Nmnat 1: a Security Guard of Retinal Ganglion Cells (RGCs) in Response to High Glucose Stress.

BACKGROUND/AIMS: Retinal neurodegeneration is an early event in the pathological process of diabetic retinopathy (DR). Retinal ganglion cell (RGC) injury is an important pathological feature during neurodegenerative process. Protecting RGCs from high glucose-induced injury is a promising strategy for delaying or hindering diabetes mellitus-related retinal neuropathy. This study aims to investigate the role of Nmnat1, an enzyme which catalyzes a key step in the biosynthesis of nicotinamide adenine dinucleotide (NAD), in high glucose-induced RGC injury. METHODS: Western blot and immunofluorescence analysis was conducted to detect Nmnat1 expression pattern in the retina and RGC-5 cell. MTT assay, Hoechst staining, trypan blue staining, and calcein-AM/ propidium iodide (PI) staining was conducted to determine the effect of Nmnat1 knockdown on RGC-5 cell function. Microarray and bioinformatics analysis was conducted to identify potential signaling pathways affected by Nmnat1 knockdown. Pharmacological intervention, molecular intervention, and in vitro experiments were conducted to reveal molecular mechanism of Nmnat1-mediated protective effect on RGC-5 cell function. RESULTS: Nmnat1 is constitutively expressed in retina and RGC-5 cells. Nmnat1 knockdown aggravates RGC injury, and accelerates the development of RGC-5 cell apoptosis upon high glucose stress. MAPK signaling is the primary signaling pathway affected by Nmnat1 knockdown. Under high glucose stress, Nmnat1 knockdown leads to p38-MAPK signaling inactivation. p38-MAPK pathway inhibitor strongly blocks Nmnat1-mediated protective effect on RGC-5 cell function. CONCLUSION: Nmnat1 protects RGC against high glucose-induced injury via p38-MAPK signaling pathway. Nmnat1 may serve as a neuroprotective target for diabetes mellitus-related retinal neuropathy.

Simultaneous single-sample determination of NMNAT isozyme activities in mouse tissues.

A novel assay procedure has been developed to allow simultaneous activity discrimination in crude tissue extracts of the three known mammalian nicotinamide mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1) isozymes. These enzymes catalyse the same key reaction for NAD biosynthesis in different cellular compartments. The present method has been optimized for NMNAT isozymes derived from Mus musculus, a species often used as a model for NAD-biosynthesis-related physiology and disorders, such as peripheral neuropathies. Suitable assay conditions were initially assessed by exploiting the metal-ion dependence of each isozyme recombinantly expressed in bacteria, and further tested after mixing them in vitro. The variable contributions of the three individual isozymes to total NAD synthesis in the complex mixture was calculated by measuring reaction rates under three selected assay conditions, generating three linear simultaneous equations that can be solved by a substitution matrix calculation. Final assay validation was achieved in a tissue extract by comparing the activity and expression levels of individual isozymes, considering their distinctive catalytic efficiencies. Furthermore, considering the key role played by NMNAT activity in preserving axon integrity and physiological function, this assay procedure was applied to both liver and brain extracts from wild-type and Wallerian degeneration slow (Wld(S)) mouse. Wld(S) is a spontaneous mutation causing overexpression of NMNAT1 as a fusion protein, which protects injured axons through a gain-of-function. The results validate our method as a reliable determination of the contributions of the three isozymes to cellular NAD synthesis in different organelles and tissues, and in mutant animals such as Wld(S).

Three-minute high-performance liquid chromatographic assay for NMN adenylyltransferase using a 20-mm-long reversed-phase column.

NMN adenylyltransferase (NAD pyrophosphorylase; NMNAT) reversibly catalyzes the synthesis of NAD from ATP and NMN. In this paper, we describe a rapid and sensitive high-performance liquid chromatographic assay for NMNAT, which uses a 20-mm-long C18 reversed-phase (RP) column. The activity was measured by separating in less than 3 min the substrates (NMN and ATP) from the product (NAD) with 0.1 M potassium phosphate, pH 6.0, at a 2 ml/min flow-rate and 22 degrees C. NAD was directly quantitated from its ultraviolet absorbance. Amounts of NAD as small as 25 pmol could be measured. The activity value closely agreed with that determined by the spectrophotometric assay. This method was successfully applied to the determination of NMNAT activity in human placental and bull testis extracts, as well as in rat pheochromocytoma (PC12) cells.

Assay methods for nicotinamide mononucleotide adenylyltransferase of wide applicability.

NMN adenylyltransferase (NMNAT) reversibly catalyzes the synthesis of NAD+ or NaAD+ from ATP and NMN or NaMN. In this work, we describe a continuous coupled spectrophotometric assay that can be rapidly and routinely used in place of the previous cumbersome two-step assay. The reaction rates measured with the coupled assay display a linear dependence with respect to enzyme concentration over the range investigated. The method yields accurate and reliable estimates of the enzyme activity in the direction of NAD+ synthesis. Furthermore, we developed an HPLC-based method suitable for the assay activity both in the forward and reverse directions of the enzymatic reaction. The method appears particularly useful for measuring the NMNAT activity when the product is not NAD+ (e.g., in studies using alternative substrates), and offers the possibility of monitoring simultaneously both the NMNAT-catalyzed reaction and interfering side reactions. This is achieved through the HPLC identification and quantitation of metabolites and derivatives produced in the reaction mixture during the assay. The two methods described here should cover most needs for the assay of NMNAT activity.

Presence of acetyl-transferases and adenylyl-transferases in gentamicin-resistant transconjugants.

UNLABELLED: The aim of this work was to test the production of aminoglycoside modifying enzymes in 20 gentamicin-resistant transconjugants obtained from clinical strains of Entero-bacteriaceae. The susceptibility to aminoglycosides was determined by disc diffusion method and agar dilution method according to European Committee for Clinical Laboratory Standards, 1988. The transfer of gentamicin-resistance R-plasmids was made by conjugation on a solid medium with recipients E. coli K12. Phosphocellulose paper binding assay with 14C acetyl. CoA and 14C.ATP by Haas and Dowding was performed to reveal the enzyme production. Four different acetyl-transferases have been found: AAC/3/I, AAC/3/-V, AAC/3/-IV which modify gentamicin, and AAC/6'/-I with activity on amikacin. Only two of the transconjugants showed adenylyl-transferase activity:AAD/2"/. Some of strains tested possessed two enzymes. The most interesting finding was that the majority of strains owned AAC/3/-IV, which modifies apramycin. This was be explained with the fact that apramycin is still in a large use for animal husbandry in Bulgaria. IN CONCLUSION: four different acetyl-transferases: AAC/3/-I, AAC/3/-V, AAC/3/-IV and AAC/6'/-I and the adenylyl-transferase AAD/2"/ were found to be the biochemical mechanisms of resistance to aminoglycosides in 20 gentamicin-resistant transconjugants.

Changes in pyridine and adenine nucleotide levels in Friend erythroleukaemia cells during growth and differentiation.

Pyridine and adenine nucleotide levels were measured in Friend erythroleukaemia cells (FELC) stimulated to growth and induced to differentiate by hexamethylene bisacetamide (HMBA) and N'-methylnicotinamide (N'-MNAM). A three- to fourfold increase in the NADP(H) was found to parallel cell growth stimulation in both the presence and absence of differentiation inducers. NAD(H) increased about twofold in control and to a minor extent in HMBA-treated FELC but did not vary significantly in N'-MNAM-treated cells. ATP was significantly higher in control cells stimulated to growth than in resting ones, but it did not vary in inducer-treated cells. These data confirm the relationship between high NADP(H) levels and cell resumption to growth; moreover they show that NAD(H) pool reduction and NAD/NADH ratio rise are associated with the process of FELC differentiation. The activities of NAD pyrophosphorylase and NAD kinase are much more enhanced in growth-stimulated FELC than in resting ones. On the other hand transition from the quiescent to the proliferative state was accompanied by a decrease in the activity of poly(ADP-ribose) polymerase. A decrease in poly(ADP-ribose) polymerase activity was also found in differentiated cells in contrast to controls.

Determination of NAD pyrophosphorylase activity in biological samples.

A sensitive and simple method was developed for the accurate measurement of NAD pyrophosphorylase (NMN adenylyltransferase; EC 2.7.7.1) activity in biological samples. The reaction product of [4-3H]NAD was separated from the substrates [4-3H]NMN and ATP by HPLC. Under the standardized conditions of the assay, the enzyme activity in human chronic myelogenous leukemia K562 cells was found mainly in the nucleus (97%) with a sp act of 183.5 +/- 3.5 nmol/h/mg protein. The Km's for substrates NMN and ATP were 0.11 +/- 0.01 mM and 0.55 +/- 0.04 mM, respectively. This technique is highly reproducible with a 5% variation (SD) in five separate determinations. The lowest number of cells used for this enzyme assay was 41,000 with a protein content of 4 micrograms. The range of NAD produced during the assay was 2 to 200 microM. NAD pyrophosphorylase activities in the mononuclear cells of leukemic patients, human ovarian carcinoma cells, and rat liver were assayed.

[The effect of the x-ray irradiation of rats on the NAD-pyrophosphorylase and poly(ADP-ribose)polymerase activities of brain nuclei and on the NAD content in nerve tissue]. / Vliianie rentgenovskogo oblucheniia krys na NAD-pirofosforilaznuiu i poli(ADP-riboza)polimeraznuiu aktivnost' iader golovnogo mozga i na soderzhanie NAD v nervnoi tkani.

A study was made of the influence of X-irradiation of rats with various doses on NAD-pyrophosphorylase and poly(ADP-ribose) polymerase activity of brain nuclei. It was shown that X-radiation was ineffective with regard to NAD-pyrophosphorylase activity of nuclei and increased their poly(ADP-ribose) polymerase activity. Stimulation of poly(ADP-ribose) polymerase activity of nuclei was a function of radiation dose and correlated with the decrease in the NAD content of nervous tissue. It was found that mainly nonhistone proteins were ADP-ribosylated in nuclei of both irradiated and nonirradiated rats.

Recent observations on the structure and the properties of yeast NMN adenylyltransferase.

A homogeneous preparation of yeast NMN adenylyltransferase (EC 2.7.7.1) showed microheterogeneity, which was revealed by FPLC (Fast Protein Liquid Chromatography) ion exchange chromatography. The resolved components have been characterized with respect to electrophoretic behavior and adenine content. The results led to a hypothesis about a possible role of poly(ADP-ribosylation) in modulating the enzyme activity.