Analysis of NIA and GSNOR family genes and nitric oxide homeostasis in response to wheat-leaf rust interaction.

Nitric oxide (NO) modulates plant response to biotic and abiotic stresses by S-nitrosylation-mediated protein post-translational modification. Nitrate reductase (NR) and S-nitrosoglutathione reductase (GSNOR) enzymes are essential for NO synthesis and the maintenance of Nitric oxide/S-nitroso glutathione (NO/GSNO) homeostasis, respectively. S-nitrosoglutathione, formed by the S-nitrosylation reaction of NO with glutathione, plays a significant physiological role as the mobile reservoir of NO. The genome-wide analysis identified nine NR (NIA) and three GSNOR genes in the wheat genome. Phylogenic analysis revealed that the nine NIA genes +were clustered into four groups and the 3 GSNORs into two groups. qRT-PCR expression profiling of NIAs and GSNORs was done in Chinese spring (CS), a leaf rust susceptible wheat line showing compatible interaction, and Transfer (TR), leaf rust-resistant wheat line showing incompatible interaction, post-inoculation with leaf rust pathotype 77-5 (121-R-63). All the NIA genes showed upregulation during incompatible interaction in comparison with the compatible reaction. The GSNOR genes showed a variable pattern of expression: the TaGSNOR1 showed little change, whereas TaGSNOR2 showed higher expression during the incompatible response. TaGSNOR3 showed a rise of expression both in compatible and incompatible reactions. Before inoculation and after 72 h of pathogen inoculation, NO localization was studied in both compatible and incompatible reactions. The S-nitrosothiol accumulation, NR, and glutathione reductase activity showed a consistent increase in the incompatible interactions. The results demonstrate that both NR and GSNOR plays significant role in defence against the leaf rust pathogen in wheat by modulating NO homeostasis or signalling.

S-nitrosoglutathione Reductase-Mediated Nitric Oxide Affects Axillary Buds Outgrowth of Solanum lycopersicum L. by Regulating Auxin and Cytokinin Signaling.

Auxin and cytokinin are two kinds of important phytohormones that mediate outgrowth of axillary buds in plants. How nitric oxide and its regulator of S-nitrosoglutathione reductase (GSNOR) take part in auxin and cytokinin signaling for controlling axillary buds outgrowth remains elusive. We investigated the roles of GSNOR during tomato axillary bud outgrowth by using physiological, biochemical and genetic approaches. GSNOR negatively regulated NO homeostasis. Suppression of GSNOR promoted axillary bud outgrowth by inhibiting the expression of FZY in both apical and axillary buds. Meanwhile, AUX1 and PIN1 were down-regulated in apical buds but up-regulated in axillary buds in GSNOR-suppressed plants. Thus, reduced IAA accumulation was shown in both apical buds and axillary buds of GSNOR-suppressed plants. GSNOR-mediated changes of NO and auxin affected cytokinin biosynthesis, transport, and signaling. And a decreased ratio of auxin: cytokinin was shown in axillary buds of GSNOR-suppressed plants, leading to bud dormancy breaking. We also found that the original NO signaling was generated by nitrate reductase (NR) catalyzing nitrate as substrate. NR-mediated NO reduced the GSNOR activity through S-nitrosylation of Cys-10, then induced a further NO burst, which played the above roles to promote axillary buds outgrowth. Together, GSNOR-mediated NO played important roles in controlling axillary buds outgrowth by altering the homeostasis and signaling of auxin and cytokinin in tomato plants.

The effects of FAR1 and TGFBRAP1 on the proliferation and apoptosis of follicular granulosa cells in goose (Anser cygnoides).

The low laying performance of geese seriously damages the growth of the poultry industry, and is related to the development of pre- hierarchical follicles. Our previous studies have revealed that FAR1 and TGFBRAP1 were involved in follicular development, but the exact regulation mechanism still kept unclear. In recent studies, the expression of FAR1 and TGFBRAP1 mRNA were detected, and we found that their expression levels were relatively higher in hierarchical follicles than in pre-hierarchical follicles (P < 0.05). Moreover, generally the level of FAR1 and TGFBRAP1 mRNA gradually increased in hierarchical follicles. In addition, the proliferation and apoptosis of granulosa cells were assayed with overexpression or knockdown technology. The results showed that by the knockdown of FAR1 mRNA level, the proliferation rate of follicular granulosa cells increased significantly, the apoptosis rate decreased (P < 0.05), and the apoptosis rate also reduced obviously by transfecting TGFBRAP1-siRNA (P < 0.05). Finally, the overexpression of FAR1 or TGFBRAP1 resulted in the inhabitation to the secretion of E2 and P4 in granulosa cells, while the knockdown of FAR1 or TGFBRAP1 enhanced the secretion of E2 and P4. In conclusion, the results indicated that FAR1 and TGFBRAP1 regulated the apoptosis of follicular granulosa cells and cut the secretion of E2 and P4 in geese, which provided basic data for the understanding of the regulating process of goose reproduction.

Aldehyde dehydrogenase 3a2 protects AML cells from oxidative death and the synthetic lethality of ferroptosis inducers.

Metabolic alterations in cancer represent convergent effects of oncogenic mutations. We hypothesized that a metabolism-restricted genetic screen, comparing normal primary mouse hematopoietic cells and their malignant counterparts in an ex vivo system mimicking the bone marrow microenvironment, would define distinctive vulnerabilities in acute myeloid leukemia (AML). Leukemic cells, but not their normal myeloid counterparts, depended on the aldehyde dehydrogenase 3a2 (Aldh3a2) enzyme that oxidizes long-chain aliphatic aldehydes to prevent cellular oxidative damage. Aldehydes are by-products of increased oxidative phosphorylation and nucleotide synthesis in cancer and are generated from lipid peroxides underlying the non-caspase-dependent form of cell death, ferroptosis. Leukemic cell dependence on Aldh3a2 was seen across multiple mouse and human myeloid leukemias. Aldh3a2 inhibition was synthetically lethal with glutathione peroxidase-4 (GPX4) inhibition; GPX4 inhibition is a known trigger of ferroptosis that by itself minimally affects AML cells. Inhibiting Aldh3a2 provides a therapeutic opportunity and a unique synthetic lethality to exploit the distinctive metabolic state of malignant cells.

Detection and Classification of Novel Renal Histologic Phenotypes Using Deep Neural Networks.

With the advent and increased accessibility of deep neural networks (DNNs), complex properties of histologic images can be rigorously and reproducibly quantified. We used DNN-based transfer learning to analyze histologic images of periodic acid-Schiff-stained renal sections from a cohort of mice with different genotypes. We demonstrate that DNN-based machine learning has strong generalization performance on multiple histologic image processing tasks. The neural network extracted quantitative image features and used them as classifiers to look for differences between mice of different genotypes. Excellent performance was observed at segmenting glomeruli from non-glomerular structure and subsequently predicting the genotype of the animal on the basis of glomerular quantitative image features. The DNN-based genotype classifications highly correlate with mesangial matrix expansion scored by a pathologist (R.E.C.), which differed in these animals. In addition, by analyzing non-glomeruli images, the neural network identified novel histologic features that differed by genotype, including the presence of vacuoles, nuclear count, and proximal tubule brush border integrity, which was validated with immunohistologic staining. These features were not identified in systematic pathologic examination. Our study demonstrates the power of DNNs to extract biologically relevant phenotypes and serve as a platform for discovering novel phenotypes. These results highlight the synergistic possibilities for pathologists and DNNs to radically scale up our ability to generate novel mechanistic hypotheses in disease.

Activin/Smad2 and Wnt/ß-catenin up-regulate HAS2 and ALDH3A2 to facilitate mesendoderm differentiation of human embryonic stem cells.

Activin and Wnt signaling are necessary and sufficient for mesendoderm (ME) differentiation of human embryonic stem cells (ESCs). In this study, we report that during ME differentiation induced by Activin and Wnt, Activin/Smad2 induces a decrease of the repressive histone modification of H3K27me3 by promoting the proteasome-dependent degradation of enhancer of zeste 2 polycomb (EZH2)-repressive complex 2 subunit. As a result, recruitment of the forkhead protein FOXH1 on open chromatin regions integrates the signals of Activin/Smad2 and Wnt/ß-catenin to activate the expression of the ME genes including HAS2 and ALDH3A2 Consistently, H3K27me3 decrease is enriched on open chromatin around regulatory regions. Furthermore, knockdown of HAS2 or ALDH3A2 greatly attenuates ME differentiation. These findings unveil a pathway from extracellular signals to epigenetic modification-mediated gene activation during ME commitment.

Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC3000.

The bacterial pathogen Pseudomonas syringae modulates plant hormone signaling to promote infection and disease development. P. syringae uses several strategies to manipulate auxin physiology in Arabidopsis thaliana to promote pathogenesis, including its synthesis of indole-3-acetic acid (IAA), the predominant form of auxin in plants, and production of virulence factors that alter auxin responses in the host; however, the role of pathogen-derived auxin in P. syringae pathogenesis is not well understood. Here we demonstrate that P. syringae strain DC3000 produces IAA via a previously uncharacterized pathway and identify a novel indole-3-acetaldehyde dehydrogenase, AldA, that functions in IAA biosynthesis by catalyzing the NAD-dependent formation of IAA from indole-3-acetaldehyde (IAAld). Biochemical analysis and solving of the 1.9 Å resolution x-ray crystal structure reveal key features of AldA for IAA synthesis, including the molecular basis of substrate specificity. Disruption of aldA and a close homolog, aldB, lead to reduced IAA production in culture and reduced virulence on A. thaliana. We use these mutants to explore the mechanism by which pathogen-derived auxin contributes to virulence and show that IAA produced by DC3000 suppresses salicylic acid-mediated defenses in A. thaliana. Thus, auxin is a DC3000 virulence factor that promotes pathogenicity by suppressing host defenses.

Phytosphingosine degradation pathway includes fatty acid α-oxidation reactions in the endoplasmic reticulum.

Although normal fatty acids (FAs) are degraded via ß-oxidation, unusual FAs such as 2-hydroxy (2-OH) FAs and 3-methyl-branched FAs are degraded via α-oxidation. Phytosphingosine (PHS) is one of the long-chain bases (the sphingolipid components) and exists in specific tissues, including the epidermis and small intestine in mammals. In the degradation pathway, PHS is converted to 2-OH palmitic acid and then to pentadecanoic acid (C15:0-COOH) via FA α-oxidation. However, the detailed reactions and genes involved in the α-oxidation reactions of the PHS degradation pathway have yet to be determined. In the present study, we reveal the entire PHS degradation pathway: PHS is converted to C15:0-COOH via six reactions [phosphorylation, cleavage, oxidation, CoA addition, cleavage (C1 removal), and oxidation], in which the last three reactions correspond to the α-oxidation. The aldehyde dehydrogenase ALDH3A2 catalyzes both the first and second oxidation reactions (fatty aldehydes to FAs). In Aldh3a2-deficient cells, the unmetabolized fatty aldehydes are reduced to fatty alcohols and are incorporated into ether-linked glycerolipids. We also identify HACL2 (2-hydroxyacyl-CoA lyase 2) [previous name, ILVBL; ilvB (bacterial acetolactate synthase)-like] as the major 2-OH acyl-CoA lyase involved in the cleavage (C1 removal) reaction in the FA α-oxidation of the PHS degradation pathway. HACL2 is localized in the endoplasmic reticulum. Thus, in addition to the already-known FA α-oxidation in the peroxisomes, we have revealed the existence of FA α-oxidation in the endoplasmic reticulum in mammals.

S-nitrosylation of the Mitochondrial Chaperone TRAP1 Sensitizes Hepatocellular Carcinoma Cells to Inhibitors of Succinate Dehydrogenase.

S-nitrosoglutathione reductase (GSNOR) represents the best-documented denitrosylase implicated in regulating the levels of proteins posttranslationally modified by nitric oxide on cysteine residues by S-nitrosylation. GSNOR controls a diverse array of physiologic functions, including cellular growth and differentiation, inflammation, and metabolism. Chromosomal deletion of GSNOR results in pathologic protein S-nitrosylation that is implicated in human hepatocellular carcinoma (HCC). Here we identify a metabolic hallmark of aberrant S-nitrosylation in HCC and exploit it for therapeutic gain. We find that hepatocyte GSNOR deficiency is characterized by mitochondrial alteration and by marked increases in succinate dehydrogenase (SDH) levels and activity. We find that this depends on the selective S-nitrosylation of Cys(501) in the mitochondrial chaperone TRAP1, which mediates its degradation. As a result, GSNOR-deficient cells and tumors are highly sensitive to SDH inhibition, namely to α-tocopheryl succinate, an SDH-targeting molecule that induced RIP1/PARP1-mediated necroptosis and inhibited tumor growth. Our work provides a specific molecular signature of aberrant S-nitrosylation in HCC, a novel molecular target in SDH, and a first-in-class therapy to treat the disease. Cancer Res; 76(14); 4170-82. ©2016 AACR.

Disruption of the Sjögren-Larsson Syndrome Gene Aldh3a2 in Mice Increases Keratinocyte Growth and Retards Skin Barrier Recovery.

The fatty aldehyde dehydrogenase (FALDH) ALDH3A2 is the causative gene of Sjögren Larsson syndrome (SLS). To date, the molecular mechanism underlying the symptoms characterizing SLS has been poorly understood. Using Aldh3a2(-/-) mice, we found here that Aldh3a2 was the major FALDH active in undifferentiated keratinocytes. Long-chain base metabolism was greatly impaired in Aldh3a2(-/-) keratinocytes. Phenotypically, the intercellular spaces were widened in the basal layer of the Aldh3a2(-/-) epidermis due to hyperproliferation of keratinocytes. Furthermore, oxidative stress-induced genes were up-regulated in Aldh3a2(-/-) keratinocytes. Upon keratinocyte differentiation, the activity of another FALDH, Aldh3b2, surpassed that of Aldh3a2 As a result, Aldh3a2(-/-) mice were indistinguishable from wild-type mice in terms of their whole epidermis FALDH activity, and their skin barrier function was uncompromised under normal conditions. However, perturbation of the stratum corneum caused increased transepidermal water loss and delayed barrier recovery in Aldh3a2(-/-) mice. In conclusion, Aldh3a2(-/-) mice replicated some aspects of SLS symptoms, especially at the basal layer of the epidermis. Our results suggest that hyperproliferation of keratinocytes via oxidative stress responses may partly contribute to the ichthyosis symptoms of SLS.

FrxA is an S-nitrosoglutathione reductase enzyme that contributes to Helicobacter pylori pathogenicity.

Helicobacter pylori is a pathogen that infects the gastric mucosa of a large percentage of the human population worldwide, and predisposes to peptic ulceration and gastric cancer. Persistent colonization of humans by H. pylori triggers an inflammatory response that leads to the production of reactive nitrogen species. However, the mechanisms of H. pylori defence against nitrosative stress remain largely unknown. In this study, we show that the NADH-flavin oxidoreductase FrxA of H. pylori, besides metabolizing nitrofurans and metronidazole, has S-nitrosoglutathione reductase activity. In agreement with this, inactivation of the FrxA-encoding gene resulted in a strain that was more sensitive to S-nitrosoglutathione. FrxA was also shown to contribute to the proliferation of H. pylori in macrophages, which are key phagocytic cells of the mammalian innate immune system. Moreover, FrxA was shown to support the virulence of the pathogen upon mouse infection. Altogether, we provide evidence for a new function of FrxA that contributes to the successful chronic colonization ability that characterizes H. pylori.

The role of regulatory proteins and S-nitrosylation of endothelial nitric oxide synthase in the human clitoris: implications for female sexual function.

INTRODUCTION: During female sexual arousal, clitoral blood flow is controlled by endothelial nitric oxide synthase (eNOS) and its product, nitric oxide (NO). The mechanisms regulating eNOS activity and NO bioavailability in the clitoris are largely unknown. AIM: To identify proteins involved in regulation of eNOS activity within the clitoris and to evaluate the effects of S-nitrosoglutathione reductase (GSNO-R) and eNOS nitrosylation/denitrosylation on clitoral blood flow. METHODS: Immunohistochemistry for eNOS, caveolin-1 (Cav1), heat shock protein-90 (Hsp90), phosphodiesterase type 5 (PDE5), GSNO-R, and soluble guanylate cyclase (sGC) was performed on human and murine clitoral tissue. Western blot analysis was performed for eNOS, phosphorylated eNOS (phospho-eNOS, Ser1177), Cav1, Hsp90, sGC, PDE5, phosphoinositide 3-kinase (PI3K), Akt (protein kinase B), and GSNO-R on protein from human clitoral tissue. A biotin switch assay was used to analyze the S-nitrosylation of eNOS, nNOS, and GSNO-R. Clitoral blood flow was measured in wild-type and GSNO-R(-/-) mice at baseline and during cavernous nerve electrical stimulation (CNES). MAIN OUTCOME MEASURES: Localization of eNOS regulatory proteins and clitoral blood flow. RESULTS: eNOS and GSNO-R co-localized to the vascular endothelium and sinusoids of human clitoral tissue. Immunohistochemistry also localized Cav1 and Hsp90 to the endothelium and PDE5 and sGC to the trabecular smooth muscle. Expression of S-nitrosylated (SNO)-eNOS and SNO-GSNO-R was detected by biotin switch assays. Wild-type control mice exhibited increased clitoral blood flow with CNES whereas GSNO-R(-/-) animals failed to show an increase in blood flow. CONCLUSIONS: Several key eNOS regulatory proteins are present in the clitoral tissue in a cellular specific pattern. S-nitrosylation of eNOS may also represent a key regulatory mechanism governing eNOS activation/deactivation since mice deficient in GSNO-R failed to increase clitoral blood flow. Additional studies are necessary to define the role of S-nitrosylation in the genital vascular response and its subsequent impact on female sexual function.

Retinaldehyde dehydrogenase enzymes regulate colon enteric nervous system structure and function.

The enteric nervous system (ENS) forms from the neural crest-derived precursors that colonize the bowel before differentiating into a network of neurons and glia that control intestinal function. Retinoids are essential for normal ENS development, but the role of retinoic acid (RA) metabolism in development remains incompletely understood. Because RA is produced locally in the tissues where it acts by stimulating RAR and RXR receptors, RA signaling during development is absolutely dependent on the rate of RA synthesis and degradation. RA is produced by three different enzymes called retinaldehyde dehydrogenases (RALDH1, RALDH2 and RALDH3) that are all expressed in the developing bowel. To determine the relative importance of these enzymes for ENS development, we analyzed whole mount preparations of adult (8-12-week old) myenteric and submucosal plexus stained with NADPH diaphorase (neurons and neurites), anti-TuJ1 (neurons and neurites), anti-HuC/HuD (neurons), and anti-S100ß (glia) in an allelic series of mice with mutations in Raldh1, Raldh2, and Raldh3. We found that Raldh1-/-, Raldh2+/-, Raldh3+/- (R1(KO)R2(Het)R3(Het)) mutant mice had a reduced colon myenteric neuron density, reduced colon myenteric neuron to glia ratio, reduced colon submucosal neuron density, and increased colon myenteric fibers per neuron when compared to the wild type (WT; Raldh1WT, Raldh2WT, Raldh3WT) mice. These defects are unlikely to be due to defective ENS precursor migration since R1(KO)R2(Het)R3(KO) mice had increased enteric neuron progenitor migration into the distal colon compared to WT during development. RALDH mutant mice also have reduced contractility in the colon compared to WT mice. These data suggest that RALDH1, RALDH2 and RALDH3 each contribute to ENS development and function.

GSNOR-mediated de-nitrosylation in the plant defence response.

A key feature of the plant defence response is the transient engagement of a nitrosative burst, resulting in the synthesis of reactive nitrogen intermediates (RNIs). Specific, highly reactive cysteine (Cys) residues of low pK(a) are a major site of action for these intermediates. The addition of an NO moiety to a Cys thiol to form an S-nitrosothiol (SNO), is termed S-nitrosylation. This redox-based post-translational modification is emerging as a key regulator of protein function in plant immunity. Here we highlight recent advances in our understanding of de-nitrosylation, the mechanism that depletes protein SNOs, with a focus on S-nitrosoglutathione reductase (GSNOR). This enzyme controls total cellular S-nitrosylation indirectly during the defence response by turning over S-nitrosoglutathione (GSNO), a major cache of NO bioactivity.

Differential Notch signaling in the epicardium is required for cardiac inflow development and coronary vessel morphogenesis.

RATIONALE: The proepicardium is a transient structure comprising epicardial progenitor cells located at the posterior limit of the embryonic cardiac inflow. A network of signals regulates proepicardial cell fate and defines myocardial and nonmyocardial domains at the venous pole of the heart. During cardiac development, epicardial-derived cells also contribute to coronary vessel morphogenesis. OBJECTIVE: To study Notch function during proepicardium development and coronary vessel formation in the mouse. METHODS AND RESULTS: Using in situ hybridization, RT-PCR, and immunohistochemistry, we find that Notch pathway elements are differentially activated throughout the proepicardial-epicardial-coronary transition. Analysis of RBPJk-targeted embryos indicates that Notch ablation causes ectopic procardiogenic signaling in the proepicardium that in turn promotes myocardial differentiation in adjacent mesodermal progenitors, resulting in a premature muscularization of the sinus venosus horns. Epicardium-specific Notch1 ablation using a Wt1-Cre driver line disrupts coronary artery differentiation, reduces myocardium wall thickness and myocyte proliferation, and reduces Raldh2 expression. Ectopic Notch1 activation disrupts epicardium development and causes thinning of ventricular walls. CONCLUSIONS: Epicardial Notch modulates cell differentiation in the proepicardium and adjacent pericardial mesoderm. Notch1 is later required for arterial endothelium commitment and differentiation and for vessel wall maturation during coronary vessel development and myocardium growth.

Expression of retinaldehyde dehydrogenase enzymes in mucosal dendritic cells and gut-draining lymph node stromal cells is controlled by dietary vitamin A.

The vitamin A metabolite retinoic acid (RA) plays a crucial role in mucosal immune responses. We demonstrate in this study that RA-producing retinaldehyde dehydrogenase (RALDH) enzymes are postnatally induced in mesenteric lymph node (MLN) dendritic cells (DCs) and MLN stromal cells. RALDH enzyme activity in lamina propria-derived CD103(+) MLN-DCs did not depend on TLR signaling. Remarkably, RA itself could directly induce RALDH2 in both DCs and stromal cells in vitro. Furthermore, upon provision of a vitamin A-deficient diet, it was found that RA-mediated signaling was strongly reduced within the small intestines, while RALDH2 mRNA and RALDH enzyme activity in lamina propria DCs and MLN-DCs, as well as RALDH2 mRNA expression in MLN stromal cells, were strongly diminished. Moreover, supply of vitamin A to vitamin A-deficient mice restored RA-mediated signaling in the intestine and RALDH activity in lamina propria-derived CD103(+) MLN-DCs. Our results show that RA-dependent signaling within the intestine is indispensable for RALDH activity in the draining MLN.

Dissection of the phytohormonal regulation of trichome formation and biosynthesis of the antimalarial compound artemisinin in Artemisia annua plants.

• Biosynthesis of the sesquiterpene lactone and potent antimalarial drug artemisinin occurs in glandular trichomes of Artemisia annua plants and is subjected to a strict network of developmental and other regulatory cues. • The effects of three hormones, jasmonate, gibberellin and cytokinin, were studied at the structural and molecular levels in two different A. annua chemotypes by microscopic analysis of gland development, and by targeted metabolite and transcript profiling. Furthermore, a genome-wide cDNA-amplified fragment length polymorphism (AFLP)-based transcriptome profiling was carried out of jasmonate-elicited leaves at different developmental stages. • Although cytokinin and gibberellin positively affected at least one aspect of gland formation, these two hormones did not stimulate artemisinin biosynthesis. Only jasmonate simultaneously promoted gland formation and coordinated transcriptional activation of biosynthetic gene expression, which ultimately led to increased sesquiterpenoid accumulation with chemotype-dependent effects on the distinct pathway branches. Transcriptome profiling revealed a trichome-specific fatty acyl- coenzyme A reductase, trichome-specific fatty acyl-CoA reductase 1 (TFAR1), the expression of which correlates with trichome development and sesquiterpenoid biosynthesis. • TFAR1 is potentially involved in cuticular wax formation during glandular trichome expansion in leaves and flowers of A. annua plants. Analysis of phytohormone-modulated transcriptional regulons provides clues to dissect the concerted regulation of metabolism and development of plant trichomes.

Lymphocyte development requires S-nitrosoglutathione reductase.

NO is critical to immunity, but its role in the development of the immune system is unknown. In this study, we show that S-nitrosoglutathione reductase (GSNOR), a protein key to the control of protein S-nitrosylation, is important for the development of lymphocytes. Genetic deletion of GSNOR in mice results in significant decrease in both T and B lymphocytes in the periphery. In thymus, GSNOR deficiency causes excessive protein S-nitrosylation, increases apoptosis, and reduces the number of CD4 single-positive thymocytes. Lymphopenia and increase in S-nitrosylation and apoptosis in GSNOR-deficient mice are largely abolished by genetic deletion of inducible NO synthase. Furthermore, the protection of lymphocyte development by GSNOR is apparently intrinsic to hematopoietic cells. Thus, GSNOR, likely through regulation of S-nitrosylation and apoptosis, physiologically plays a protective role in the development of the immune system.

Insights into the organization of dorsal spinal cord pathways from an evolutionarily conserved raldh2 intronic enhancer.

Comparative studies of the tetrapod raldh2 (aldh1a2) gene, which encodes a retinoic acid (RA) synthesis enzyme, have led to the identification of a dorsal spinal cord enhancer. Enhancer activity is directed dorsally to the roof plate and dorsal-most (dI1) interneurons through predicted Tcf- and Cdx-homeodomain binding sites and is repressed ventrally via predicted Tgif homeobox and ventral Lim-homeodomain binding sites. Raldh2 and Math1/Cath1 expression in mouse and chicken highlights a novel, transient, endogenous Raldh2 expression domain in dI1 interneurons, which give rise to ascending circuits and intraspinal commissural interneurons, suggesting roles for RA in the ontogeny of spinocerebellar and intraspinal proprioceptive circuits. Consistent with expression of raldh2 in the dorsal interneurons of tetrapods, we also found that raldh2 is expressed in dorsal interneurons throughout the agnathan spinal cord, suggesting ancestral roles for RA signaling in the ontogenesis of intraspinal proprioception.