Novel potent antagonists of human neuropeptide Y Y5 receptor. Part 1: 2-oxobenzothiazolin-3-acetic acid derivatives

Novel NPY-Y5 antagonist FR73966 was discovered by screening of our in-house chemical library. The analogues were prepared by application of parallel synthesis techniques. Some of the resulting 2-oxobenzothiazolin-3-acetic acid derivatives exhibited nanomolar binding affinity for human NPY-Y5 receptors.     

Novel potent antagonists of human neuropeptide Y Y5 receptors. Part 2: substituted benzo[a]cycloheptene derivatives

Novel benzo[a]cycloheptene derivatives were prepared for the purpose of searching new neuropeptide Y-Y5 (NPY-Y5) receptor antagonists. The structure-activity relationships are described and compound 2o (FR226928) showed the most potent affinity for Y5 receptor of all we prepared and was found to have higher potency and better selectivity for Y5 over Y1 receptor affinities when compared with the known lead compound 1.     

Novel potent antagonists of human neuropeptide Y Y5 receptors. Part 3: 7-methoxy-1-hydroxy-1-substituted tetraline derivatives

As a part of our continuing research on NPY-Y5 receptor antagonists in the series of novel 6-methoxybenzo[a]cycloheptene derivatives, we discovered a novel skeleton, 7-methoxy-1-hydroxytetraline 7 which had been used as an intermediate, to be more suitable for increasing potencies leading to compound 3 (FR230481). Additionally, we discovered that the naphthalenesulfonamide moiety which was thought to be an essential pharmacophore could be replaced by the 5-chlorobenzothiazolin-3-acetic acid moiety to lead to potent compound 4 (FR233118). The structure-activity relationships on compounds 3,4 and their related derivatives are described. Unfortunately, although compounds 3 and 4 had very high affinities for Y5 receptors, their poor permeabilities to brain were shown by exo-vivo binding assays when orally administered.     

Novel cell line selectively expressing neuropeptide Y-Y2 receptors

Neuropeptide Y (NPY) recognition by the human neuroblastoma cell lines SiMa, Kelly, SH-SY5Y, CHP-234, and MHH-NB-11 was analyzed in radioactive binding assays using tritiated NPY. For the cell lines CHP-234 and MHH-NB-11 binding of [3H]propionyl-NPY was observed with Kd-values of 0.64 +/- 0.07 nM and 0.53 +/- 0.12 nM, respectively, determined by saturation analysis with non-linear regression. The receptor subtype was determined by competition analysis using the subtype selective NPY analogues [Leu31, Pro34]-NPY (NPY-Y1, NPY-Y5), [Ahx(5-24)]-NPY (NPY-Y2), [Ala31, Aib32]-NPY (NPY-Y5), NPY [3-36] (NPY-Y2, NPY-Y5), and NPY [13-36] (NPY-Y2). Both cell lines, CHP-234 and MHH-NB-11, the latter one being characterized for NPY receptors for the first time, showed exclusive expression of NPY-Y2 receptors. In both cell lines binding of NPY induced signal transduction, which was monitored as reduction of forskolin-induced cAMP production in an ELISA.     

PYY(3-36) into the arcuate nucleus inhibits food deprivation-induced increases in food hoarding and intake

Central administration of neuropeptide Y (NPY) increases food intake in laboratory rats and mice, as well as food foraging and hoarding in Siberian hamsters. The NPY-Y1 and Y5 receptors (Rs) within the hypothalamus appear sufficient to account for these increases in ingestive behaviors. Stimulation of NPY-Y2Rs in the Arcuate nucleus (Arc) has an anorexigenic effect as shown by central or peripheral administration of its natural ligand peptide YY (3-36) and pharmacological NPY-Y2R antagonism by BIIE0246 increases food intake. Both effects on food intake by NPY-Y2R agonism and antagonism are relatively short-lived lasting ∼4 h. The role of NPY-Y2Rs in appetitive ingestive behaviors (food foraging/hoarding) is untested, however. Therefore, Siberians hamsters, a natural food hoarder, were housed in a semi-natural burrow/foraging system that had (a) foraging requirement (10 revolutions/pellet), no free food (true foraging group), (b) no running wheel access, free food (general malaise control) or (c) running wheel access, free food (exercise control). We microinjected BIIE0246 (antagonist) and PYY_(3-36) (agonist) into the Arc to test the role of NPY-Y2Rs there on ingestive behaviors. Food foraging, hoarding, and intake were not affected by Arc BIIE0246 microinjection in fed hamsters 1, 2, 4, and 24 h post injection. Stimulation of NPY-Y2Rs by PYY_(3-36) inhibited food intake at 0–1 and 1–2 h and food hoarding at 1–2 h without causing general malaise or affecting foraging. Collectively, these results implicate a sufficiency, but not necessity, of the Arc NPY-Y2R in the inhibition of food intake and food hoarding by Siberian hamsters.     

神经肽受体Y5亚型在大鼠胃的表达

应用RT—PCR、Western印迹和免疫组化技术研究神经肽受体亚型Y5在正常大鼠和胃轻瘫大鼠胃的表达,并进行组织学定位。发现Y5受体在大鼠胃存在着表达,主要表达定位在胃窦的黏膜层,可能与胃动力的调控有关。半定量分析显示胃轻瘫大鼠较正常大鼠表达水平下调。     

Design, synthesis and SAR of a series of 2-substituted 4-amino-quinazoline neuropeptide Y Y5 receptor antagonists

The design of a novel series of NPY-Y5 receptor antagonists is described. Key elements for the design were the identification of weak Y5 hits from a Y1 program, results from a combinatorial approach and database mining. This led to the discovery of the quinazoline 4 and the aryl-sulphonamide moiety as major components of the pharmacophore for Y5 affinity. The synthesis and SAR towards CGP71683A is described.     

Novel dihydropyrazine analogues as NPY antagonists

The dihydropyridine is currently one of the lead compounds in the neuropeptide-Y(1) (NPY-Y(1)) receptor antagonist program. Compound is a selective, high affinity ligand at the NPY-Y(1) receptors (IC(50)=4.2 nM) in SK-N-MC cells. To further expand the SAR study surrounding this dihydropyridine core structure we succeeded in synthesizing an analogous series of dihydropyrazine derivatives. This structural modification yielded compounds substantially different from the parent molecules in terms of molecular polarization and electron distribution while the overall molecular structure was generally preserved. This altered property should therefore provide us with additional SAR information on the optimal binding requirement with NPY receptors.     

Interstrand cross-linking of DNA by FK317 and its deacetylated metabolites FR70496 and FR157471

FR900482 (1) and FR66979 (2) are structurally novel natural products isolated by Fujisawa in 1987 and have been shown to be highly potent antitumor antibiotics structurally related to the mitomycins. Studies on the mode of action have established that these new agents form covalent DNA interstrand cross-links both in vitro and in vivo as a result of the reactive mitosene intermediate generated upon bioreductive activation. Semisynthetic analogues such as FK973 (3) and FK317 (4) were developed in the search for potentially superior clinical candidates. Although FK317 has been shown to be a potent compound, to date no direct evidence of DNA interstrand cross-link sequence specificity has been reported. In this study, DNA interstrand cross-links were generated by treatment of a synthetic duplex DNA substrate with FK317 (4) and its deacetylated metabolites FR70496 (5) and FR157471 (6). Analysis by gel electrophoresis revealed the formation of orientation isomers displaying electrophoretic mobility vastly greater than the mobilities of those generated from FR900482 (1). Despite these differences, it was established by Fe(II)-EDTA footprinting that FK317 (4) as well as 5 and 6 forms DNA interstrand cross-links within the expected 5'CpG3' step, clearly demonstrating that the phenolic hydrogen in 1 and 2 is not a prerequisite for efficient DNA interstrand cross-linking by the FR class of compounds.     

Physical and physiological demands of field and assistant soccer referees during America's cup

The aim of this study was to examine the physical and physiological demands of top-class soccer field (FR; n = 7) and assistant (AR; n = 7) referees during America's Cup. The FR and AR were equipped with a global positioning system (GPS) and a heart rate (HR) monitor for the recording of displacements and cardiovascular stress, respectively, every 5 and 15 minutes, halves, and entire match. High-intensity accelerations (ACs) and a new index of performance efficiency (Effindex) were also examined during each match. The FR completed longer distances for all walking and running activities and exhibited a greater match HR compared with the AR. The FR exhibited a significant reduction in the mean number of ACs during the second half (40 vs. 28), with a significantly worse Effindex at the end of both halves for FR and at the end of the second half for AR. A significant correlation between FR and AR for the number (r = 0.336; p = 0.03) and distance (r = 0.367; p = 0.017) of ACs performed each 5-minute period indicated a synchronization of officials' activities. In conclusion, top-class American FR and AR exhibited similar physical demands as previously reported by their European counterparts during international tournaments. The high number of AC observed, the effect of fatigue on these actions over the match, and the AC's synchronization between FR and AR suggest that ACs should be considered for the preparation of referees and team sport athletes. The Effindex may provide an important tool for the evaluation of referees' and players' performance that requires further investigation.     

The effects of food restriction and hypophysectomy on numbers of primordial follicles and concentrations of hormones in rats

Three experiments were done to examine the effects of food restriction, beginning at 21 days of age, on loss of primordial follicles and on concentrations of gonadotropins and sex steroids in rats. In Experiment 1, food restriction (FR) from 21 to 51-55 days of age had no effect on number of primordial follicles, but increased the plasma concentration (p less than 0.05) of follicle stimulating hormone (FSH). (p less than 0.05). In Experiment 2, comparisons were made of groups of rats (1) fed ad libitum (AL) (2) hypophysectomized at 21 days of age and fed ad libitum (AL-HY), (3) food restriction from 21 to 52-58 days of age (FR), and (4) food restriction with twice-daily injections of follicular fluid (FR-FF). Hypophysectomy was the only treatment that decreased the loss of primordial follicles (p less than 0.001). Concentrations of FSH were decreased in AL-HY and increased in FR and FR-FF rats (144 +/- 13, 53 +/- 15, 275 +/- 30 and 359 +/- 56 ng/ml in AL, AL-HY, FR and FR-FF rats, respectively). Concentrations of luteinizing hormone (LH) were lower (p less than 0.05) in AL-HY, FR and FR-FF rats than in AL rats. In Experiment 3, AL and FR rats were unilaterally ovariectomized (ULO) at 30 days of age. Blood samples were taken 5 days prior to ULO, at ULO and at 12 h, 5 days, and 22-28 days after ULO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fructose feeding in the suckling-weaning transition in rats: effects on hyperlipidemia in adulthood

The sucking-weaning transition is characterized by high rates of growth and development and may be a sensitive period during which dietary intake could program metabolism to increase the risk of cardiovascular disease and diabetes in adulthood. Intake of a high fructose (FR) diet is known to induce hypertriglyceridemia and insulin resistance in rats when they are consuming this diet. We examined whether a FR diet fed early in life produces detrimental changes in lipid and glucose metabolism that persist to adulthood. Weanling rats were fed 65% FR (wt/wt), a purified control diet (CNTL) or standard chow (CHOW) for 5 weeks. Beyond 9 weeks of age, all rats were fed CHOW. During FR feeding, plasma triglycerides (TG) were significantly elevated in the FR group (FR = 217 +/- 20; CNTL = 163 +/- 17; chow = 156 +/- 10). At 21 wks of age, TG's were similar in rats fed FR or CNTL versus CHOW at weaning (p > 0.87). Hepatic fatty acid synthase (FAS) activity was elevated in FR and CNTL groups vs. CHOW (65 +/- 7, 72 +/- 6 vs. 48 +/- 4 nmol NADPH/mg protein/min, p < 0.01). There were no differences in indices of glucose homeostasis at 21 weeks of age. Early exposure to a diet high in simple sugars (FR or CNTL) and/or low in fiber during the suckling-weaning transition may contribute to modest dyslipidemia later in life. Together, changes observed in this study may increase the risk of cardiovascular disease in adulthood.     

Cell Cycle Responses to Red or Far-red Light, or Darkness, in the Shoot Apex of Silene coeli-rosa L. During Floral Induction

Twenty-eight-day-old plants of Silene coeli-rosa L. were maintainedin short days (SD) for 9 d (0–8) or exposed to 7 longdays (LD), or 7 SD with a 5 min exposure at 1700 h of each dayto far-red (FR), red (R) or 5 min FR/5 min R, or 7 dark-interrupted(di = 1700–1720 h) LD. Treatments were followed by twofurther SD. The mitotic index and G1 and G2 proportions weremeasured in the shoot apices of plants sampled at 2000 h ofeach day of each replicated treatment. Exposure to 7 LD (= 100per cent flowering) resulted in significant increases, relativeto the SD controls, in both the G2 proportion and the mitoticindex on d 0 to 3, 7 and 8. Five minute FR (= 0 per cent flowering)resulted in cell cycle responses similar to those in LD onlyfrom d 0 to 2. R and FR/R (both = 0 per cent flowering) didnot result in any increases in the G2 proportion in the apexapart from d 3 of FR/R. However 5 min FR/5 min R, and to a lesserextent 5 min R, did result in significant increases in the mitoticindex on d 0, 1, 7, and 8. diLD (= 8–10 per cent flowering)also prevented any significant increases in the G2 proportionon d 0 to 3, and 5 to 8 but the mitotic index was again higheron these days compared with control data. Thus the transitionto floral growth for 90 per cent of the plants is associatedwith changes in the cell cycle in the shoot apex measured asincreases in the G2 proportion at 2000 h of LD 0 to 3 and 7to 8. Silene coeli-rosa L., cell cycle, flowering, phytochrome, shoot apex     

Monoalkylation of DNA by reductively activated FR66979

The antitumor antibiotic FR66979 has previously been shown to form interstrand cross-links in duplex DNA at the sequence [5'-d(CG)]2, linking the exocyclic amino groups (N2) of deoxyguanosine (dG) residues. During the reaction of reductively activated FR66979 with DNA. products are formed which have electrophoretic mobility in denaturing polyacrylamide gels which is intermediate between that of unmodified and interstrand cross-linked DNA. We show here that these products are monoadducts between FR66979 and DNA and provide strong evidence for the site of alkylation being N2 of dG. Moreover, the sequence selectivity of monoalkylation reactions between FR66979 and DNA containing either 5'-d(CG).5'-d(CI) or [5'-d(CG)]2 was observed to be ca. 5-fold less than for the related antitumor antibiotic mitomycin C (MC). The mechanistic implications of this result are discussed. Furthermore, it was demonstrated that contrary to a previous report, FR66979 requires DNA to be in duplex form for efficient monoadduct formation.     

Laparoscopic insemination technique with low numbers of spermatozoa in superovulated prepuberal gilts for biotechnological application

New biotechnologies, such as sperm-mediated gene transfer (SMGT), spermatozoa freezing and spermatozoa sorting have improved the possibilities to produce animals with desirable features. The main problem associated with these technologies is the scarce availability of spermatozoa for insemination. The objective of this study was to develop a laparoscopic insemination (LI) technique in gilt that allows the use of low semen doses resulting in high fertilization rates (FR) and minimal distress to the animal; the efficiency of this technique was compared to conventional artificial insemination (AI). Ten gilts were inseminated 36 h post hCG treatment near both utero-tubal junctions (UTJ) with 1.5 x 10(9)spermatozoa/5 mL per horn and 10 gilts (C) underwent conventional AI. Embryos were collected either at two to four cell stage (LI, n = 5; C, n = 5) for determination of fertilization rate or at day 6 for evaluation of developmental competence (LI, n = 5; C, n = 5). LI gilts showed a slightly higher FR than control animals. In a second trial, 24 gilts underwent LI with varying doses (1.5 x 10(8), 1.5 x 10(7), 1 x 10(7), 5 x 10(6) or 1 x 10(6)) of semen. Two to four stage embryos were collected and FR was evaluated in each tube. FR obtained with the lowest dose was significantly different from that with other dosages (P < 0.05). Embryos were cultured in vitro to blastocyst stages (percentage of blastocysts: 79.2 +/- 3.6%). In a third trial, five gilts were inseminated with semen processed by SMGT technique; both FR (86.1 +/- 9.9%) and transgene protein expression were satisfactory. In conclusion, this study shows that LI can be a useful tool for reducing doses of insemination, without affecting the efficiency of fertilization; this technique could have a wide range of biotechnological applications.     

FIN5 positively regulates far-red light responses in Arabidopsis thaliana

We report the characterization of a semi-dominant mutation fin5-1 (far-red insensitive 5-1) of Arabidopsis, which was isolated from genetic screening of phytochrome A (phyA) signaling components. Plants with the fin5-1 mutation exhibited a long hypocotyl phenotype when grown under far-red (FR) light, but not under red light. Physiological analyses implied that FIN5 might be differentially involved in diverse responses that are regulated by phyA under continuous FR light. Anthocyanin accumulation, gravitropic response of hypocotyl growth, and FR light-preconditioned blocking of greening were also impaired in the fin5-1 mutant, whereas photoperiodic floral induction was not, if at all, significantly affected. Moreover, light-regulated expression of the CHS, PORA and PsbS genes was attenuated in fin5-1 mutant plants, while the light-induced expression of CAB was normal. The mutation exhibited semi-dominance regarding control of hypocotyl growth in FR light. We suggest that FIN5 defines a novel branch in the network of phyA signaling in Arabidopsis.     

海马NMDA受体与神经肽Y在慢性应激性抑郁发生中的作用及其关系

本文旨在探讨N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid,NMDA)受体与神经肽Y(neuropeptide Y,NPY)在慢性应激抑郁发生中的作用与关系。建立慢性不可预见性温和应激(chronic unpredictable mild stress,CUMS)抑郁模型,海马单侧分别微量注射非竞争性NMDA受体拮抗剂MK-801、NPY-Y1受体阻断剂GR231118和NMDA后,利用体重测量及糖水偏爱测试、强迫游泳及敞箱实验等方法观察动物行为变化,运用免疫组织化学方法检测海马CA3区和齿状回(dentate gyrus,DG)内NPY的表达。结果显示,CUMS组大鼠表现出抑郁样行为变化,海马NPY表达显著降低;海马微量注射NMDA或NPY-Y1受体阻断剂GR231118,动物行为学表现均与CUMS组相同,注射NMDA可使NPY表达显著降低;海马微量注射MK-801能明显改善应激引起的抑郁样行为表现,并使海马NPY表达增加。联合注射GR231118与MK-801后,GR231118可以显著减弱MK-801的抗抑郁样行为的效应。以上结果表明,CUMS可能使谷氨酸(glutamic acid,Glu)过量释放,NMDA受体过度激活,抑制NPY表达,导致抑郁发生。NPY抗抑郁作用主要是通过NPY-Y1受体实现。     

Interaction of gibberellins and phytochrome in the control of cowpea epicotyl elongation

The physiological response of cowpea ( Vigna sinensis L.) epicotyl explants to far‐red light (FR) and its interaction with gibberellins (GAs) have been investigated. The effect of FR and GA₁ varied with the age of the seedlings from which the explants were made: for FR, it decreased progressively with age (though the sensitivity of the epicotyls to FR did not change significantly until at least day 11), whereas it remained essentially constant for applied GA₁ between days 5 and 9 after sowing. This indicates that the loss of response to FR may be due to a decrease in endogenous GA levels in the epicotyl. For a range of GA₁ and GA₂₀ (0.01–1 µg explant⁻¹), both hormones were more active in FR than in R irradiated epicotyls, suggesting that phytochrome may affect GA sensitivity besides GA metabolism. The location of the epicotyl region most sensitive to FR (between 5 and 20 mm below the apex) was different from that to GAs (the upper 10 mm). Nevertheless, FR extended the region responsive to applied GAs, even in paclobutrazol‐treated epicotyls where elongation was due entirely to exogenous GAs. This means that modulation of epicotyl elongation by phytochrome, that occurs in a zone different from though overlapping with the GA‐sensitive subapical zone, is also mediated by GAs. Growth in the most FR‐sensitive region of the epicotyl stimulated by FR or GA₁ was due to cell elongation, and in the most GA‐sensitive region to both cell division and elongation. The effect of FR and GA₁ was negated by colchicine, indicating that microtubules may be involved in the response to both factors.     

Influence on adipocyte plasma membrane bound protein kinase by feedback regulator.

Protein kinase, phosphodiesterase and adenylate cyclase of plasma membrane of adipocytes and the effect of the feedback regulator (FR) on these three enzymes was measured and compared. The basal level ratio of adenylate cyclase to phosphodiesterase to protein kinase was 1:1.9:3.0. Epinephrine and/or FR alters this ratio. FR stimulated protein kinase activity up to 3 fold in the presence of a wide range of enzyme concentrations, 5-50 mug membrane protein/tube. The concentration of FR effective for stimulation of membrane protein kinase was much greater than that needed for inhibition of adenylate cyclase and phosphodiesterases. The inhibition by FR on adenylate cyclase was the most potent effect among the 3 enzymes. 1 U (or 2 U/ml) of FR inhibited 50% of the adenylate cyclase activity in a defined system. The maximum effective concentration of FR for stimulation of membrane protein kinase was greater than 10 U/ml. Histone type 11A was the best substrate for protein phosphorylation so far observed. The FR stimulatory effect was observed at all substrate concentrations used ranging from 1-5 mg/ml. A NaF concentration curve shows that 15 mM NaF gave maximum phosphorylation. The stimulatory effect of FR was observed both in the presence and absence of NaF. Protein kinase of adipocyte plasma membrane was mainly cAMP-independent. The effect of FR (20 U/ml) in stimulation of protein phosphorylation was much greater than that of cAMP (1 X 10(-6) M). The cAMP and FR effects seemed to be additive. Preincubation of plasma membrane with FR in the absence of ATP resulted in no decrease but slight increase in protein kinase activity. A shift in protein kinase, phosphodiesterase and adenylate cyclase ratios by FR suggests the regulatory role of FR in cAMP metabolism in adipocytes.     

Stimulation of neurons in rat ARC inhibits gastric acid secretion via hypothalamic CRF1/2- and NPY-Y1 receptors

Neuropeptide Y (NPY) neuronal projections from the arcuate nucleus (ARC) have been proposed to target corticotropin-releasing factor (CRF)-positive neurons in the paraventricular nucleus (PVN) as part of the ARC-PVN axis. The existence of a positive feedback loop involving CRF receptors in the PVN has been suggested. Exogenous NPY and CRF in the PVN have been shown to inhibit gastric acid secretion. Recently, we have demonstrated that activation of ARC neurons inhibits gastric acid secretion via vagal pathways. To what extent NPY- and CRF-mediated mechanisms in the PVN contribute to the CNS modulation of gastric acid secretion is still an open question. In the present study, we performed consecutive bilateral microinjections of antagonists to NPY receptor subtypes Y1 and Y2 and to CRF1/2 receptors in the PVN and of the excitatory amino acid kainate in the ARC to assess the role of NPY- and CRF-mediated mechanisms in the kainate-induced effects on gastric acid secretion. Gastric acid secretion was measured at the basal condition and during pentagastrin (16 microg/kg body wt) stimulation. Microinjection of vehicle in the PVN and kainate in the ARC decreased gastric acid secretion. Microinjection of the specific NPY-Y1 receptor antagonist BIBP-3226 (200 pmol) and the nonspecific CRF1/2 antagonist astressin (30 pmol) in the PVN abolished the inhibitory effect of neuronal activation in the ARC by kainate on gastric acid secretion. The CRF antagonist astressin was more effective. Pretreatment with the NPY-Y2 receptor antagonist BIIE-0246 (120 pmol) in the PVN had no significant effect. Our results indicate that activation of neurons in the ARC inhibits gastric acid secretion via CRF1/2 and NPY-Y1 receptor-mediated pathways in the PVN.