Secondary NAD+ deficiency in the inherited defect of glutamine synthetase.

Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.

Retrocyclins and their activity against HIV-1.

Primate theta-defensins are physically distinguished as the only known fully-cyclic peptides of animal origin. Humans do not produce theta-defensin peptides due to a premature stop codon present in the signal sequence of all six theta-defensin pseudogenes. Instead, since the putative coding regions of human theta-defensin pseudogenes have remained remarkably intact, their corresponding peptides, called "retrocyclins", have been recreated using solid-phase synthetic approaches. Retrocyclins exhibit an exceptional therapeutic index both as inhibitors of HIV-1 entry and as bactericidal agents, which makes retrocyclins promising candidates for further development as topical microbicides to prevent sexually transmitted diseases. This review presents the evolution, antiretroviral mechanism of action, and potential clinical applications of retrocyclins to prevent sexual transmission of HIV-1.

Transcriptomic analysis of the zebrafish inner ear points to growth hormone mediated regeneration following acoustic trauma.

BACKGROUND: Unlike mammals, teleost fishes are capable of regenerating sensory inner ear hair cells that have been lost following acoustic or ototoxic trauma. Previous work indicated that immediately following sound exposure, zebrafish saccules exhibit significant hair cell loss that recovers to pre-treatment levels within 14 days. Following acoustic trauma in the zebrafish inner ear, we used microarray analysis to identify genes involved in inner ear repair following acoustic exposure. Additionally, we investigated the effect of growth hormone (GH) on cell proliferation in control zebrafish utricles and saccules, since GH was significantly up-regulated following acoustic trauma. RESULTS: Microarray analysis, validated with the aid of quantitative real-time PCR, revealed several genes that were highly regulated during the process of regeneration in the zebrafish inner ear. Genes that had fold changes of ≥ 1.4 and P -values ≤ 0.05 were considered significantly regulated and were used for subsequent analysis. Categories of biological function that were significantly regulated included cancer, cellular growth and proliferation, and inflammation. Of particular significance, a greater than 64-fold increase in growth hormone (gh1) transcripts occurred, peaking at 2 days post-sound exposure (dpse) and decreasing to approximately 5.5-fold by 4 dpse. Pathway Analysis software was used to reveal networks of regulated genes and showed how GH affected these networks. Subsequent experiments showed that intraperitoneal injection of salmon growth hormone significantly increased cell proliferation in the zebrafish inner ear. Many other gene transcripts were also differentially regulated, including heavy and light chain myosin transcripts, both of which were down-regulated following sound exposure, and major histocompatability class I and II genes, several of which were significantly regulated on 2 dpse. CONCLUSIONS: Transcripts for GH, MHC Class I and II genes, and heavy- and light-chain myosins, as well as many others genes, were differentially regulated in the zebrafish inner ear following overexposure to sound. GH injection increased cell proliferation in the inner ear of non-sound-exposed zebrafish, suggesting that GH could play an important role in sensory hair cell regeneration in the teleost ear.

Pharmacological targeting of IDO-mediated tolerance for treating autoimmune disease.

Cells at the maternal-fetal interface express indoleamine 2,3 dioxygenase (IDO) to consume all local tryptophan for the express purpose of starving adjacent maternal T cells of this most limiting and essential amino acid. This stops local T cell proliferation to ultimately result in the most dramatic example of immune tolerance, acceptance of the fetus. By contrast, inhibition of IDO using 1-methyl-tryptophan causes a sudden catastrophic rejection of the mammalian fetus. Immunomodulatory factors including IFNgamma, TNFalpha, IL-1, and LPS use IDO induction in responsive antigen presenting cells (APCs) also to transmit tolerogenic signals to T cells. Thus it makes sense to consider IDO induction towards tolerance for autoimmune diseases in general. Approaches to cell specific therapeutic IDO induction with NAD precursor supplementation to prevent the collateral non-T cell pathogenesis due to chronic TNFalpha-IDO activated tryptophan depletion in autoimmune diseases are reviewed. Tryptophan is an essential amino acid most immediately because it is the only precursor for the endogenous biosynthesis of nicotinamide adenine dinucleotide (NAD). Both autoimmune disease and the NAD deficiency disease pellagra occur in women at greater than twice the frequency of occurrence in men. The importance of IDO dysregulation manifest as autoimmune pellagric dementia is genetically illustrated for Nasu-Hakola Disease (or PLOSL), which is caused by a mutation in the IDO antagonizing genes TYROBP/DAP12 or TREM2. Loss of function leads to psychotic symptoms rapidly progressing to presenile dementia likely due to unchecked increases in microglial IDO expression, which depletes neurons of tryptophan causing neurodegeneration. Administration of NAD precursors rescued entire mental hospitals of dementia patients literally overnight in the 1930's and NAD precursors should help Nasu-Hakola patients as well. NAD depletion mediated by peroxynitrate PARP1 activation is one of the few established mechanisms of necrosis. Chronic elevation of TNFalpha leading to necrotic events by NAD depletion in autoimmune disease likely occurs via combination of persistent IDO activation and iNOS-peroxynitrate activation of PARP1 both of which deplete NAD. Pharmacological doses of NAD precursors repeatedly provide dramatic therapeutic benefit for rheumatoid arthritis, type 1 diabetes, multiple sclerosis, colitis, other autoimmune diseases, and schizophrenia in either the clinic or animal models. Collectively these observations support the idea that autoimmune disease may in part be considered as localized pellagra manifesting symptoms particular to the inflamed target tissues. Thus pharmacological doses of NAD precursors (nicotinic acid/niacin, nicotinamide/niacinamide, or nicotinamide riboside) should be considered as potentially essential to the therapeutic success of any IDO-inducing regimen for treating autoimmune diseases. Distinct among the NAD precursors, nicotinic acid specifically activates the g-protein coupled receptor (GPCR) GPR109a to produce the IDO-inducing tolerogenic prostaglandins PGE(2) and PGD(2). Next, PGD(2) is converted to the anti-inflammatory prostaglandin, 15d-PGJ(2). These prostaglandins exert potent anti-inflammatory activities through endogenous signaling mechanisms involving the GPCRs EP2, EP4, and DP1 along with PPARgamma respectively. Nicotinamide prevents type 1 diabetes and ameliorates multiple sclerosis in animal models, while nothing is known about the therapeutic potential of nicotinamide riboside. Alternatively the direct targeting of the non-redox NAD-dependent proteins using resveratrol to activate SIRT1 or PJ34 in order to inhibit PARP1 and prevent autoimmune pathogenesis are also given consideration.

Isolation of the B3 transcription factor of the Xenopus TFIIIA gene.

The selective expression of the Xenopus TFIIIA gene in immature oocytes is principally regulated by a single 5'-flanking DNA sequence element, termed element 3 (i.e. E3). We describe the isolation and characterization of a cDNA for a protein present in immature Xenopus ooctyes, termed B3.65, which appears to bind to and activate E3-mediated expression. The approximate molecular weight of the E3 binding protein(s) was determined by ultraviolet light cross-linking analysis. B3.65, a protein of the appropriate molecular weight, was purified biochemically from immature Xenopus ooctye extracts by affinity chromatography. Antiserum to purified B3.65 super-shifted the E3 activator complex. In addition, B3.65 mRNA was found to be highly enriched in immature oocytes. All of these data are consistent with B3.65 either being the E3 activator, or antigenically related to the specific activator required for XenopusTFIIIA gene transcription. B3.65 is a member of the K-homologous (KH) domain family of proteins, with almost absolute identity to Xenopus Vg1 RBP/VERA (97%) and significant similarity to human koc (82%). The koc mRNA is over-expressed in human pancreatic cancer tissues, and B3.65 mRNA was detected in Xenopus pancreas and kidney. Interestingly, KH proteins, like Vg1RBP/VERA, are most commonly associated with RNA metabolism, in their capacity to regulate RNA localization, stability, and translation. Our results suggest that B3.65 is a key regulator of both RNA- and DNA metabolism.

The potential role of indoleamine 2,3 dioxygenase (IDO) as a predictive and therapeutic target for diabetes treatment: a mythical truth.

Type 1 diabetes (T1D) is an autoimmune disease in which a T-cell-mediated reaction demolishes insulin-producing cells of pancreatic islets. Inadequacy of insulin therapy has motivated research focused on mechanisms by which autoimmune reactions can be suppressed. In recent years, the role of indoleamine 2,3 dioxygenase (IDO) in regulation of immune system has been extensively investigated. Initially, IDO was recognized as a host defense mechanism. However, recent studies have suggested an immunomodulatory role for IDO which may contribute to the induction of immune tolerance. In this review, we concentrate on the role of IDO in several pathologic conditions with a focus on T1D to rationalize our hypothesis regarding the potential for inclusion of IDO in certain therapeutic strategies aimed at early detection, treatment or ideally cure of chronic and autoimmune diseases such as T1D.

Nicotinic acid-mediated activation of both membrane and nuclear receptors towards therapeutic glucocorticoid mimetics for treating multiple sclerosis.

Acute attacks of multiple sclerosis (MS) are most commonly treated with glucocorticoids, which can provide life-saving albeit only temporary symptomatic relief. The mechanism of action (MOA) is now known to involve induction of indoleamine 2,3-dioxygenase (IDO) and interleukin-10 (IL-10), where IL-10 requires subsequent heme oxygenase-1 (HMOX-1) induction. Ectopic expression studies reveal that even small changes in expression of IDO, HMOX-1, or mitochondrial superoxide dismutase (SOD2) can prevent demyelination in experimental autoimmune encephalomyelitis (EAE) animal models of MS. An alternative to glucocorticoids is needed for a long-term treatment of MS. A distinctly short list of endogenous activators of both membrane G-protein-coupled receptors and nuclear peroxisome proliferating antigen receptors (PPARs) demonstrably ameliorate EAE pathogenesis by MOAs resembling that of glucocorticoids. These dual activators and potential MS therapeutics include endocannabinoids and the prostaglandin 15-deoxy-Δ¹²,¹4-PGJ2. Nicotinamide profoundly ameliorates and prevents autoimmune-mediated demyelination in EAE via maintaining levels of nicotinamide adenine dinucleotide (NAD), without activating PPAR nor any G-protein-coupled receptor. By comparison, nicotinic acid provides even greater levels of NAD than nicotinamide in many tissues, while additionally activating the PPARγ-dependent pathway already shown to provide relief in animal models of MS after activation of GPR109a/HM74a. Thus nicotinic acid is uniquely suited for providing therapeutic relief in MS. However nicotinic acid is unexamined in MS research. Nicotinic acid penetrates the blood brain barrier, cures pellagric dementia, has been used for over 50 years clinically without toxicity, and raises HDL concentrations to a greater degree than any pharmaceutical, thus providing unparalleled benefits against lipodystrophy. Summary analysis reveals that the expected therapeutic benefits of high-dose nicotinic acid administration far outweigh any known adverse risks in consideration for the treatment of multiple sclerosis.

The importance of NAD in multiple sclerosis.

The etiology of multiple sclerosis (MS) is unknown but it manifests as a chronic inflammatory demyelinating disease in the central nervous system (CNS). During chronic CNS inflammation, nicotinamide adenine dinucleotide (NAD) concentrations are altered by (T helper) Th1-derived cytokines through the coordinated induction of both indoleamine 2,3-dioxygenase (IDO) and the ADP cyclase CD38 in pathogenic microglia and lymphocytes. While IDO activation may keep auto-reactive T cells in check, hyper-activation of IDO can leave neuronal CNS cells starving for extracellular sources of NAD. Existing data indicate that glia may serve critical functions as an essential supplier of NAD to neurons during times of stress. Administration of pharmacological doses of non-tryptophan NAD precursors ameliorates pathogenesis in animal models of MS. Animal models of MS involve artificially stimulated autoimmune attack of myelin by experimental autoimmune encephalomyelitis (EAE) or by viral-mediated demyelination using Thieler's murine encephalomyelitis virus (TMEV). The Wld(S) mouse dramatically resists razor axotomy mediated axonal degeneration. This resistance is due to increased efficiency of NAD biosynthesis that delays stress-induced depletion of axonal NAD and ATP. Although the Wld(S) genotype protects against EAE pathogenesis, TMEV-mediated pathogenesis is exacerbated. In this review, we contrast the role of NAD in EAE versus TMEV demyelinating pathogenesis to increase our understanding of the pharmacotherapeutic potential of NAD signal transduction pathways. We speculate on the importance of increased SIRT1 activity in both PARP-1 inhibition and the potentially integral role of neuronal CD200 interactions through glial CD200R with induction of IDO in MS pathogenesis. A comprehensive review of immunomodulatory control of NAD biosynthesis and degradation in MS pathogenesis is presented. Distinctive pharmacological approaches designed for NAD-complementation or targeting NAD-centric proteins (SIRT1, SIRT2, PARP-1, GPR109a, and CD38) are outlined towards determining which approach may work best in the context of clinical application.

A high throughput live transparent animal bioassay to identify non-toxic small molecules or genes that regulate vertebrate fat metabolism for obesity drug development.

BACKGROUND: The alarming rise in the obesity epidemic and growing concern for the pathologic consequences of the metabolic syndrome warrant great need for development of obesity-related pharmacotherapeutics. The search for such therapeutics is severely limited by the slow throughput of animal models of obesity. Amenable to placement into a 96 well plate, zebrafish larvae have emerged as one of the highest throughput vertebrate model organisms for performing small molecule screens. A method for visually identifying non-toxic molecular effectors of fat metabolism using a live transparent vertebrate was developed. Given that increased levels of nicotinamide adenine dinucleotide (NAD) via deletion of CD38 have been shown to prevent high fat diet induced obesity in mice in a SIRT-1 dependent fashion we explored the possibility of directly applying NAD to zebrafish. METHODS: Zebrafish larvae were incubated with daily refreshing of nile red containing media starting from a developmental stage of equivalent fat content among siblings (3 days post-fertilization, dpf) and continuing with daily refreshing until 7 dpf. RESULTS: PPAR activators, beta-adrenergic agonists, SIRT-1 activators, and nicotinic acid treatment all caused predicted changes in fat, cholesterol, and gene expression consistent with a high degree of evolutionary conservation of fat metabolism signal transduction extending from man to zebrafish larvae. All changes in fat content were visually quantifiable in a relative fashion using live zebrafish larvae nile red fluorescence microscopy. Resveratrol treatment caused the greatest and most consistent loss of fat content. The resveratrol tetramer Vaticanol B caused loss of fat equivalent in potency to resveratrol alone. Significantly, the direct administration of NAD decreased fat content in zebrafish. Results from knockdown of a zebrafish G-PCR ortholog previously determined to decrease fat content in C. elegans support that future GPR142 antagonists may be effective non-toxic anti-obesity therapeutics. CONCLUSION: Owing to the apparently high level of evolutionary conservation of signal transduction pathways regulating lipid metabolism, the zebrafish can be useful for identifying non-toxic small molecules or pharmacological target gene products for developing molecular therapeutics for treating clinical obesity. Our results support the promising potential in applying NAD or resveratrol where the underlying target protein likely involves Sirtuin family member proteins. Furthermore data supports future studies focused on determining whether there is a high concentration window for resveratrol that is effective and non-toxic in high fat obesity murine models.

Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.

The zebrafish is firmly established as a genetic model for the study of vertebrate blood development. Here we have characterized the blood-forming system of adult zebrafish. Each major blood lineage can be isolated by flow cytometry, and with these lineal profiles, defects in zebrafish blood mutants can be quantified. We developed hematopoietic cell transplantation to study cell autonomy of mutant gene function and to establish a hematopoietic stem cell assay. Hematopoietic cell transplantation can rescue multilineage hematopoiesis in embryonic lethal gata1-/- mutants for over 6 months. Direct visualization of fluorescent donor cells in embryonic recipients allows engraftment and homing events to be imaged in real time. These results provide a cellular context in which to study the genetics of hematopoiesis.