We report the findings of the analysis of 75 different nutritional supplements bought through the internet. Seven products (all from the class of prohormones) contained other hormone substances than ...indicated on the labels, and two further products contained ephedrine and caffeine without a clear indication on the labels.
We previously reported the existence of a descending multisynaptic, pituitary-independent, neural pathway between the hypothalamus and the testes in the male rat. Stimulation of this pathway by the ...intracerebroventricular (icv) injection of IL-1β or corticotropin-releasing factor blunts the testosterone (T) response to human chorionic gonadotropin (hCG). This response is mediated at least in part by catecholamine β-adrenergic receptor activation. The present work was performed to further investigate the role of brain catecholamines and testicular blood flow in this pathway. The icv injection of 5 μl of 200 proof ethanol (EtOH; 86 μmol) did not result in detectable levels of the drug in the general circulation and did not induce neuronal damage, but rapidly blunted hCG-induced T release while not decreasing LH levels or altering testicular blood flow. EtOH significantly up-regulated transcripts of the immediate-early gene c-fos in the paraventricular nucleus (PVN) of the hypothalamus. Lesions of the PVN blocked the inhibitory effect of IL-1β on T, but only partially interfered with the influence of EtOH. PVN catecholamine turnover significantly increased after icv injection of IL-1β, but not EtOH. Brain catecholamine depletion due to the neurotoxin 6-hydroxydopamine did not alter the ability of hCG to induce T release, but significantly reversed the inhibitory effect of icv EtOH or IL-1β on this response. Collectively, these results indicate that icv-injected IL-1β or EtOH blunts hCG-induced T secretion through a catecholamine-mediated mechanism that does not depend on either peripherally mediated effects or pituitary LH, and that the PVN plays a role in these effects.
Careful analysis of the NMR structures of cyclo(4−10)Ac-Δ3Pro,dFpa,dTrp,Asp,dNal6,Dpr10GnRH, dicyclo(4−10/5−8)Ac-dNal1,dCpa2,dTrp3, Asp4,Glu,dArg,Lys8,Dpr10GnRH, and ...dicyclo(4−10/5,5‘−8)Ac-dNal1,dCpa2,dPal3,Asp4, Glu5(Gly),dArg6,Dbu8,Dpr10GnRH showed that, in the N-terminal tripeptide, a type II β-turn around residues 1 and 2 was probable along with a γ-turn around dTrp3/dPal3. This suggested the possibility of constraining the N-terminus by the introduction of a cyclo(1−3) scaffold. Optimization of ring size and composition led to the discovery of cyclo(1−3)Ac-dAsp1,dCpa2,dLys3,dNal6,dAla10GnRH (5, K i = 0.82 nM), cyclo(1,1‘−3)Ac-dAsp1(Gly),dCpa2,dOrn3,dNal6,dAla10GnRH (13, K i = 0.34 nM), cyclo(1,1‘−3)Ac-dAsp1(Gly),dCpa2,dLys3,dNal6,dAla10GnRH (20, K i = 0.14 nM), and cyclo(1,1‘−3)Ac-dAsp1(βAla), dCpa2,dOrn3,dNal6,dAla10GnRH (21, K i = 0.17 nM), which inhibited ovulation significantly at doses equal to or lower than 25 μg/rat. These results were particularly unexpected in view of the critical role(s) originally ascribed to the side chains of residues 1 and 3. Other closely related analogues, such as those where the dAsp1(βAla), dOrn3 cycle of 21 was changed to dOrn1(βAla), dAsp3 of cyclo(1,1‘−3)Ac-dOrn1(βAla),dCpa2,dAsp3,dNal6,dAla10GnRH (22, K i = 2.2 nM) or where the size of the cycle was conserved and dAsp1(βAla), dOrn3 was replaced by dGlu1(Gly), dOrn3 as in cyclo(1,1‘−3)Ac-dGlu1(Gly),dCpa2,dOrn3,dNal6,dAla10GnRH (23, K i = 4.2 nM), were approximately 100 and 25 times less potent in vivo, respectively. Analogues with ring sizes of 18 {cyclo(1,1‘−3)Ac-dGlu1(Gly),dCpa2,dLys3,dNal6,dAla10GnRH (24)} and 19 {cyclo(1,1‘−3)Ac-dGlu1(βAla),dCpa2,dLys3,dNal6,dAla10GnRH (25)} atoms were also less potent than 21 with slightly higher K i values (1.5 and 2.2 nM, respectively). These results suggested that the N-terminal tripeptide was likely to assume a folded conformation favoring the close proximity of the side chains of residues 1 and 3. The dicyclic analogue dicyclo(1−3/4−10)Ac-dAsp1,dCpa2,dLys3,Asp4,dNal6,Dpr10GnRH (26) was fully active at 500 μg, with a K i value of 1 nM. The in vivo potency of 26 was at least 10-fold less than that of monocyclic cyclo(1−3)Ac-dAsp1,dCpa2,dLys3,dNal6,dAla10GnRH (5); this suggested the existence of unfavorable interactions between the now optimized and constrained (1−3) and (4−10) cyclic moieties that must interact as originally hypothesized. Tricyclo(1−3/4−10/5−8)Ac-dGlu1,dCpa2, dLys3,Asp4,Glu5,dNal6,Lys8,Dpr10 GnRH (27) was inactive at 500 μg/rat with a corresponding low affinity (K i = 4.6 nM) when compared to those of the most potent analogues (K i < 0.5 nM).
With the ultimate goal of identifying a consensus bioactive conformation of GnRH antagonists, the compatibility of a number of side chain to side chain bridges in bioactive analogues was ...systematically explored. In an earlier publication, cycloAsp4-Dpr10GnRH antagonists with high potencies in vitro and in vivo had been identified. Independently from Dutta et al. and based on structural considerations, the cyclic Glu5-Lys8 constraint was also found to be tolerated in GnRH antagonists. We describe here a large number of cyclic (4−10) and (5−8) and dicyclic (4−10/5−8) GnRH antagonists optimized for affinity to the rat GnRH receptor and in vivo antiovulatory potency. The most potent monocyclic analogues were cyclo(4−10)Ac-dNal1,dFpa2,dTrp,Asp,dArg6,Xaa10GnRH with Xaa = D/LAgl (1, K i = 1.3 nM) or Dpr (2, K i = 0.36 nM), which completely blocked ovulation in cycling rats after sc administration of 2.5 μg at noon of proestrus. Much less potent were the closely related analogues with Xaa = Dbu (3, K i = 10 nM) or cyclo(4−10)Ac-dNal1,dFpa2,dTrp3,Glu4,dArg6,d/lAgl10GnRH (4, K i = 1.3 nM). Cyclo(5−8)Ac-dNal1,dCpa2,dTrp3,Glu,dArg,Lys8,dAla10GnRH (13), although at least 20 times less potent in the AOA than 1 or 2 with similar GnRHR affinity (K i = 0.84 nM), was found to be one of the most potent in a series of closely related cyclo(5−8) analogues with different bridge lengths and bridgehead chirality. The very high affinity of cyclo(5,5‘−8)Ac-dNal1,dCpa2,dPal3,Glu5(βAla),dArg6,(d or l)Agl, dAla10GnRH 14 (K i = 0.15 nM) correlates well with its high potency in vivo (full inhibition of ovulation at 25 μg/rat). Dicyclo(4−10/5−8)Ac-dNal1,dCpa2,dTrp3,Asp4,Glu5,dArg6,Lys8,Dpr10GnRH (24, K i = 0.32 nM) is one-fourth as potent as 1 or 2, in the AOA; this suggests that the introduction of the (4−10) bridge in 13, while having little effect on affinity, restores functional/conformational features favorable for stability and distribution. To further increase potency of dicyclic antagonists, the size and composition of the (5−8) bridge was varied. For example, the substitution of Xbb5‘ by Gly (30, K i = 0.16 nM), Sar (31, K i = 0.20 nM), Phe (32, K i = 0.23 nM), dPhe (33, K i = 120 nM), Arg (36, K i = 0.20 nM), Nal (37, K i = 4.2 nM), His (38, K i = 0.10 nM), and Cpa (39, K i = 0.23 nM) in cyclo(4−10/5,5‘−8)Ac-dNal1,dCpa2,dPal3,Asp4,Glu5(Xbb5‘),dArg6,Dbu,8Dpr10GnRH yielded several very high affinity analogues that were 10, ca. 10, 4, >200, 1, ca. 4, >2, and 2 times less potent than 1 or 2, respectively. Other scaffolds constrained by disulfide (7, K i = 2.4 nM; and 8, K i = 450 nM), cycloGlu5-Aph8 (16, K i = 20 nM; and 17, K i = 0.28 nM), or cycloAsp5-/Glu5-/Asp5(Gly5‘)-Amp8 (19, K i = 1.3 nM; 22, K i = 3.3 nM; and 23, K i = 3.6 nM) bridges yielded analogues that were less potent in vivo and had a wide range of affinities. The effects on biological activity of substituting dCpa or dFpa at position 2, dPal or dTrp at position 3, and dArg, dNal, or dCit at position 6 are also discussed. Interestingly, monocyclo(5−8)Glu5,dNal6,Lys8GnRH (18, K i = 1.0 nM) and dicyclo(4−10/5−8)Asp4,Glu5,dNal6,Lys8,Dpr10GnRH (28, K i = 1.2 nM) contain the native N-terminal pGlu-His-Trp- and are antagonists with relatively high affinity but very low antagonist potency in vivo, illustrating an earlier observation that structural constraints alone may lead to partial agonism or competitive antagonism. All of these observations suggest very rigorous requirements for ligand/receptor recognition and binding as well as a distinct effect of some substitutions on pharmacokinetics.
A series of antagonists of gonadotropin-releasing hormone (GnRH) homologous to azaline B (Ac-DNal1,DCpa2,DPal3,Aph5(Atz),DAph6+ ++(Atz),ILys8,DAla10GnRH) was synthesized, characterized, and tested in ...a rat antiovulatory assay (AOA). Selected analogues were also tested in both an in vitro dispersed rat pituitary cell culture assay for inhibition of GnRH-stimulated luteinizing hormone release and an in vitro histamine release assay. The duration of action of some of the most potent and safest analogues in those assays was also determined in the castrated male rat in order to measure the extent (efficacy and duration of action) of inhibition of luteinizing hormone release. Structurally, this series of analogues has novel substitutions (X and Y) in the structure of the azaline B precursor: Ac-DNal1,DCpa2,DPal3,-Aph5(X),DAph6(Y),++ +ILys8,DAla10GnRH. These substitutions were designed to confer increased hydrophilicity as compared to that of azaline B (determined by relative retention times on a C18 reverse phase column using a triethylammonium phosphate buffer at pH 7.3) or to make them more easily accessible synthetically. Some bulky substituents were introduced in order to probe the spatial limitations of the receptor's cavity. These substitutions include acylated 4-aminophenylalanine at positions 5 and/or 6 (29 analogues), N alpha-methylated backbone substitutions (six analogues), N omega-isopropylaminophenylalanine at position 8, and hydrophilic amino acids at position 1. Out of 20 novel analogues tested for long duration of action in this series, only seven (Ac-DNal1,DCpa2,DPal3,Aph5,DAph6,ILys8 ,DAla10GnRH, Ac-DNal1,DCpa2,DPal3,Aph5(For),DAph6(For) ,ILys8,DAla10GnRH, Ac-DNal1,DCpa2,DPal3,Aph5(Ac),DAph6(Ac),- ILys8,DAla10GnRH (acyline), Ac-DNal1,DCpa2,DPal3,Aph5(Pio),DAph6++ +(Pio),ILys8,DAla10GnRH, Ac-DNal1,DCpa2,DPal3,Aph5(Atz),DAph6++ +(Ac),ILys8,DAla10GnRH, Ac-DNalDCpa2,DPal3,Aph5(Atz-beta Ala),DAph6(Atz-beta Ala),ILys8, DAla10GnRH, Ac-DNal1,DCpa2,DPal3,Aph5(Atz-Gab), DAph6(Atz-Gab),ILys8,DAla10GnRH) had relative potencies and/or duration of action comparable to those of azaline B. The others were one-half to one-tenth as effective as azaline B. N alpha-Methylated backbone substitutions at position 5 yielded analogues that were significantly more hydrophilic presumably because of the breakage of the NH alpha-Tyr5 to Arg8-CO hydrogen bond reported to stabilize a beta-turn encompassing residues 5-8 and which favored beta-sheet formation as shown earlier by Haviv et al. This substitution resulted, however, in an increased potency in the histamine release assay and in significantly shorter duration of action. Similarly, attempts at replacing isopropyllysine in position 8 by either isopropyl-4-aminophenylalanine or isopropyl-4-(aminomethyl)phenylalanine resulted in loss of potency in the AOA. Changes in chirality at position 1 or 10 resulted in analogues that were one-tenth and one-half as potent, respectively, as acyline.
The purpose of this work was to compare the plasma adrenocorticotropin (ACTH), corticosterone and interleukin-6 (IL-6) responses that rats of the outbred Sprague-Dawley strain obtained from two ...different vendors: Charles River (CR) and Harlan (HSD). Basal plasma ACTH and IL-6 concentrations were similar in rats from either vendor (HSD or CR), while CR animals exhibited slightly elevated corticosterone levels in late afternoon. Inflammatory stimuli such as lipopolysaccharide (LPS) (1 microgram/kg, i.v.) or turpentine (50 microliter/100 g, i.m.) which induce the production of endogenous cytokines, produced a significantly larger ACTH response in CR, compared to HSD rats, while the overall corticosterone responses were comparable in both rat groups. This could probably not be accounted for by a greater ACTH responsiveness in CR rats per se because CR and HSD rats showed similar peak ACTH responses to electrofootshock. Furthermore, in contrast to when the stimulus was one that induced endogenous cytokine production, the administration of exogenous interleukin-1beta (IL-1beta, 200 ng/kg, i.v.) produced a 2-fold greater rise in plasma ACTH concentrations in HSD rats compared to CR rats. The plasma IL-6 responses to the inflammatory stimuli showed a similar pattern to ACTH, with LPS and turpentine tending to pruduce greater IL-6 responses in CR rats, though these differences were not statistically significant. In contrast HSD rats had a significantly greater IL-6 response to IL-1beta than did CR rats. Collectively, these results show that Sprague-Dawley rats obtained from different commercial sources can differ in immune-neuroendocrine responses to inflammatory stimuli.
Salvia divinorum Epling & Jativa is an hallucinogenic mint traditionally used for curing and divination by the Mazatec Indians of Oaxaca, Mexico. Young people from Mexican cities were reported to ...smoke dried leaves of
S. divinorum as a marijuana substitute. Recently, two
S. divinorum specimens were seized in a large-scale illicit in-door and out-door hemp plantation. Salvinorin A also called divinorin A, a
trans-neoclerodane diterpene, was identified in several organic solvent extracts by gas chromatography–mass spectrometry. The botanical identity of the plant was confirmed by comparing it to an authentic herbarium specimen. More plants were then discovered in Swiss horticulturists greenhouses. All these data taken together suggest that many attempts exist in Switzerland to use
S. divinorum as a recreational drug. This phenomenon may be enhanced because neither the magic mint, nor its active compound are banned substances listed in the Swiss narcotic law.
During the summer of 2018, a widespread drought developed over Northern and Central Europe. The increase in temperature and the reduction of soil moisture have influenced carbon dioxide (CO 2) ...exchange between the atmosphere and terrestrial ecosystems in various ways, such as a reduction of photosynthesis, changes in ecosystem respiration, or allowing more frequent fires. In this study, we characterize the resulting perturbation of the atmospheric CO 2 seasonal cycles. 2018 has a good coverage of European regions affected by drought, allowing the investigation of how ecosystem flux anomalies impacted spatial CO 2 gradients between stations. This density of stations is unprecedented compared to previous drought events in 2003 and 2015, particularly thanks to the deployment of the Integrated Carbon Observation System (ICOS) network of atmospheric greenhouse gas monitoring stations in recent years. Seasonal CO 2 cycles from 48 European stations were available for 2017 and 2018. Earlier data were retrieved for comparison from international databases or national networks. Here, we show that the usual summer minimum in CO 2 due to the surface carbon uptake was reduced by 1.4 ppm in 2018 for the 10 stations located in the area most affected by the temperature anomaly, mostly in Northern Europe. Notwithstanding, the CO 2 transition phases before and after July were slower in 2018 compared to 2017, suggesting an extension of the growing season, with either continued CO 2 uptake by photosynthesis and/or a reduction in respiration driven by the depletion of substrate for respiration inherited from the previous months due to the drought. For stations with sufficiently long time series, the CO 2 anomaly observed in 2018 was compared to previous European droughts in 2003 and 2015. Considering the areas most affected by the temperature anomalies, we found a higher CO 2 anomaly in 2003 (+3 ppm averaged over 4 sites), and a smaller anomaly in 2015 (+1 ppm averaged over 11 sites) compared to 2018. This article is part of the theme issue 'Impacts of the 2018 severe drought and heatwave in Europe: from site to continental scale'.
In three earlier papers, the structures and biological potencies of numerous mono- and dicyclic antagonists of GnRH were reported. Among these, two families, each containing two to four members were ...identified that had very high antagonist potencies in an antiovulatory assay (within a factor of 2 of those of the most potent linear analogues) and high affinities (K i < 0.5 nM) for the rat GnRH receptor (rGnRHR). The most favored cycles bridged the side chains of residues (4−10), , (5−8), (4−10/5−8), (1−3), and (1−3/4−10). Our goal was to identify a consensus model of bioactive conformations of GnRH antagonists, yet these biocompatible constraints did not sufficiently restrain the spatial location of the N-terminal tripeptide with respect to the C-terminal heptapeptide, due largely to the rotational freedom about the bonds connecting these regions. Examination of models derived from NMR studies of cyclo(4−10) analogues suggested a large number of possible cyclic constraints such as cyclo (0−8), (1−8), or (2−8). All analogues tested with these substitutions were inactive as antiovulatory agents at 1 mg/rat (5−9) and had low affinity for rGnRHR. On the other hand, bridging positions 3 and 8 with a dAsp3 to Dbu8 (12, K i = 13 nM) or Orn8 (13, K i = 14 nM) in the parent compound cyclo(3−8)Ac-dNal1,dCpa2,dXaa3,Arg5,dNal6,Xbb8,dAla10GnRH yielded analogues that blocked ovulation at 250 μg/rat. Analogue 14 (K i = 2.3 nM), with a dAsp3, Lys8 bridge, was fully active at 50 μg/rat. Loss of potency (>20-fold) was observed with the substitution of dAsp3 in 14 by dGlu3 in 15 (K i = 23 nM). Dicyclic analogues possessing the (4−10) cycle and selected (1−6), (2−6), and (2−8) cycles led to analogues that were inactive at doses of 500 μg/rat or larger. Two analogues with (1−8/4−10) cycles (16, K i = 1.1 nM) or (3−8/4−10) cycles (22, K i = 17 nM) showed full antiovulatory potency at 250 μg/rat. None of these substitutions yielded analogues potent enough (>80% inhibition of ovulation at 5 μg/rat or less and K i < 0.5 nM) to be candidates for structural analysis by NMR. On the other hand, four dicyclic (1,1‘−5/4−10) analogues met this criterion: dicyclo(1,1‘−5/4−10)Ac-Asp1(Gly),dCpa2,dTrp3,Asp,Dbu, dNal,Dpr10GnRH (32, K i = 0.22 nM), dicyclo(1,1‘−5/4−10)Ac-Asp1(Gly),dCpa2,dNal3,Asp4,Dbu5, dNal6,Dpr10GnRH (34, K i = 0.38 nM), dicyclo(1,1‘−5/4−10)Ac-Asp1(βAla),dCpa2, dTrp3,Asp4,Dbu5,dNal6,Dpr10GnRH (40, K i = 0.15 nM), and dicyclo(1,1‘−5/4−10)Ac-Glu1(Gly), dCpa2,dTrp3,Asp4,Dbu5,dNal6,Dpr10GnRH (41, K i = 0.24 nM). Since they differed slightly in terms of the (1,1‘−5) bridge length (21 and 22 atoms) and bridgehead configuration, we may hypothesize that they assume similar bioactive conformations that satisfy a very discriminating receptor, since many other closely related analogues were significantly less potent.
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