Catabolism and disposition of exogenous 5-hydroxytryptamine in cockroach,Periplaneta americana,tissues

The concentrations of tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, N-acetyl 5-hydroxytryptamine and N-acetyl dopamine were determined in the cerebral ganglia, haemolymph and Malpighian tubules of the cockroach Periplaneta americana, using high performance liquid chromatography with electrochemical detection. Injected 5-hydroxytryptamine was rapidly removed from the haemolymph with a concomitant elevation of circulating N-acetyl 5-hydroxytryptamine and little accumulation of 5-hydroxytryptamine in the cerebral ganglia. N-acetyl 5-hydroxytryptamine and N-acetyl dopamine were also rapidly removed from the haemolymph. Incubation of haemolymph from 5-hydroxytryptamine-injected insects and glucosidase or phosphatase, indicated that most of the injected 5-hydroxytryptamine had been converted to a sugar conjugate of N-acetyl 5-hydroxytryptamine. Whole haemolymph did not catabolize 5-hydroxytryptamine or N-acetyl 5-hydroxytryptamine whereas Malpighian tubules N-acetylated both 5-hydroxytryptamine and dopamine and metabolized N-acetyl 5-hydroxytryptamine. Injection of p-chlorophenylalanine (200 and 500 μg/g) had no effect on 5-hydroxytryptamine concentrations in the cockroach cerebral ganglia.     

Influence of crowding on biogenic amine levels in the nervous system of the female cockroach Blaberus craniifer burm. (Dictyoptera,blaberidae)

1. Tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, a 5-hydroxyindolacetic acid-like substance, N-acetyl-5-hydroxytryptamine, dopamine, N-acetyldopamine and octopamine were assayed simultaneously in the central nervous system of virgin and fed females Blaberus craniifer Burm.2. Amounts were quantified by HPLC and electrochemical detection in both crowded and isolated females during the first weeks of imaginal life, from days 0–30.3. Amine contents were modified by housing conditions, and in general, higher levels were found in the isolated individuals, with the most pronounced differences observed at D10 and D30.4. The relationship between these fluctuations and the speeding up of ovarian maturation by isolation is discussed.     

Presence of males affects the incidence of ovulation after pouch young removal in brushtail possums (Trichosurus vulpecula)

The traditional method for inducing and synchronising oestrus in the brushtail possum (Trichosurus vulpecula) is by removal of their suckling pouch young (RPY). However, our studies have recently shown that, in addition to wide variation between animals in the time of ovulation after RPY, a proportion of animals failed to ovulate. Evidence from several mammalian species indicates that the presence of males can stimulate ovarian activity and synchronise oestrus in females. The aim of this study was to determine the effect of the male on the oestrous cycle of the female brushtail possum after RPY. A total of 67 adult female brushtail possums were treated as three replicates. In order to observe the day of preovulatory follicle emergence and ovulation, animals underwent laparoscopic examination at 1–4 day intervals over a period from 0–21 days after RPY. The first replicate (N=18, May/June 1995) involved only animals kept in isolation from males, whereas the two remaining replicates compared ovarian responses between animals kept with (N=10, July 1995; N=14, June 1996) or in isolation from (N=10, July 1995; N=15, June 1996) males. The incidence of ovulation after RPY was significantly higher in females that were housed with males than in those kept in isolation from males (100%, 92.8% vs. 50.0%, 66.7%, 14.3%; P<0.001). Every animal that ovulated, had previously had a preovulatory follicle present at the site where the corpus luteum formed. Conversely, none of the animals that failed to ovulate, developed a preovulatory follicle during the period of study. The range of mean day of preovulatory follicle emergence (6.00–6.86 days), of ovulation (11.80–12.20 days) and the synchrony of ovulation between animals (range 8–17 days) after RPY, were not significantly affected by the presence of males. This study demonstrates for the first time, that the presence of males significantly increases the incidence of ovulation after RPY in the brushtail possum. However neither the timing of reproductive events nor the synchrony of ovulation were affected by presence of the male.     

Identification of N5,N10-methylene tetrahydrofolate reductase as the enzyme involved in the 5-methyl tetrahydrofolate-dependent formation of a beta-carboline derivative of 5-hydroxytryptamine in human platelets.

An enzyme in human platelets or rat brain incubated with 5-methyl tetrahydrofolate (5MeH₄folate) yields formaldehyde (4, 13), which will combine with biogenic amines to form β-carbolines (5) or tetrahydroisoquinolines. This activity was purified 500-fold from human platelets which are the main storage site for 5-hydroxytryptamine in man. This enzyme was identical to N⁵, N¹⁰-methylene tetrahydrofolate (N⁵,N¹⁰-methylene H₄folate) reductase by the following criteria: (i) co-purification, (ii) heat denaturation, (iii) pH response, (iv) molecular weight, (5) cofactor requirements. A mechanism involving the enzymatic generation of formaldehyde followed by adduct formation with a biogenic amine is proposed.     

Effects of sex hormones on enzymic esterification of 2-(N-hydroxyacetamido)-fluorene by rat liver cytosol

1. Enzymic esterifications of 2-(N-hydroxyacetamido)fluorene and several other hydroxamic acids by liver cytosol were studied. Determination of 2-acetamido-3-methylthiofluorene was used for the assay. 2. With rat liver cytosol, requirement for ATP, Mg²⁺ and SO₄²⁻ suggested formation of phosphate and sulphate esters of 2-(N-hydroxyacetamido)fluorene. 3. Rats showed sex and age differences in their activity. Liver from adult male rat was at least twice as active as liver from adult female rat. No such sex differences were found in mice, hamsters and guinea pigs. 4. Administration of testosterone (300μg/day) subcutaneously for 8 days increased the activity in the female rat by 100%, whereas diethylstilboestrol (100μg/day) had no effect. In the male rat diethylstilboestrol treatment decreased the activity by 60%, whereas testosterone pretreatment was without any effect. 5. Among various endocrine ablations such as adrenalectomy, castration, adrenalectomy–castration and hypophysectomy in the adult male rat, hypophysectomy was found to be the most effective in decreasing the activity of the liver to about 50% of control values. 6. Like 2-(N-hydroxyacetamido)fluorene, various other N-hydroxy derivatives of 2-acetamido-7-fluorofluorene, 2-acetamidophenanthrene, 4-acetamidobiphenyl and 4-acetamidostilbene were also shown to be esterified to different extents by rat liver cytosol.     

Reassessing the role of YUCCAs in auxin biosynthesis

It is remarkable that although auxin was the first growth-promoting plant hormone to be discovered, and although more researchers work on this hormone than on any other, we cannot be definitive about the pathways of auxin synthesis in plants. In 2001, there appeared to be a dramatic development in this field, with the announcement of a new gene,¹ and a new intermediate, purportedly from the tryptamine pathway for converting tryptophan to the main endogenous auxin, indole-3-acetic acid (IAA). Recently, however, we presented evidence challenging the original and subsequent identifications of the intermediate concerned.²Key words: auxin synthesis, YUCCA, tryptamine, N-hydroxytryptamineThe new gene was termed YUC, and the putative intermediate is N-hydroxytryptamine. It was claimed that the YUC protein, a flavin-containing monooxygenase, converts tryptamine (formed from tryptophan by decarboxylation) to N-hydroxytryptamine, which is converted via other intermediates to IAA. When the YUC gene was expressed in E. coli and the resulting protein incubated with tryptamine, a weak TLC spot resulted, which produced a mass spectrum said to match that expected from N-hydroxytryptamine.¹ However, the authors did not report mass spectral data from authentic N-hydroxytryptamine, and their suggested fragmentation pattern breaks a fundamental rule of mass spectrometry (the even-electron rule).² Nevertheless, N-hydroxytryptamine has been added to virtually all IAA synthesis schemes published since 2001.³In 2010, LeClere et al. expressed a maize YUC gene in E. coli,⁴ and again claimed that the resulting protein converted tryptamine to N-hydroxytryptamine. This time, the mass spectrum was of better quality, but we have shown that it does not match that of authentic N-hydroxytryptamine, synthesised in our laboratory.^2,5 We have demonstrated by electrospray tandem mass spectrometry that the protonated molecule of N-hydroxytryptamine (m/z 177) fragments to give an abundant ion at m/z 144. This was the crucial piece of evidence that the product obtained by LeClere et al. was not N-hydroxytryptamine, since their compound gave an abundant ion at m/z 160, and no ion at m/z 144.⁴The m/z 144 ion is formed by loss of NH₂OH (hydroxylamine), as shown by accurate mass determinations (Fig. 1). In other words, it is the alkyl-amine bond that is broken; this is also the case for tryptamine and serotonin. In the latter case, an m/z 160 ion results through loss of ammonia, because the hydroxyl group on the indole ring (at position 5) is retained in the fragment. The compound obtained by LeClere et al. when protonated, also had a mass of 177, consistent with a hydroxylated tryptamine, and the abundant m/z 160 ion indicates that this fragment, as in serotonin, retains the hydroxyl group.⁴ However, we believe that the LeClere et al. product is not serotonin, because of dissimilar behavior on thin layer chromatography. Apart from the probability that it is a hydroxylated tryptamine, the identity of the LeClere et al. product is not known.Open in a separate windowFigure 1Fragmentation of (A) N-hydroxytryptamine, (B) tryptamine and (C) 5-hydroxytryptamine (serotonin), as determined by MS/MS analysis.² The m/z ratios of the fragments produced are indicated. The loss of neutral hydroxylamine (A) or ammonia (B and C) involves heterolytic cleavage and/or hydrogen atom rearrangement, and consequent retention of the positive charge on the remaining indole-containing fragment.¹⁴It is interesting to contrast the previous “identifications” of N-hydroxytryptamine1,⁴ with the identification of gibberellins, during the period when most of the gibberellins were identified (1970–1990). There were rigorous criteria for the identification of these compounds, imposed by a triumvirate of “Gibberellin Godfathers”: Jake MacMillan, Nobatuka Takahashi and the late Bernard Phinney, and more latterly by Caporegimes such as Peter Hedden and Yuji Kamiya. Three of the present authors (James B. Reid, Noel W. Davies and John J. Ross) experienced at first hand the rigour with which these criteria were applied.Essentially, any identification of an endogenous gibberellin was viewed with suspicion unless a synthesized form of that compound (a standard, confirmed by NMR) was available for comparison. For a firm identification, the retention time on GC should be identical between the standard and the putative compound, on the same GC instrument. Next, the electron ionization fragmentation patterns of the compound of interest and the standard should match, again on the one GC-MS system. It was not considered adequate to compare a spectrum of the compound of interest with published spectra from another laboratory. Often a spectrum from a plant extract might contain extra ions, contributed by “interfering” compounds and this was sometimes acceptable. However, the absence of ions that should be present was usually sufficient to render the identification unconvincing. Electrospray mass spectra are intrinsically much poorer in information than electron ionisation spectra since most or all of the signal is concentrated in the protonated molecule, and tandem mass spectrometry (MS/MS) is required to create diagnostic fragment ions. The MS/MS spectrum of another hydroxylated tryptamine that we have examined is dominated by a strong m/z 160 ion, and discrimination between hydroxylated tryptamines on the basis of MS alone could be problematic. N-Hydroxytryptamine is the exception in this regard, and it can be easily distinguished.Another technique that has been used extensively in gibberellin research, and in early auxin research as well, is “feeding” labelled compounds and determining the fate of the label concerned (often deuterium or ¹³C). This technique contributed strongly to the identification of most of the candidate auxin pathways.⁶ Its power should not be underestimated, and yet in the auxin field, it was under-utilised during much of the later 1990s and the 2000s. We have used this technique to demonstrate that tryptamine is not converted to N-hydroxytryptamine in pea roots or seeds.^2,5 In fact, to our knowledge, N-hydroxytryptamine has not yet been identified in any plant species.N-Hydroxytryptamine has been the main link between the YUC genes and the tryptamine pathway, and this link is now called into question. In fact, there are some indications that YUCCAs might not be concerned with IAA synthesis at all. The floozy mutant of petunia has a strong phenotype but normal levels of IAA.⁷ The yuc1 yuc2 yuc4 yuc6 quadruple mutant of Arabidopsis also exhibits a whole-plant phenotype but again its IAA content is not reduced compared with WT plants.⁸ Indeed, as yet there is not a single instance where knocking out YUC function has been shown to significantly reduce IAA content. We should note also that while overexpression of YUC genes does lead to elevated IAA content, the increase is small (up to about 2-fold^1,9) compared with the increases recorded for other IAA over-producing mutants; for example, sur1 (also known as rty) and sur2, which can contain 5 to 20 times more IAA than the WT.¹⁰-12Therefore, it is possible that YUC catalyses a reaction or reactions in another pathway leading to another compound that is required for normal plant development; hence the phenotypic consequences of loss-of-function yuc mutants. This compound might be another auxin or auxinlike compound, which might explain why elevating auxin content genetically sometimes rescues yuc phenotypes.¹³ The suggestion that YUC controls the synthesis of another compound was made as early as 2002,⁷ but has attracted little attention from auxin biologists. There seems little doubt that YUCCAs play essential roles in plant development, as evidenced by the phenotypes of knockout mutants, even though it is sometimes necessary to construct multiple mutants to observe strong phenotypes.¹³ Furthermore, YUC genes are found in a wide range species, and we recently extended the list to include pea.² However, the almost universal placement of N-hydroxytryptamine in auxin synthesis schemes since 2001 is now called into question by the recently published evidence.²     

Further characterization and quantitative determination of 5-methoxy-N-methyltryptamine in Phalaris arundinacea

The 100 MHz PMR spectrum of 5-methoxy-N-methyltryptamine (5MMT) isolated from Phalaris arundinacea is elucidated, acid-induced fluorescence characteristics of 5-methoxy- and 5-hydroxytryptamine alkaloids on Avicel and silica gel TLC are described, and the concentration of 5MMT in P. arundinacea is determined by fluorometric TLC scanning.     

Schistosoma mansoni: neurotransmitters, longitudinal musculature and effects of electrical stimulation

Longitudinal muscle shortening of adult male Schistosoma mansoni is produced by electrical stimulation. Responses are frequency and strength dependent. Neither 5-hydroxytryptamine (5-HT) antagonists nor dopamine interfere with the response. 5-HT does not enhance it. Carbachol (10^?4M), eserine (10^?6M), and metrifonate (10^?5M) block the response but acetylcholine alone has no affect. Atropine (10^?4M) partially counteracts the effects of carbachol. Hyperosmotic sucrose but not urea blocks the stimulation response. It is concluded that the response is nonneuronally conducted via a pathway involving gap junctions and that neurotransmitters probably act as modulators of motor activity rather than as initiators of it.     

Thermal preferences of hatchling saltwater crocodiles (Crocodylus porosus) in response to time of day,social aggregation and feeding

Three month old hatchling Crocodylus porosus with data loggers in their stomachs were placed in thermal gradients, in isolation (N=16) and in groups of 4 (N=8 groups; 32 individuals). Mean T_b and variation in T_b (SD) was not different whether individual crocodiles in isolation were fasted or fed, or if individuals were housed in isolation (I) or in groups (G). However, individuals in isolation (N=16) maintained slightly lower T_bs than those in groups (N=32) during the early morning (06:00–11:00 h). The overall mean T_b recorded for fasted individuals in the isolated and group treatments (N=48) was 30.9±2.3 °C SD, with 50% of T_bs (T_set) between 29.4 °C and 32.6 °C, and a voluntary maximum and minimum of 37.6 °C and 23.2 °C respectively. During the day (11:00–17:00 h), individuals in isolation and in groups selected the warmer parts of the gradient on land, where they moved little. Outside of this quiescent period (QP), activity levels were much higher and they used the water more. There was a strong diurnal cycle for fasted individuals in isolation and in groups, with T_b during the QP (31.9±2.09 °C; N=48) significantly higher than during the non-quiescent period (NQP: 30.6±2.31 °C). Thermal variation (SD) in T_b was relatively stable throughout the day, with the highest variation at around dusk and early evening (18:00–20:00 h), which coincided with a period of highest activity. The diurnal activity cycle appears innate, and may reflect the need to engage in feeding activity at the water's edge in the early evening, despite ambient temperatures being cooler, with reduced activity and basking during the day. If so, preferred T_b may be more accurately defined as the mean T_b during the QP rather than the NQP. Implications for the thermal environment best suited for captive C. porosus hatchlings are discussed.     

A demonstration that 5-hydroxytryptamine administered peripherally can affect sexual behavior in male rats

In the male rat, subcutaneous injections for 7 days of 20 mg/Kg B.W./day of 5-hydroxytryptamine creatinin sulphate (5-HT), caused remarkable i$\text{n}$hibitory effects on sexual behavior.The mount and intromission latencies were incre$\text{a}$sed in rats treated with 5-HT, whereas ejacul$\text{a}$tion latency in the few rats treated with 5-HT that it achieved, was similar to that obtained in control rats.The mount and intromission frequencies were decreased in the rats treated with 5-HT.The mean inter-intromission interval (MII) and post-ejaculatory interval were prolonged in rats treated with 5-HT.These data provide evidence for the role of peripheral 5-HT in regulating sexual behavior of - male rats.     

The N-Reductive System Composed of Mitochondrial Amidoxime Reducing Component (mARC), Cytochrome b5 (CYB5B) and Cytochrome b5 Reductase (CYB5R) Is Regulated by Fasting and High Fat Diet in Mice

The mitochondrial amidoxime reducing component mARC is the fourth mammalian molybdenum enzyme. The protein is capable of reducing N-oxygenated structures, but requires cytochrome b5 and cytochrome b5 reductase for electron transfer to catalyze such reactions. It is well accepted that the enzyme is involved in N-reductive drug metabolism such as the activation of amidoxime prodrugs. However, the endogenous function of the protein is not fully understood. Among other functions, an involvement in lipogenesis is discussed. To study the potential involvement of the protein in energy metabolism, we tested whether the mARC protein and its partners are regulated due to fasting and high fat diet in mice. We used qRT-PCR for expression studies, Western Blot analysis to study protein levels and an N-reductive biotransformation assay to gain activity data. Indeed all proteins of the N-reductive system are regulated by fasting and its activity decreases. To study the potential impact of these changes on prodrug activation in vivo, another mice experiment was conducted. Model compound benzamidoxime was injected to mice that underwent fasting and the resulting metabolite of the N-reductive reaction, benzamidine, was determined. Albeit altered in vitro activity, no changes in the metabolite concentration in vivo were detectable and we can dispel concerns that fasting alters prodrug activation in animal models. With respect to high fat diet, changes in the mARC proteins occur that result in increased N-reductive activity. With this study we provide further evidence that the endogenous function of the mARC protein is linked with lipid metabolism.     

Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter

The rat serotonin transporter (rSERT) is an N-glycosylated integral membrane protein with 12 transmembrane regions; the N-glycans improve the ability of the SERT polypeptide chain to fold into a functional transporter, but they are not required for the transmembrane transport of serotonin per se. In order to define the best system for the expression, purification and structural analysis of serotonin transporter (SERT), we expressed SERT in Escherichia coli, Pichia pastoris, the baculovirus expression system and in four different stable mammalian cell lines. Two stable cell lines that constitutively expressed SERT (Imi270 and Coca270) were constructed using episomal plasmids in HEK293 cells expressing the EBNA-1 antigen. SERT expression in the three different inducible stable mammalian cell lines was induced either by a decrease in temperature (cell line pCytTS-SERT), the addition of tetracycline to the growth medium (cell line T-REx-SERT) or by adding DMSO which caused the cells to differentiate (cell line MEL-SERT). All the mammalian cell lines expressed functional SERT, but SERT expressed in E. coli or P. pastoris was nonfunctional as assessed by 5-hydroxytryptamine uptake and inhibitor binding assays. Expression of functional SERT in the mammalian cell lines was assessed by an inhibitor binding assay; the cell lines pCytTS-SERT, Imi270 and Coca270 contained levels of functional SERT similar to that of the standard baculovirus expression system (250,000 copies per cell). The expression of SERT in induced T-REx-SERT cells was 400,000 copies per cell, but in MEL-SERT it was only 80,000 copies per cell. All the mammalian stable cell lines expressed SERT at the plasma membrane as assessed by [³H]-5-hydroxytryptamine uptake into whole cells, but the V_max for the T-Rex-SERT cell line was 10-fold higher than any of the other cell lines. It was noticeable that the cell lines that constitutively expressed SERT grew extremely poorly, compared to the inducible cell lines whose growth rates were similar to the parental cell lines when not induced. In addition, the cell lines MEL-SERT, Imi270 and T-REx-SERT all expressed fully N-glycosylated SERT and no unglycosylated inactive protein, in contrast to the baculovirus expression system where the vast majority of expressed SERT was unglycosylated and nonfunctional.     

5-Hydroxytryptamine stimulates 86Rb+ efflux from pancreatic β-cells

The effects of 5-hydroxytryptamine and 5-hydroxytryptophan on ⁸⁶Rb⁺ efflux from prelabelled ob/ob-mouse islets were studied to better understand the cellular mechanisms underlying the effects of 5-hydroxytryptamine and 5-hydroxytryptophan on insulin release. 5-Hydroxytryptophan (4 mM) had no effect on ⁸⁶Rb⁺ efflux either at a low (3 mM) or at a high (20 mM) d-glucose concentration, whereas 5-hydroxytryptamine (4 mM) stimulated ⁸⁶Rb⁺ efflux at both glucose concentrations. These results indicate that 5-hydroxytryptamine may reduce glucose-induced insulin release by inhibiting early steps in the β-cell stimulus-secretion coupling.     

Saccharopine and 2-aminoadipic acid in Reseda odorata

2(S),2′(S)-N⁶-(2′-Glutaryl)lysine (l-saccharopine) and 2(S)-2-aminoadipic acid have been isolated from Reseda odorata. When traditional isolation procedures are used l-pyrosaccharopine (5(S),5′(S)-N-(5′-amino-5′-carboxy-pentyl)-2-pyrrolidone-5-carboxylic acid) is formed from l-saccharopine by lactamisation. The degree of lactamisation during various isolation steps has been studied, The amino acids were identified by IR and PMR spectroscopy and the configurations established by enzymic and polarimetric analyses. The contents of saccharopine and 2-amino-adipic acid have been determined relative to the total nitrogen content at various stages in the growth cycle of R. odorata.     

Substrate-dependent activation energy of the reaction catalyzed by monoamine oxidase

The effect of temperature on the deamination of 5-hydroxytryptamine, tyramine, and phenethylamine by monoamine oxidase (MAO) of human placenta, beef liver, and rat liver has been studied. Both MAO A and MAO B activities are influenced by the lipid-phase transition and, in some cases, another type of transition. The estimates of activation energy (E_act) for the deamination of 5-hydroxytryptamine, phenethylamine, tyramine, dopamine, and pentylamine at 5–20 °C show that a given substrate is associated with a particular value irrespective of the source of MAO acting upon it. The substrate dependence of E_act is explained by the differences in lipophilicity of the various substrates. The interaction of enzyme and the lipids in the environment of its active site would differ with each substrate, and would give rise to different activated complexes, each corresponding to a given substrate. The E_act values are presumably related to these complexes, rather than to enzyme alone.     

Biogenic amines in the two-spotted spider mite

Whole body extracts of the two-spotted spider mite (Tetranychus urticae Koch) were analyzed using a 16-channel electrochemical array high performance liquid chromatography (HPLC)-based detection system that allows the simultaneous isolation and identification of a variety of biogenic amines. The spider mite extracts were found to contain the biogenic amines octopamine, dopamine, and 5-hydroxytryptamine (5-HT), as well as several precursors and metabolites including tyrosine, tyramine, tryptophan, and N-acetyl octopamine. Differences in the levels of biogenic amines were observed between eggs and the adult stages and between males and females. This is the first direct determination of biogenic amines in the Tetranychidae and the first demonstration of 5-HT in any mite species. © 1994 Wiley-Liss, Inc.     

5-Hydroxytryptamine metabolism by the nuclear fraction of rat-liver homogenate

1. The metabolism of 5-hydroxy[1′-¹⁴C]tryptamine creatinine sulphate in the nuclear fraction of rat-liver homogenate was studied. In the incubation mixture five metabolites were found. 2. Two metabolites were not radioactive; one of them was identified as 5-hydroxyindole-3-carboxylic acid and the second tentatively as 5-hydroxyindole-3-aldehyde. 3. 5-Hydroxyindol-3-ylacetic acid, 1′-N-acetyl-5-hydroxytryptamine and 5-hydroxytryptophol were not precursors of 5-hydroxyindolealdehyde and 5-hydroxyindolecarboxylic acid. 4. It was shown that the metabolism of 5-hydroxytryptamine in the nuclear fraction involves monoamine oxidase, the precursor of 5-hydroxyindolealdehyde and 5-hydroxyindolecarboxylic acid being most probably 5-hydroxyindol-3-ylacetaldehyde.     

Differential modulation of white adipose tissue endocannabinoid levels by n-3 fatty acids in obese mice and type 2 diabetic patients

n-3 polyunsaturated fatty acids (n-3 PUFA) might regulate metabolism by lowering endocannabinoid levels. We examined time-dependent changes in adipose tissue levels of endocannabinoids as well as in parameters of glucose homeostasis induced by n-3 PUFA in dietary-obese mice, and compared these results with the effect of n-3 PUFA intervention in type 2 diabetic (T2DM) subjects. Male C57BL/6J mice were fed for 8, 16 or 24?weeks a high-fat diet alone (cHF) or supplemented with n-3 PUFA (cHF?+?F). Overweight/obese, T2DM patients on metformin therapy were given for 24?weeks corn oil (Placebo; 5?g/day) or n-3 PUFA concentrate as above (Omega-3; 5?g/day). Endocannabinoids were measured by liquid chromatography-tandem mass-spectrometry. Compared to cHF-fed controls, the cHF?+?F mice consistently reduced 2-arachidonoylglycerol (up to ~2-fold at week 24) and anandamide (~2-fold) in adipose tissue, while the levels of endocannabinoid-related anti-inflammatory molecules N-eicosapentaenoyl ethanolamine (EPEA) and N-docosahexaenoyl ethanolamine (DHEA) increased more than ~10-fold and ~8-fold, respectively. At week 24, the cHF?+?F mice improved glucose tolerance and fasting blood glucose, the latter being positively correlated with adipose 2-arachidonoylglycerol levels only in obese cHF-fed controls, like fasting insulin and HOMA-IR. In the patients, n-3 PUFA failed to reduce 2-arachidonoylglycerol and anandamide levels in adipose tissue and serum, but they increased both adipose tissue and serum levels of EPEA and DHEA. In conclusion, the inability of n-3 PUFA to reduce adipose tissue and serum levels of classical endocannabinoids might contribute to a lack of beneficial effects of these lipids on glucose homeostasis in T2DM patients.     

Effect of prenatal androgens on click-evoked otoacoustic emissions in male and female sheep (Ovis aries)

Otoacoustic emissions (OAEs) were measured in male and female Suffolk sheep (Ovis aries). Some sheep had been administered androgens or estrogens during prenatal development, some were gonadectomized after birth, and some were allowed to develop normally. As previously reported for spotted hyenas, gonadectomy did not alter the OAEs for either sex; accordingly, the untreated/intact and the untreated/gonadectomized animals were pooled to form the control groups. The click-evoked otoacoustic emissions (CEOAEs) exhibited by the female control group (N = 12) were slightly stronger (effect size = 0.42) than those in the male control group (N = 15), which is the same direction of effect reported for humans and rhesus monkeys. Females administered testosterone prenatally (N = 16) had substantially weaker (masculinized) CEOAEs than control females (effect size = 1.15). Both of these outcomes are in accord with the idea that prenatal exposure to androgens weakens the cochlear mechanisms that underlie the production of OAEs. The CEOAEs of males administered testosterone prenatally (N = 5) were not different from those of control males, an outcome also seen in similarly treated rhesus monkeys. Males administered dihydrotestosterone (DHT) prenatally (N = 3) had slightly stronger (hypo-masculinized) CEOAEs than control males. No spontaneous otoacoustic emissions (SOAEs) were found in any ears, a common finding in non-human species. To our knowledge, this is the first ruminant species measured for OAEs.     

Modification of female onion fly,Delia antiqua (Meigen), reproductive behavior by male paragonial gland extracts (Diptera: Anthomyiidae)

Egg depositional rates of onion flies, Delia antiqua(Meigen), injected thoracically with extracts of male paragonial glands were identical (14.5 eggs/female/ day) to those of normally mated females. Moreover, when continuously exposed to males, extract-injected females refused to mate and produced unfertilized eggs for the duration of the > 15- day experiment. For this normally monocoitic dipteran, <1 male equiv of paragonial secretion completely reproduced the ovipositional responses characteristic of normal mating, and this effect required no involvement of the genitalia or genital chamber. We suggest that the receptor for the active chemical (s) (sex peptide?) would be an excellent target for biorational insect control by sterilization. Moreover, these primer sex pheromones might play an important role in insect reproductive isolation and evolution.