIs, including RNA binding proteins, mitochondrial proteins, and protein kinases. One of such unconventional DNA-binding proteins, MAPK1, acts as a transcriptional repressor for interferon gamma-induced genes.

PURPOSE. To characterize the effect of HIF-2 haploinsufficiency on retinal neovascularization and angiogenic signaling in neonatal mice. METHODS. Retinal samples were obtained from HIF-2-haploinsufficient (Epas1 / ) and wild-type (Epas1 / ) neonatal mice subjected to an oxygen-induced retinopathy (OIR) protocol. Histologic and molecular studies were performed immediately, 12 hours, or 5 days after initiation of the hypoxia phase of the OIR protocol. Molecular profiling was performed in mouse brain endothelial cells maintained in normoxia or hypoxia. Transfection studies assessed the response of isolated promoter regions from proangiogenic genes to HIF-1 or -2 overexpression. RESULTS. Epas1 / mice exhibited no significant differences in retinal vasculature during normal development but had reduced retinal neovascularization in an OIR protocol. Multiple proangiogenic factors were induced during the hypoxia phase in Epas1 / OIR retinal samples, whereas Epas1 / OIR retinal samples had absent or blunted induction of these same factors. Several, but not all, proangiogenic factors were induced in mouse brain endothelial cells after hypoxia. In transfection assays, most proangiogenic promoter regions were preferentially activated by HIF-2 relative to HIF-1. CONCLUSIONS. HIF-2 deficiency results in reduced neovascularization and blunted inducibility of multiple proangiogenic factors in the retinas of mice with OIR. The authors propose that HIF-2 is a master regulator of proangiogenic factors in retinal vascular endothelial cells, the predominant cell type of the retina in which HIF-2 is expressed. Future studies will address whether the molecular and functional roles for HIF-2 identified from these studies can be generalized to other pathophysiological states involving neovascularization. (Invest Ophthalmol Vis Sci. 2008;49:2714‐2720) DOI:10.1167/iovs.07-1469