Adenine Diphosphate Research Articles

VITAMIN B2 AND ITS STATUS IN VEGETARIANS AND VEGANS

The main goal of this review was to briefly highlight the key points of today’s knowledge about the role and metabolism of vitamin B2 as well as to compare its status in vegans, vegetarians, and omnivores according to present data. Biologically active forms of water-soluble vitamin B2 (riboflavin) are represented by flavin mononucleotide (FMN) and flavin adenine diphosphate (FAD), which are coenzymes in the reactions of the electron transport chain and the pyruvate dehydrogenase complex. Riboflavin is also involved in the metabolism of fatty acids, the synthesis of cholesterol and steroid hormones. Riboflavin also participates in the folate cycle. Vitamin B2 is crucial for the metabolism of other vitamins. FMN is necessary for the for-mation of the active form of vitamin B6, while FAD is necessary for the synthesis of niacin. Severe clinical insufficiency of vitamin B2 is not common. Besides insufficient riboflavin consumption, hypothyroidism can also be a cause of vitamin B2 deficiency as thyroxine regulates the conversion of ribo-flavin to its active forms  FMN and FAD. Lack of riboflavin can cause tissue damage; especially of the epithelial, reproductive, and nervous system. The variety of its manifestations is related to the fact that riboflavin deficiency also disrupts the metabolism of vitamins B6, or PP. Similarly, riboflavin deficiency can also disrupt the folate cycle, increasing the level of homocysteine which damages vascular intima and leads to endothelial dysfunction and atherosclerosis as a result. Rapid urinary excretion of riboflavin makes it safe even if taken in a high dose, changing the urine colour into bright-yellow. This safety of riboflavin makes it unharmful to be used as an oral tracer for monitoring compliance in clinical research. Ultra-high doses of ribo-flavin (more than 400 mg/day) can cause diarrhoea or polyuria. The content of vitamin B2 in animal-derived products is higher than in plant-based ones. This also determines its lower intake in vegans and vegetarians compared to omnivores. While there is a shred of evidence that plant-based diets in-crease the production of bioavailable riboflavin by the gut microbiota, this does not appear to be sufficient to completely compensate for the vegetari-ans’/vegans’ reduced dietary intake of vitamin B2, as most of the researches point the fact that vegans and vegetarians are more prone to lack vita-min B2 than omnivores. At the same time, it should be noted that there exists no universally adopted optimal method for assessing the status of vit-amin B2 in the body. Further research and standardization of methods may ease to assess the prevalence and risks of riboflavin deficiency in various dietary groups

Protecting-Group-Free Synthesis of ADP-Ribose and Dinucleoside Di-/Triphosphate Derivatives via P(V)-P(V) Coupling Reaction.

Biomolecules containing adenosine di- or triphosphate (ADP or ATP) are crucial for diverse biological processes. Synthesis of these biomolecules and development of their chemical probes are important to elucidate their functions. Enabling reproducible and high-yielding access to these ADP- and ATP-containing molecules via conventional P(III)-P(V) and P(V)-P(V) coupling reactions is challenging owing to water content in highly polar phosphate-containing substrates. Herein, we report an efficient and reliable method for protecting-group-free P(V)-P(V) coupling reaction through in situ activation of phosphates using hydrolysis-stable 2-[N-(2-methylimidazoyl)]-1,3-dimethylimidazolinium chloride (2-MeImIm-Cl), providing the corresponding electrophilic P(V) intermediates for subsequent nucleophilic attack using their coupling partners. This P(V)-P(V) coupling reaction proceeded even in a wet reaction medium and showed a broad substrate scope, accommodating protecting-group-free synthesis of ADP-ribose and nicotinamide adenine diphosphate analogs, ATP-containing biomolecules, and ADP-ribosyl peptides.

High dose NAD+ boosting as pan-sirtuin activation increases atherosclerotic plaques and systemic inflammation in Apoe knockout mice

Abstract Background NAD+ (nicotinamide adenine diphosphate) is a cosubstrate of sirtuins (SIRT) that are activated upon caloric restriction. The nuclear SIRT1 and SIRT6 exert protective effects on atherothrombosis through immunometabolic pathways. By supplementing its precursors, an increase in the NAD+ pool has been reported to extend life span in mice. However, supplementing NAD+ precursors may also stimulate inflammation depending on the context. Nicotinamide riboside (NR) is a precursor of NAD+, whose supplementation has been used to combat metabolic syndromes and liver steatosis. Notably, the effects of pan-sirtuin activation using NR on atherosclerosis remain unknown. Purpose We hypothesized that NR supplementation confers atheroprotection by activating the NAD+ sirtuin axis. We aimed to test this hypothesis by investigating the effects of NR supplementation on plaque formation using an atherosclerosis-prone mouse model. Methods 8-week-old male apolipoprotein E (ApoE) knockout (KO) mice were fed for 12 weeks a high-cholesterol diet (HCD, 1.25 wt%) supplemented with three NR doses: 0, 1.2, and 2.4 g/kg diet. The groups were designated as NR-, NR+, and NR++, respectively (n=14/group). After overnight fasting, aortae, liver, and blood were collected. Cytokines were analyzed using a multiplex ELISA. Triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) levels were determined by colorimetry and the Friedewald equation. Cross sections of the aortic root were stained with Oil-Red O. Results High-dose (HD) NR supplementation increased plaque thickness in NR++ ApoE KO mice, compared with NR+ mice (Figure 1A and B). In parallel, plasma expression levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 as well as TG and LDL-C levels were increased in NR++ mice (Figure 1C-F) suggesting that NR supplementation induced systemic inflammation and disturbed lipid metabolism. Analyses of liver lysates showed that expression levels of SIRT1 were decreased and CD38 increased in NR++ mice. Full length PARP1 remained unchanged, while both cleaved PARP1 and total PARylation in liver decreased. NAD+ concentrations in liver and plasma remained unchanged. These data suggest that an increase in hepatic CD38 might consume more NAD+ in the atherosclerotic environment and could be the culprits for worsening atherosclerotic progression and systemic inflammation (Figure 2). Conclusions HD NR supplementation increased plaque thickness, as well as IL-6 and LDL-C plasma levels, moderate NR dose had neutral effects. Elevated hepatic expression levels of CD38 and decreased SIRT1 may at least in part explain the atherosclerotic phenotype. Thus, HD NR supplementation in mice does not decrease but worsen atherosclerosis and induce systemic inflammation. A cautious approach should be considered when applying NAD+ boosting in patients with atherosclerotic disease.

A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain.

Viral macrodomains, which can bind to and/or hydrolyze adenine diphosphate ribose (ADP-ribose or ADPr) from proteins, have been suggested to counteract host immune response and be viable targets for the development of antiviral drugs. Therefore, developing high-throughput screening (HTS) techniques for macrodomain inhibitors is of great interest. Herein, using a novel tracer TAMRA-ADPr, an ADP-ribose compound conjugated with tetramethylrhodamine, we developed a robust fluorescence polarization assay for various viral and human macrodomains including SARS-CoV-2 Macro1, VEEV Macro, CHIKV Macro, human MacroD1, MacroD2, and PARP9 Macro2. Using this assay, we validated Z8539 (IC50 6.4 μM) and GS441524 (IC50 15.2 μM), two literature-reported small-molecule inhibitors of SARS-CoV-2 Macro1. Our data suggest that GS441524 is highly selective for SARS-CoV-2 Macro1 over other human and viral macrodomains. Furthermore, using this assay, we identified pNP-ADPr (ADP-ribosylated p-nitrophenol, IC50 370 nM) and TFMU-ADPr (ADP-ribosylated trifluoromethyl umbelliferone, IC50 590 nM) as the most potent SARS-CoV-2 Macro1 binders reported to date. An X-ray crystal structure of SARS-CoV-2 Macro1 in complex with TFMU-ADPr revealed how the TFMU moiety contributes to the binding affinity. Our data demonstrate that this fluorescence polarization assay is a useful addition to the HTS methods for the identification of macrodomain inhibitors.

Novel carbon skeletons activate human NicotinAMide Phosphoribosyl Transferase (NAMPT) enzyme in biochemical assay.

Nicotinamide adenine dinucleotide (NAD) is a central molecule in cellular metabolism that has been implicated in human health, the aging process, and an array of human diseases. NAD is well known as an electron storage molecule, cycling between NAD and the reduced NADH. In addition, NAD is cleaved into nicotinamide and Adenine diphosphate ribose by NAD-consuming enzymes such as sirtuins, PARPs and CD38. There are numerous pathways for the biosynthesis of NAD to maintain a baseline concentration and thus avoid cellular death. The NAD salvage pathway, a two-step process to regenerate NAD after cleavage, is the predominant pathway for humans. Nicotinamide PhosphribosylTransferase (NAMPT) is the rate-limiting enzyme within the salvage path. Exposure to pharmacological modulators of NAMPT has been reported to either deplete or increase NAD levels. This study used a curated set of virtual compounds coupled with biochemical assays to identify novel activators of NAMPT. Autodock Vina generated a ranking of the National Cancer Institute's Diversity Set III molecular library. The library contains a set of organic molecules with diverse functional groups and carbon skeletons that can be used to identify lead compounds. The target NAMPT surface encompassed a novel binding location that included the NAMPT dimerization plane, the openings to the two active site channels, and a portion of the known binding location for NAMPT substrate and product. Ranked molecules were evaluated in a biochemical assay using purified recombinant NAMPT enzyme. Two novel carbon skeletons were confirmed to stimulate NAMPT activity. Compound 20 (NSC9037) is a polyphenolic xanthene derivative in the fluorescein family, while compound 2 (NSC19803) is the polyphenolic myricitrin nature product. Micromolar quantities of compound 20 or compound 2 can double NAMPT's product formation. In addition, natural products that contain high concentrations of polyphenolic flavonoids, similar to myricitrin, also stimulate NAMPT activity. Confirmation of a novel binding site for these compounds will further our understanding of the cellular mechanism leading to NAD homeostasis and better human health outcomes.

Xrn1 is a deNADding enzyme modulating mitochondrial NAD-capped RNA

The existence of non-canonical nicotinamide adenine diphosphate (NAD) 5′-end capped RNAs is now well established. Nevertheless, the biological function of this nucleotide metabolite cap remains elusive. Here, we show that the yeast Saccharomyces cerevisiae cytoplasmic 5′-end exoribonuclease Xrn1 is also a NAD cap decapping (deNADding) enzyme that releases intact NAD and subsequently degrades the RNA. The significance of Xrn1 deNADding is evident in a deNADding deficient Xrn1 mutant that predominantly still retains its 5′-monophosphate exonuclease activity. This mutant reveals Xrn1 deNADding is necessary for normal growth on non-fermenting sugar and is involved in modulating mitochondrial NAD-capped RNA levels and may influence intramitochondrial NAD levels. Our findings uncover a contribution of mitochondrial NAD-capped RNAs in overall NAD regulation with the deNADding activity of Xrn1 fulfilling a central role.

Navigating Hemostasis of Bleeding Among Children With β-Thalassemia.

Bleeding phenotype is reported in β-thalassemia patients. However, the underlying etiology remains elusive. We aimed to assess coagulation profile and the platelet aggregation in β-thalassemia children with bleeding diathesis. Fifty β-thalassemia children with a positive bleeding history were recruited. Bleeding phenotype was explored through full history taking and thorough clinical examination. Complete blood count, prothrombin time, international normalized ratio, and platelets aggregometry were performed for children with negative workup. Mucosal bleeding was manifest among most of our patients (96%). Two-third of patients had decreased aggregation with ristocetin (68%), adenine di-phosphate (64%), and arachidonic acid (64%). While half of the patients (48%) had deficient response to epinephrine. Collagen, ristocetin, and arachidonic acid induced aggregation were negatively correlated to frequency of blood transfusion (P=0.021, r=-0.325; P<0.001, r=-0.465; P=0.018, r=-0.333, respectively). Aggregation to collagen and epinephrine demonstrated a negative correlation with age (P=0.04, r=-0.287; P=0.03, r=-0.315). Deferiprone was associated with a deficient response to ristocetin and collagen when compared with deferasirox or no chelation (P=0.021 and 0.006, respectively). Impaired ristocetin response was linked to hydroxyurea (P=0.035). Platelets function defect should be considered in β-thalassemia patients with bleeding symptoms.

Potential Pathogenic Genes and Mechanism of Ankylosing Spondylitis: A Study Based on WGCNA and Bioinformatics Analysis

The purpose of this study is to explore the high-risk pathogenic driver genes for the occurrence and development of ankylosing spondylitis (AS) based on the bioinformatics method at the molecular level, to further elaborate the molecular mechanism of the pathogenesis of AS, and to provide potential biological targets for the diagnosis and treatment of clinical AS. The gene expression profile data GSE16879 were downloaded from the GEO (Gene Expression Omnibus) database, and weighted gene coexpression network analysis was performed. Highly correlated genes were divided into 14 modules, and 582 genes contained in the yellow (classic module) and 59 genes contained in grey60 (hematologic module) modules had the strongest correlation with AS. After protein-protein interaction (PPI) analysis, the top 20 genes with the highest scores were obtained from classic module and hematologic module, respectively. The DAVID (Database for Annotation, Visualization, and Integrated Discovery) database was used for Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis to analyze the biological functions of high-risk genes related to AS. The results showed that the process of signal recognition particle-dependent cotranslational protein targeting to membrane, ribosome, nicotinamide adenine diphosphate hydride dehydrogenase (ubiquinone) activity, platelet activation, integrin complex, and extracellular matrix binding were enriched. In this study, weighted gene coexpression network analysis, an efficient system biology algorithm, was used to analyze the high-risk pathogenic driver gene of AS. We provide new targets for the diagnosis and treatment of clinical AS and new ideas for further study.

Modulating Sirtuin Biology and Nicotinamide Adenine Diphosphate Metabolism in Cardiovascular Disease-From Bench to Bedside.

Sirtuins (SIRT1–7) comprise a family of highly conserved deacetylases with distribution in different subcellular compartments. Sirtuins deacetylate target proteins depending on one common substrate, nicotinamide adenine diphosphate (NAD+), thus linking their activities to the status of cellular energy metabolism. Sirtuins had been linked to extending life span and confer beneficial effects in a wide array of immune-metabolic and cardiovascular diseases. SIRT1, SIRT3, and SIRT6 have been shown to provide protective effects in various cardiovascular disease models, by decreasing inflammation, improving metabolic profiles or scavenging oxidative stress. Sirtuins may be activated collectively by increasing their co-substrate NAD+. By supplementing NAD+ precursors, NAD+ boosters confer pan-sirtuin activation with protective cardiometabolic effects in the experimental setting: they improve endothelial dysfunction, protect from experimental heart failure, hypertension and decrease progression of liver steatosis. Different precursor molecules were applied ranging from nicotinamide (NAM), nicotinamide mononucleotide (NMN) to nicotinamide riboside (NR). Notably, not all experimental results showed protective effects. Moreover, the results are not as striking in clinical studies as in the controlled experimental setting. Species differences, (lack of) genetic heterogeneity, different metabolic pathways, dosing, administration routes and disease contexts may account for these challenges in clinical translation. At the clinical scale, caloric restriction can reduce the risks of cardiovascular disease and raise NAD+ concentration and sirtuin expression. In addition, antidiabetic drugs such as metformin or SGLT2 inhibitors may confer cardiovascular protection, indirectly via sirtuin activation. Overall, additional mechanistic insight and clinical studies are needed to better understand the beneficial effects of sirtuin activation and NAD+ boosters from bench to bedside.

Curcumin Alleviates Aβ42-Induced Neuronal Metabolic Dysfunction via the Thrb/SIRT3 Axis and Improves Cognition in APPTG Mice.

Curcumin has been reported to have a therapeutic effect on Alzheimer's disease (AD), but the specific mechanism remains to be elucidated. In the present research, we aimed to investigate the effect and molecular mechanism of curcumin on AD. Mouse primary hippocampal neuron cells were treated with various concentrations of beta-amyloid 42 (Aβ42) and the results found that Aβ42 inhibited cell viability in a dose-dependent manner. Compared with 50ng/mL Aβ42, 500ng/mL Aβ42 could further promote cell apoptosis, reduce the ratio of Nicotinamide adenine dinucleotide (NAD(+))/Nicotinamide adenine diphosphate hydride (NADH) and Adenosine 5'-triphosphate (ATP) level, and inhibit Sirtuins 3 (SIRT3) deacetylation activity and protein expression of Thyroid hormone receptor beta (Thrb) and SIRT3. Hence, 500ng/mL Aβ42 was used to establish a cell model of AD. Curcumin significantly reversed the inhibitory effects of Aβ42 on cell viability, SIRT3 deacetylation activity, the ratio of NAD+/NADH, ATP level and the protein expression of Thrb and SIRT3, and the promotive effect on apoptosis. ChIPBase was used to predict the binding region of Thrb and SIRT3. Dual luciferase reporter gene and Chromatin immune precipitation (ChIP) assays were employed to verify the relationship between Thrb and promoter of SIRT3 mRNA. Overexpression of Thrb recovered Aβ42 induced metabolic dysfunction, while Thrb silence aggravated Aβ42 induced metabolic dysfunction. Moreover, Thrb silence or 3-TYP (a selective inhibitor of SIRT3) treatment abolished the amelioration of curcumin on Aβ42 induced metabolic dysfunction. Additionally, curcumin attenuated memory deficits in Amyloid precursor protein transgenic (APPTG) mice. Collectively, curcumin alleviated Aβ42-induced neuronal metabolic dysfunction through increasing Thrb expression and SIRT3 activity and improved cognition in APPTG mice.

SPAAC-NAD-seq, a sensitive and accurate method to profile NAD+-capped transcripts

Nicotinamide adenine diphosphate (NAD+) is a novel messenger RNA 5' cap in Escherichia coli, yeast, mammals, and Arabidopsis Transcriptome-wide identification of NAD+-capped RNAs (NAD-RNAs) was accomplished through NAD captureSeq, which combines chemoenzymatic RNA enrichment with high-throughput sequencing. NAD-RNAs are enzymatically converted to alkyne-RNAs that are then biotinylated using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Originally applied to E. coli RNA, which lacks the m7G cap, NAD captureSeq was then applied to eukaryotes without extensive verification of its specificity for NAD-RNAs vs. m7G-capped RNAs (m7G-RNAs). In addition, the Cu2+ ion in the CuAAC reaction causes RNA fragmentation, leading to greatly reduced yield and loss of full-length sequence information. We developed an NAD-RNA capture scheme utilizing the copper-free, strain-promoted azide-alkyne cycloaddition reaction (SPAAC). We examined the specificity of CuAAC and SPAAC reactions toward NAD-RNAs and m7G-RNAs and found that both prefer the former, but also act on the latter. We demonstrated that SPAAC-NAD sequencing (SPAAC-NAD-seq), when combined with immunodepletion of m7G-RNAs, enables NAD-RNA identification with accuracy and sensitivity, leading to the discovery of new NAD-RNA profiles in Arabidopsis Furthermore, SPAAC-NAD-seq retained full-length sequence information. Therefore, SPAAC-NAD-seq would enable specific and efficient discovery of NAD-RNAs in prokaryotes and, when combined with m7G-RNA depletion, in eukaryotes.

Biocompatible fabrication of TiO2 nanoparticles: Antimicrobial, anticoagulant, antiplatelet, direct hemolytic and cytotoxicity properties

In the present report, biocompatible fabrication of TiO2 nanoparticles (NPs) are fabricated from biomass of Alternaria solani. The prepared TiO2 NPs are well characterized by sophisticated techniques such as, powder X-ray diffraction (PXRD), diffuse reflectance spectra (DRS), Fourier infrared spectroscopy (FTIR) and Scanning electron microscopy (SEM) Techniques. The anatase phase of the TiO2 NPs is confirmed by the PXRD patterns. The DRS studies revealed the wide, intense peak centered at ~330 nm and band gap energy are found to be ~3.5 eV. Ti-O vibration modes centered at 648 cm−1 are confirmed by the FTIR spectra. Morphological features of biosynthesized TiO2 NPs are depicted in the SEM images and it is found that agglomeration in the prepared particles is observed due to the binding between the biomolecules of the biomass. The antimicrobial activity of TiO2 NPs are tested using the well-known agar well diffusion method and it is found that, the significant zone of inhibition in Klebsiella aerogenes, Candida albicans and Escherichia coli is found to be superior is when compared to Staphylococcus aureus and Pseudomonas desmolyticum. It is observed that the augmented clotting time for the bio-synthesized TiO2 NPs ranges between the 165–440 s in platelet-rich plasma (PRP). Likewise, the platelet-poor plasma (PPP) exhibiting the clotting factor is increased from control 175 to 450 s, which reveals the superior anticoagulant properties. Further, the antiplatelet activity of the prepared NPs is tested using the adenine diphosphate (ADP) as agonists and the platelet aggregation and inhibition is found to be ~68.18% and 31.82% respectively. In addition to this, the haemolytic activity of the prepared NPs did not hydrolyse the RBCs. The fabricated NPs also showed significant cytotoxicity against cancer cell lines, both human lung cancer cell lines (A549) when compared to human breast cancer (MCF-7). The obtained results indicate that, the fungal mediated synthesis of TiO2 NPs is simple, eco-friendly and exhibits multi-functional properties.

Study on the time-effectiveness of exercise preconditioning on heart protection in exhausted rats.

To investigate the persistence time and the effectiveness of exercise preconditioning (EP) on myocardial protection in exhausted rats from myocardial enzymes, electrocardiogram (ECG), cardiac function, and mitochondrial respiratory function after cessation of exercise training. One hundred and twelve healthy male Sprague-Dawley rats were randomly divided into seven groups (n = 16): control group (CON), exhaustive exercise (EE) group, EP group, and EE after EP (EP + EE); furthermore, EP + EE group was randomly divided into 1D, 3D, 9D, and 18D groups (1D, 3D, 9D, and 18D) and performed exhaustive treadmill exercise at a speed of 30 m/min on the 1st, 3rd, 9th, and 18th days separately after EP exercise stopped. We detected the serum contents of N-terminal pro B type natriuretic peptide (NT-proBNP) and cardiac troponin I (cTnI) by the enzyme-linked immunosorbent assays method, recorded ECG, detected heart function by pressure volume catheter, measured the respiratory rates of rat myocardial mitochondria state 3 and 4 of complex I, complex II, and IV by high-resolution breathing apparatus. EP could decrease the serum content of NT-proBNP and cTnI, improved the electrical derangement and the left ventricular function in exhausted rats. Moreover, the protective effect was more obvious in the 9th day after EP stopped, whereas it would disappear when EP stopped for more than 18 days. Compared with EE group, the respiratory rate value of myocardial mitochondrial complex increased in 1D, 3D, and 9D groups. Therefore, the protective effect of EP on the heart of exhausted rats decreased with the prolongation of stopping training time, and the effect was significant within 3 days of discontinuing training, then decreased gradually, and completely disappeared in the 18th day. EP enhanced the cardiac function in exhausted rats through raising the nicotinamide adenine diphosphate hydride (NADH) electron transport chain and increased the respiration rates of mitochondrial respiratory complex I and IV state 3, thereby improved myocardial mitochondrial respiratory function and energy metabolism.

Allosteric discrimination at the NADH/ADP regulatory site of glutamate dehydrogenase.

Glutamate dehydrogenase (GDH) is a target for treating insulin-related disorders, such as hyperinsulinism hyperammonemia syndrome. Modeling native ligand binding has shown promise in designing GDH inhibitors and activators. Our computational investigation of the nicotinamide adenine diphosphate hydride (NADH)/adenosine diphosphate (ADP) site presented in this paper provides insight into the opposite allosteric effects induced at a single site of binding inhibitor NADH versus activator ADP to GDH. The computed binding free-energy difference between NADH and ADP using thermodynamic integration is -0.3 kcal/mol, which is within the -0.275 and -1.7 kcal/mol experimental binding free-energy difference range. Our simulations show an interesting model of ADP with dissimilar binding conformations at each NADH/ADP site in the GDH trimer, which explains the poorly understood strong binding but weak activation shown in experimental studies. In contrast, NADH showed similar inhibitory binding conformations at each NADH/ADP site. The structural analysis of the important residues in the NADH/ADP binding site presented in this paper may provide potential targets for mutation studies for allosteric drug design.

Gene expression of 49 kDa apyrase, cytoskeletal proteins, ATPase, ADPase and amino acid contents of Pisum sativum (L.) cells germinated in Euryops arabicus (Steud. ex Jaub. & Spach) water extract

The present research reports of quick and marked changes induced by plant extract of Euryops arabicus in the gene expression of 49-kDa apyrases, cytoskeletal proteins, ATPases, ADPase and amount of amino acid of pea (Pisum sativum L. var. Alaska). Pellets of cytoskeletals proteins (27000 xg) were probed with anti-apyrase antibody, biotinylated anti-rat, actin and alpha and beta-tubulin for Western blotting. ATPase and ADPase activities were determined based on the hydrolytic efficacy of adenine triphosphate and adenine diphosphate. By 72 hours, the abundance of apyrases, cytoskeletal proteins and amount of amino acid in pellets of 27000 xg of germinated pea seeds in E. arabicus extracts were sharply increased than those sown in distilled water. All the samples exhibited that the stems had more amount from apyrases, cytoskeletal proteins, amino acids and ATPase and ADPase activities than primary leaves and primary roots that were germinated either on E. arabicus water extract or in distilled water. Based on the enzyme’s capability to hydrolyse nucleotide triphosphate and nucleotide diphosphate as well as the direct association between expression of 49-kDa apyrase and cytoskeletal proteins, E. arabicus water extract had an important effect on plant germinations.

NAD+-capped RNAs are widespread in the Arabidopsis transcriptome and can probably be translated

As the most common RNA cap in eukaryotes, the 7-methylguanosine (m7G) cap impacts nearly all processes that a messenger RNA undergoes, such as splicing, polyadenylation, nuclear export, translation, and degradation. The metabolite and redox agent, nicotinamide adenine diphosphate (NAD+), can be used as an initiating nucleotide in RNA synthesis to result in NAD+-capped RNAs. Such RNAs have been identified in bacteria, yeast, and human cells, but it is not known whether they exist in plant transcriptomes. The functions of the NAD+ cap in RNA metabolism or translation are still poorly understood. Here, through NAD captureSeq, we show that NAD+-capped RNAs are widespread in Arabidopsis thaliana NAD+-capped RNAs are predominantly messenger RNAs encoded by the nuclear and mitochondrial genomes, but not the chloroplast genome. NAD+-capped transcripts from the nuclear genome appear to be spliced and polyadenylated. Furthermore, although NAD+-capped transcripts constitute a small proportion of the total transcript pool from any gene, they are enriched in the polysomal fraction and associate with translating ribosomes. Our findings implicate the existence of as yet unknown mechanisms whereby the RNA NAD+ cap interfaces with RNA metabolic processes as well as translation initiation. More importantly, our findings suggest that cellular metabolic and/or redox states may influence, or be regulated by, mRNA NAD+ capping.

TPC2-mediated Ca2+ signaling is required for the establishment of synchronized activity in developing zebrafish primary motor neurons

During the development of the early spinal circuitry in zebrafish, spontaneous Ca2+ transients in the primary motor neurons (PMNs) are reported to transform from being slow and uncorrelated, to being rapid, synchronized and patterned. In this study, we demonstrated that in intact zebrafish, Ca2+ release via two-pore channel type 2 (TPC2) from acidic stores/endolysosomes is required for the establishment of synchronized activity in the PMNs. Using the SAIGFF213A;UAS:GCaMP7a double-transgenic zebrafish line, Ca2+ transients were visualized in the caudal PMNs (CaPs). TPC2 inhibition via molecular, genetic or pharmacological means attenuated the CaP Ca2+ transients, and decreased the normal ipsilateral correlation and contralateral anti-correlation, indicating a disruption in normal spinal circuitry maturation. Furthermore, treatment with MS-222 resulted in a complete (but reversible) inhibition of the CaP Ca2+ transients, as well as a significant decrease in the concentration of the Ca2+ mobilizing messenger, nicotinic acid adenine diphosphate (NAADP) in whole embryo extract. Together, our new data suggest a novel function for NAADP/TPC2-mediated Ca2+ signaling in the development, coordination, and maturation of the spinal network in zebrafish embryos.

An activity transition from NADH dehydrogenase to NADH oxidase during protein denaturation.

A decrease in the specific activity of an enzyme is commonly observed when the enzyme is inappropriately handled or is stored over an extended period. Here, we reported a functional transition of an FMN-bound diaphorase (FMN-DI) that happened during the long-term storage process. It was found that FMN-DI did not simply lose its β-nicotinamide adenine diphosphate (NADH) dehydrogenase activity after a long-time storage, but obtained a new enzyme activity of NADH oxidase. Further mechanistic studies suggested that the alteration of the binding strength of an FMN cofactor with a DI protein could be responsible for this functional switch of the enzyme.

Proteome Analysis of Human Follicular Thyroid Cancer Cells Exposed to the Random Positioning Machine

Several years ago, we detected the formation of multicellular spheroids in experiments with human thyroid cancer cells cultured on the Random Positioning Machine (RPM), a ground-based model to simulate microgravity by continuously changing the orientation of samples. Since then, we have studied cellular mechanisms triggering the cells to leave a monolayer and aggregate to spheroids. Our work focused on spheroid-related changes in gene expression patterns, in protein concentrations, and in factors secreted to the culture supernatant during the period when growth is altered. We detected that factors inducing angiogenesis, the composition of integrins, the density of the cell monolayer exposed to microgravity, the enhanced production of caveolin-1, and the nuclear factor kappa B p65 could play a role during spheroid formation in thyroid cancer cells. In this study, we performed a deep proteome analysis on FTC-133 thyroid cancer cells cultured under conditions designed to encourage or discourage spheroid formation. The experiments revealed more than 5900 proteins. Their evaluation confirmed and explained the observations mentioned above. In addition, we learned that FTC-133 cells growing in monolayers or in spheroids after RPM-exposure incorporate vinculin, paxillin, focal adhesion kinase 1, and adenine diphosphate (ADP)-ribosylation factor 6 in different ways into the focal adhesion complex.

Using Clickable NAD+ Analogs to Label Substrate Proteins of PARPs.

ADP-ribosylation has been well known as an important posttranslational modification, which is catalyzed by a family of enzymes called poly(ADP-ribose) polymerases (PARPs). PARPs transfer of a single or multiple adenine diphosphate ribose (ADP-ribose) units from nicotinamide adenine dinucleotide (NAD+) to specific amino acids on substrate proteins. Through mono- or poly-ADP-ribosylation enzymatic activities, PARPs regulate various biological processes, including DNA damage repair, chromatin remodeling, transcriptional regulation, RNA processing and metabolism. Notably, PARP inhibitors are in clinical trials to treat human diseases, in particular cancer. To further investigate the biological function of PARPs, and to achieve better therapeutic effect of PARP inhibitors, it is important to identify the physiological substrates of PARPs. Here we describe a protocol to use clickable analog of nicotinamide adenine dinucleotide (NAD+) that can be applied for the detection, affinity purification and identification of substrate proteins of PARPs.