Interaction of chromatid lesions induced by N-nitroso-N-methylurea and N-nitroso-N-ethylurea inVicia faba

After treatment of primary roots ofVicia faba with N-nitroso-N-methylurea (NMU) and afterwards with N-nitroso-N-ethylurea (NEU) only an additive effect has been found. When NEU was used for the first treatment and NMU was applied afterwards an interaction of induced lesions was observed in the production of interchanges. These results might be interpreted as follows: NMU induces primary chromatid lesions in a later stage of interphase than NEU, so that these lesions cannot be induced in the same cell, if NMU is applied first. Because of the complexity of events leading to aberrations, a lot of which are unknown, our interpretation should be accepted with due reserve and cannot be more than a working hypothesis for further experimental tests.     

Effect of nitrosourea in small doses on the frequency of mutation in Salmonella typhimurium

The effect of N-nitroso-N-methylurea (NMU), N-nitroso-N,N'-dimethylurea (NDMU) and N-nitroso-N-ethylurea (NEU) at doses less than 100 mkg/ml on mutability of Salmonella typhimurium strains of Ames' system (G-46, TA-1950, TA-1535, TA-100, TA-1538) has been studied. NMU and NEU at doses of 5-10 mkg/ml have been found to increase the survival and decrease the number of reversions from auxotrophity in histidine to prototrophity. The effect of given doses of NMU and NEU on bacteria repair activity has been shown. The role of pk M101 plasmide in this process is being discussed. NDMU in contrast to NMU and NEU induces read frome shift mutations and exhibits high mutagenous activity at all doses examined.     

Distribution and localisation of induced breaks in the chromosomes of Bellevalia. 3. Sodium nitrite

Summary Nitrous acid induces fragmentation of chromosomes and only very few translocations in Bellevalia romana (2n=8). A total of 1098 breaks is induced by 1×10^-3, 1,5×10^-4 and 1,5×10^-5 M NaNO₂ at pH 4,5. Root tips were fixed at 24, 48 and 72 hours after treatment for 1 hour.The percentage of breaks increases with time between treatment and fixation; it reaches its highest level at 1,5×10^-4 M, which dose coincides with the lowest mitotic rate.A dose influence on the distribution of breaks among the centromeres and the chromosome arms was observed. Consistent with the effect of other agents the highest number of breaks was in the A- the lowest, however, in the D-chromosome.The distribution of breaks on the different chromosome arms was homogeneous for all fixations. As observed previously the breakage frequency was not proportional to the arm length.The distribution of breaks on the proximal, median and distal sections of the chromosome arms is significantly different from random distribution.The nitrous acid-induced pattern of breaks is significantly different from that induced by methylating (MMS+NMU) and ethylating agents (DES+NEU).The known primary chemical reaction of nitrous acid and alkylating agents with DNA are inadequate alone in explaining the different patterns of breaks. More information is needed regarding the processes following these primary reactions.In conclusion, several possible objections against the technique of localizing breaks are shown to be non-valid.     

Comutagenic effects exerted by N-nitroso compounds.

Mutagenesis induced by dimethylnitrosamine (DMN) and N-methyl-N-nitrosourea (NMU) in Salmonella typhimurium TA100 and TA1530 is characterized by biphasic dose and time response curves. At low doses or short incubation times mutagenic response is minimal, but increases rapidly when an apparent threshold dose or threshold incubation time is exceeded. Bacteria pretreated with subthreshold doses of DMN or NMU were many times more sensitive to the mutagenic effects of methylating and ethylating N-nitroso compounds than were untreated bacteria. The growth phase of the bacteria had little effect on the percentage enhancement of mutagenesis caused by pretreatment with NMU although exponentially growing cells were more sensitive to mutagenesis induced by NMU or diethylnitrosamine. Mutagenesis induced by methylmethanesulfonate and N-propyl-N'-nitro-N-nitrosoguanidine was not significantly enhanced by pretreatment of bacteria with NMU or NEU suggesting that the former mutagens act by different mechanisms than NMU or NEU.     

Design,synthesis and biological activity of flavonoid derivatives as selective agonists for neuromedin U 2 receptor

Central neuromedin U 2 receptor (NMU2R) plays important roles in the regulation of food intake and body weight. Identification of NMU2R agonists may lead to the development of pharmaceutical agents to treat obesity. Based on the structure of rutin, a typical flavonoid and one of the NMU2R agonists we previously identified from an in-house made natural product library, 30 flavonoid derivatives have been synthesized and screened on a cell-based reporter gene assay. A number of compounds were found to be selective and highly potent to NMU2R. For example, the EC₅₀ value of compound NRA 4 is very close to that of NMU, the endogenous peptide ligand of NMU2R. Structure–activity relationship analysis revealed that a 3-hydroxyl group in ring C and a 2′-fluoride group in ring B were essential for this class of compounds to be active against NMU2R.     

Phase Shifting of the Circadian Clock by Diethylstilbestrol and Related Compounds in Neurospora crassa

Phase shifts of the circadian conidiation rhythm in Neurospora crassa were induced by 3-hour treatments of mycelia in liquid medium with diethylstilbestrol (DES), dienestrol (DIE), hexestrol (HEX), diethylstilbestroldipropionate (DESP), and dienestroldiacetate (DIEA). Over a 24-hour period beginning 24 hours after the transition from light to constant dark, maximum phase shifts occurred about 36 hours. DES was the most effective of the drugs tested, giving 10-hour phase advances at 20 micromolar. DIE and HEX caused similar phase shifts as DES at 40 micromolar. The two derivatives of the last, DESP and DIEA, were much less effective in shifting phase; only a few hours of phase advance result from treatments at 80 micromolar concentrations.

The activity of isolated plasma membrane ATPase was inhibited by DES and partially by HEX, but not by DIE, DESP, or DIEA. O₂ consumption of the mycelia was inhibited equally by DES, DIE, and HEX, while DIEA and DESP had little effect. Phase-shifts by DES cannot be interpreted as evidence that plasma membrane ATPase is a component of the circadian clock.

     

Effect of pH and temperature on nitrosamide-induced mutation in Escherichia coli

N-Nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) induced reversions in four mutant auxotropic strains of E. coli. Among other nitroso compounds tested only N-methyl-N′-nitro-N-nitrosoguanidine (MNG) was an active mutagen in the system used.     

Mutagen specificity in the induction of karyotic versus cytoplasmic respiratory deficient mutants in yeast by nitrous acid and alkylating nitrosamides

Summary In the haploid eukaryotic organism Saccharomyces cerevisiae the induction of cytoplasmic and genic (karyotic) RD mutants was studied, using nitrous acid, nitrosomethylurethane (NMU) and nitrosoimidazolidone (NIL).The cytoplasmic or genic origin of the induced RD mutants was determined by prescreening in complementation tests with ^– and wild type tester strains. Among the mutants of all three agents we could thus score the incidence of three RD mutant types: genic, suppressive and cytoplasmic (both primary and secondary). The final identification of the cytoplasmic type was only possible through tetrad analysis, performed in the cases of HNO₂ and NMU.A distinct difference in cytoplasmic versus genic mutagen specificity was observed between HNO₂ and NMU. HNO₂ was unable to induce cytoplasmic RD mutants but it proved to be highly efficient in the induction of genic RD mutants. In contrast, NMU induced more cytoplasmic effects was it possible to detect mutagenic specificities which, solely on the basis of karyotic action, were not detectable.     

Activation of neuromedin U type 1 receptor inhibits L-type Ca2+ channel currents via phosphatidylinositol 3-kinase-dependent protein kinase C epsilon pathway in mouse hippocampal neurons

Neuromedin U (NMU) plays very important roles in the central nervous system. However, to date, any role of NMU in hippocampal neurons and the relevant mechanisms still remain unknown. In the present study, we report that NMU selectively inhibits L-type high-voltage-gated Ca²⁺ channels (HVGCC) in mouse hippocampal neurons, in which NMU type 1 receptor (NMUR1), but not NMUR2, is endogenously expressed. In wild type mice, NMU (0.1 μM) reversibly inhibited HVGCC barium currents (I_Ba) by ～ 28%, while in NMUR1^?/? mice NMU had no significant effects. Intracellular infusion of GDP-β-S or a selective antibody raised against the G_oα, as well as pretreatment of the neurons with pertussis toxin, blocked the inhibitory effects of NMU, indicating the involvement of G_o-protein. This NMUR1-mediated effect did not display the characteristics of a direct interaction between G-protein βγ subunit (G_βγ) and L-type HVGCC, but was abolished by dialyzing cells with QEHA peptide or an antibody to the G_β. The classical and novel protein kinase C (PKC) antagonist calphostin C, as well as phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, abolished NMU responses, whereas the classical PKC antagonist Gö6976 had no such effects. Cells dialyzed with a PKC epsilon isoform (PKCε) specific inhibitor peptide, GAVSLLPT, abolished NMU responses. In contrast, in cells dialyzed with an inactive PKCε control scramble peptide, LSGTLPAV, no significant effects were observed. In summary, these results suggest that NMU inhibits L-type HVGCC via activation of NMUR1 and downstream G_βγ, PI3K, and a novel PKCε signaling pathway.     

Endogenous prostaglandin production by established cultures of neoplastic rat mammary epithelial cells

Summary The production and release of prostaglandins (PGs) into the growth medium by established cultures of neoplastic, mammary epithelial cells derived from (a)N-nitrosomethyl-urea (NMU)-induced and (b) 7,12-dimethylbenz(a) anthracene (DMBA)-induced mammary tumors, was assessed using radioimmunoassay techniques. Prostaglandin production was determined, to a considerable extent, by in vitro conditions and the tumor line analyzed. In medium supplemented with bovine calf serum (10%), NMU cells synthesized and released nanogram quantities of PGF₂, PGE₁, and PGF_2α (6.7, 4.7, and 1.7 ng/10⁶ cells per 48 h, respectively). Concentrations of the two stable protanoid metabolites, 6-keto-PGF_1α and TXB₂, were indistinguishable from controls. In cells derived from the DMBA-induced tumor (RBA cells), no net production of immunoreactive PGs was detected. In contrast, in media supplemented with fetal bovine serum (10%), both RBA and NMU cells synthesized and released nanogram quantities of PGE₂ (1 and 4 ng/10⁶ cells per 48 h, respectively). PGE₂ production by both NMU and RBA cells was inhibited by ibuprofen, an inhibitor of cyclooxygenase (EC 1. 14. 99.1). The pattern of PG inhibition by ibuprofen differed in the two cell lines. In NMU cells, a linear dose-response inhibitory pattern was discernable, whereas in RBA cells a biphasic pattern was observed; PGE₂ levels incresed at low concentration of ibuprofen and then decreased at higher concentration. At 100 μg/ml ibuprofen, PG synthesis and release was inhibited by 90 and 100% and cell growth by 64 and 66% in NMU and RBA cells, respecively. There was no obvinous dse-response relationship between ibuprofen concentration and cell growth inhibition in either cell line. These results underline the importance of the serum component of growth medium when analyzing PG production in vitro and suggest that the epithelial cell components of experimental mammary tumors are capable of producing physiogically relevant amounts of PGs. This work was supported by Grant CA 29602 from the National Cancer Institute, Bethesda, MD, and Grant PDT-208 from the American Cancer Society.     

Increased removal of 3-alkyladenine reduces the frequencies of hprt mutations induced by methyl- and ethylmethanesulfonate in Chinese hamster fibroblast cells.

We have previously reported the isolation of mammalian cell lines expressing the 3-methyladenine DNA glycosylase I (tag) gene from E. coli. These cells are 2-5 fold more resistant to the toxic effects of methylating agents than normal cells (15). Kinetic measurements of 3-methyladenine removal from the genome in situ show a moderate (3-fold) increase in Tag expressing cells relative to normal as compared to a high (50-fold) increase in exogenous alkylated DNA in vitro by cell extracts. Excision of 7-methylguanine is as expected, unaffected by the tag+ gene expression. The frequency of mutations formed in the hypoxanthine phosphoribosyl transferase (hprt) locus was investigated after methylmethanesulfonate (MMS), ethylmethanesulfonate (EMS), N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) exposure. Tag expression reduced the frequency of MMS and EMS induced mutations to about half the normal rate, whereas the mutation frequency in cells exposed to NMU or NEU is not affected by the tag+ gene expression. These results indicate that after exposure to compounds which produce predominantly N-alkylations in DNA, a substantial proportion of the mutations induced is formed at 3-alkyladenine residues in DNA.     

Removal by Yeast Extract of Inhibition by Diethylstilbestrol of Growth and Light-Induced Phase-Shifting in Neurospora crassa

Effects of yeast extract on the inhibitory effects of diethylstilbestrol(DES) were examined in Neurospora crassa The inhibition by DESof mycelial growth and lightinduced phase-shifting of the conidiationrhythm was overcome by the concomitant addition of yeast extractin which the active agents were substances of low molecularweight. However, shifting of the phase of the conidiation rhythmby DES was not affected by addition of the yeast extract. Theseresults suggest that DES exerts an effect at two different sites,at least, in Neurospora: one site being the system which connectsthe perception of light to the clock mechanism and is relatedto mycelial growth; the other site being the system which controlsthe clock mechanism itself. (Received May 27, 1987; Accepted September 4, 1987)     

Novel pH-dependent regulation of human cytosolic sialidase 2 (NEU2) activities by siastatin B and structural prediction of NEU2/siastatin B complex

Human cytosolic sialidase (Neuraminidase 2, NEU2) catalyzes the removal of terminal sialic acid residues from glycoconjugates. The effect of siastatin B, known as a sialidase inhibitor, has not been evaluated toward human NEU2 yet. We studied the regulation of NEU2 activity by siastatin B in vitro and predicted the interaction in silico. Inhibitory and stabilizing effects of siastatin B were analyzed in comparison with DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) toward 4-umbelliferyl N-acetylneuraminic acid (4-MU-NANA)- and α2,3-sialyllactose-degrading activities of recombinant NEU2 produced by E. coli GST-fusion gene expression. Siastatin B exhibited to have higher competitive inhibitory activity toward NEU2 than DANA at pH 4.0. We also revealed the stabilizing effect of siastatin B toward NEU2 activity at acidic pH. Docking model was constructed on the basis of the crystal structure of NEU2/DANA complex (PDB code: 1VCU). Molecular docking predicted that electrostatic neutralization of E111 and E218 residues of the active pocket should not prevent siastatin B from binding at pH 4.0. The imino group (¹NH) of siastatin B can also interact with D46, neutralized at pH 4.0. Siastatin B was suggested to have higher affinity to the active pocket of NEU2 than DANA, although it has no C7–9 fragment corresponding to that of DANA. We demonstrated here the pH-dependent affinity of siastatin B toward NEU2 to exhibit potent inhibitory and stabilizing activities. Molecular interaction between siastatin B and NEU2 will be utilized to develop specific inhibitors and stabilizers (chemical chaperones) not only for NEU2 but also the other human sialidases, including NEU1, NEU3 and NEU4, based on homology modeling.     

Effect of N-nitroso-N-methylurea on viability and mutagenic response of repair-deficient strains of Escherichia coli

Various E. coli mutants, deficient in DNA repair, differed in their response to increasing concentrations of N-nitroso-N-methylurea (NMU).Loss of viability due to exposure to NMU was greatest in those strains with a reduced capacity for repair of single-strand breaks. Viability of wild-type and uvrA^? strains was not affected by NMU concentrations up to 3.0 mM. Some loss of viability occurred, at the higher NMU concentrations, in both strains carrying exrA^? while strains carrying uvrA^?polA^? or recA^? were the most sensitive. The results support the hypothesis that the lethal effect of NMU on repair-deficient E. coli was due to its ability to induce single-strand breaks.Induction of mutations by NMU was observed in all the strains used and the results suggested that NMU damage per se was the major mutational event. The dose response curve for induction of revertants by NMU was, however, influenced by the repair system(s) present. The number of revertants scored at the higher NMU concentrations was greater in those strains lacking the recA and polA dependent repair functions than in the wild-type strain. However, at NMU concentrations below 2.0 mM the numbers of revertants induced in exrA^? carrying strains, prossessing accurate rec-dependent repair, were lower than the comparable wild-type values. The evidence suggests that the uvrA gene product also acts on some, possibly non-mutagenic, types of NMU damage and that error-prone repair of these lesions increases the number of potential revertants.     

Development of a neuromedin U–human serum albumin conjugate as a long‐acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide

Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half‐life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half‐life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA–NMU displayed long‐lasting, potent anorectic, and glucose‐normalizing activity. When compared side by side with a previously described PEG conjugate, HSA–NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU‐based therapeutics are promising candidates for the treatment of obesity and diabetes. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Diethylstilbestrol-Induced Estrogen Receptor-Dependent [Ca2+]i Rises and Apoptosis in Chinese Hamster Ovary (CHO) Cells

The effect of the synthetic estrogen diethylstilbestrol (DES) on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and cell viability was explored in Chinese hamster ovary (CHO-K1). [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. DES at concentrations ≥ 1∝ increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. In Ca²⁺-free medium, after pretreatment with 50∝ DES, 1∝ thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)-induced [Ca²⁺]_i rises were abolished. Conversely, thapsigargin pretreatment abolished DES-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not alter DES-induced [Ca²⁺]_i rises. At a concentration of 5∝, DES increased cell viability. At concentrations of 100–200 μ M, DES decreased viability in a concentration-dependent manner. The effect of 5 and 100 μM DES on viability was partly reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′ -tetraacetic acid (BAPTA). DES-induced cell death was induced via apoptosis as demonstrated by propidium iodide staining. DES (100 μ M)-induced [Ca²⁺]_i rises were largely inhibited by pretreatment with the estrogen receptor antagonist ICI-182,780 (100 μ M). ICI-182,780 did not affect 5 μ M DES-induced increase in viability but partly reversed 100 μ M DES-induced cell death. Collectively, in CHO-K1 cells, DES induced [Ca²⁺]_i rises by stimulating estrogen receptors leading to Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx. DES-caused cytotoxicity was mediated by an estrogen receptor- and Ca²⁺-dependent pathway.     

Effects of supplementation with corn distillers’ dried grains on animal performance,nitrogen balance,and enteric CH4 emissions of young Nellore bulls fed a high-tropical forage diet

The inclusion of corn-dried distillers’ grains (DDG) could be an alternative supplement to increase animal performance, nitrogen efficiency usage (NEU), and decrease enteric methane (CH₄) emissions. Our goal was to determine whether DDG could replace a traditional supplement (cottonseed meal) without affecting animal performance, N balance, and CH₄ emissions. The experiment was conducted during the forage growing season (December to April), with 15 d adaptation, and a 112 d experimental period. The experimental design was completely randomized with four treatments: a mineral supplement (MS), cottonseed meal supplement (CS), 50% replacement of CS by DDG (50DDG), and 100% replacement of CS by DDG (100DDG). Cottonseed meal and DDG were used as protein supplement. A total of 12 paddocks, 3 per treatment, were used to measure forage mass: morphological and chemical composition of forage, forage allowance, and animal performance. Six animals per treatment were used to evaluate DM intake, digestibility, CH₄ emissions, microbial protein production (MCP), and NEU of each treatment. Eighty-one Young Nellore bulls (48 testers, 12 per treatments and 33 adjusters) with initial BW of 255 ± 5 kg (10–12 months old) were supplemented with each supplement type at a level of 0.3% of BW. Pasture management was continuous stocking with a variable stocking rate (put-and-take). Enteric CH₄ was measured using the gas tracer technique. The MCP was quantified using purine derivatives and the NEU mass balance. No differences were found in nutrient intake (P > 0.228). Individual animal performance and gain per area were higher in the treatments with concentrates compared with that of MS; however, there was no difference among treatments CS, 50DDG, and 100DDG. The ADG was 0.83 for MS and 1.08 kg/animal/d when supplemented (P < 0.05). Gain per hectare was 709 kg/ha for MS and 915 kg/ha when supplemented with concentrates (P < 0.05). There was no difference in CH₄ production among treatments that average 180 g/animal/d; however, CH₄ per kg of gain was reduced with CS. The CH₄ conversion factor averaged 5.91%. There was no difference in the synthesis of MCP and NEU. Corn DDG can replace 100% of cottonseed meal as a protein source for supplementation of young Nellore bulls grazing in tropical pastures without affecting animal performance, NEU, MCP, and CH₄ emissions.     

Quasi-Adaptive Response to Alkylating Agents in <Emphasis Type="Italic">Escherichia coli</Emphasis>: A New Phenomenon

An original hypothesis of a quasi-adaptive response to nitrosomethylurea (NMU) in Escherichia coli cells was verified experimentally. In contrast to the true Ada response, which is induced in cells pretreated with a sublethal dose of NMU, a quasi-adaptive response was induced using NO-containing dinitrosyl iron complex with glutathione (DNIC_glu). Quasi-adaptation increased expression of the Ada regulon and cell resistance to the cytotoxic and mutagenic effects of NMU. The levels of alkA, alkB, and aidB gene expression in quasi-adaptation were higher than in the true Ada response. Thus, experimental evidence was obtained for the alternative mechanism regulating the function of the Ada sensory protein in controlling expression of the Ada regulon during the adaptive response. The free iron—chelating agent o-phenanthroline (OP) facilitated degradation of DNIC_glu (by electron paramagnetic resonance (EPR) spectra) and considerably or completely inhibited gene expression in the quasi-adaptive response. The new phenomenon extends the functional range of NO compounds to include a role in genetic signal transduction within the Ada response system in addition to similar roles in the SoxRS, SOS, and OxyR systems in E. coli.__________Translated from Genetika, Vol. 41, No. 5, 2005, pp. 607–613.Original Russian Text Copyright © 2005 by Vasilieva, Moschkovskaya.     

NEU1 Sialidase Regulates the Sialylation State of CD31 and Disrupts CD31-driven Capillary-like Tube Formation in Human Lung Microvascular Endothelia

The highly sialylated vascular endothelial surface undergoes changes in sialylation upon adopting the migratory/angiogenic phenotype. We recently established endothelial cell (EC) expression of NEU1 sialidase (Cross, A. S., Hyun, S. W., Miranda-Ribera, A., Feng, C., Liu, A., Nguyen, C., Zhang, L., Luzina, I. G., Atamas, S. P., Twaddell, W. S., Guang, W., Lillehoj, E. P., Puché, A. C., Huang, W., Wang, L. X., Passaniti, A., and Goldblum, S. E. (2012) NEU1 and NEU3 sialidase activity expressed in human lung microvascular endothelia. NEU1 restrains endothelial cell migration whereas NEU3 does not. J. Biol. Chem. 287, 15966–15980). We asked whether NEU1 might regulate EC capillary-like tube formation on a Matrigel substrate. In human pulmonary microvascular ECs (HPMECs), prior silencing of NEU1 did not alter tube formation. Infection of HPMECs with increasing multiplicities of infection of an adenovirus encoding for catalytically active WT NEU1 dose-dependently impaired tube formation, whereas overexpression of either a catalytically dead NEU1 mutant, NEU1-G68V, or another human sialidase, NEU3, did not. NEU1 overexpression also diminished EC adhesion to the Matrigel substrate and restrained EC migration in a wounding assay. In HPMECs, the adhesion molecule, CD31, also known as platelet endothelial cell adhesion molecule-1, was sialylated via α2,6-linkages, as shown by Sambucus nigra agglutinin lectin blotting. NEU1 overexpression increased CD31 binding to Arachis hypogaea or peanut agglutinin lectin, indicating CD31 desialylation. In the postconfluent state, when CD31 ectodomains are homophilically engaged, NEU1 was recruited to and desialylated CD31. In postconfluent ECs, CD31 was desialylated compared with subconfluent cells, and prior NEU1 silencing completely protected against CD31 desialylation. Prior CD31 silencing and the use of CD31-null ECs each abrogated the NEU1 inhibitory effect on EC tube formation. Sialyltransferase 6 GAL-I overexpression increased α2,6-linked CD31 sialylation and dose-dependently counteracted NEU1-mediated inhibition of EC tube formation. These combined data indicate that catalytically active NEU1 inhibits in vitro angiogenesis through desialylation of its substrate, CD31.     

Synthesis and evaluation of novel lipidated neuromedin U analogs with increased stability and effects on food intake

Neuromedin U (NMU) is a 25 amino acid peptide expressed and secreted in the brain and gastrointestinal tract. Data have shown that peripheral administration of human NMU decreases food intake and body weight and improves glucose tolerance in mice, suggesting that NMU receptors constitute a possible anti‐diabetic and anti‐obesity drug target. However, the clinical use of native NMU is hampered by a poor pharmacokinetic profile. In the current study, we report in vitro and in vivo data from a series of novel lipidated NMU analogs. In vitro plasma stability studies of native NMU were performed to investigate the proteolytic stability and cleavage sites using LC–MS. Native NMU was found to be rapidly cleaved at the C‐terminus between Arg²⁴ and Asn²⁵, followed by cleavage between Arg¹⁶ and Gly¹⁷. Lipidated NMU analogs were generated using solid‐phase peptide synthesis, and in vitro potency was investigated using a human embryonic kidney 293‐based inositol phosphate accumulation assay. All lipidated analogs had preserved in vitro activity on both NMU receptors with potency improving as the lipidation site was moved away from the receptor‐interacting C‐terminal octapeptide segment. In vivo efficacy was assessed in lean mice as reduction in food intake after acute subcutaneous administration of 1, 0.3, 0.1, and 0.03 µmol/kg. These lipidated NMU analogs prolonged the anorectic effect of NMU in a dose‐dependent manner. This was likely an effect of improved pharmacokinetic properties because of improved vitro plasma stability. Accordingly, the data demonstrate that lipidated NMU analogs may represent drug candidates for the treatment of obesity. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.