Bridging the age gap in breast cancer: cluster randomized trial of two decision support interventions for older women with operable breast cancer on quality of life, survival, decision quality, and treatment choices.

BACKGROUND: Rates of surgery and adjuvant therapy for breast cancer vary widely between breast units. This may contribute to differences in survival. This cluster RCT evaluated the impact of decision support interventions (DESIs) for older women with breast cancer, to ascertain whether DESIs influenced quality of life, survival, decision quality, and treatment choice. METHODS: A multicentre cluster RCT compared the use of two DESIs against usual care in treatment decision-making in older women (aged at least ≥70 years) with breast cancer. Each DESI comprised an online algorithm, booklet, and brief decision aid to inform choices between surgery plus adjuvant endocrine therapy versus primary endocrine therapy, and adjuvant chemotherapy versus no chemotherapy. The primary outcome was quality of life. Secondary outcomes included decision quality measures, survival, and treatment choice. RESULTS: A total of 46 breast units were randomized (21 intervention, 25 usual care), recruiting 1339 women (670 intervention, 669 usual care). There was no significant difference in global quality of life at 6 months after the baseline assessment on intention-to-treat analysis (difference -0.20, 95 per cent confidence interval (C.I.) -2.69 to 2.29; P = 0.900). In women offered a choice of primary endocrine therapy versus surgery plus endocrine therapy, knowledge about treatments was greater in the intervention arm (94 versus 74 per cent; P = 0.003). Treatment choice was altered, with a primary endocrine therapy rate among women with oestrogen receptor-positive disease of 21.0 per cent in the intervention versus 15.4 per cent in usual-care sites (difference 5.5 (95 per cent C.I. 1.1 to 10.0) per cent; P = 0.029). The chemotherapy rate was 10.3 per cent at intervention versus 14.8 per cent at usual-care sites (difference -4.5 (C.I. -8.0 to 0) per cent; P = 0.013). Survival was similar in both arms. CONCLUSION: The use of DESIs in older women increases knowledge of breast cancer treatment options, facilitates shared decision-making, and alters treatment selection. Trial registration numbers: EudraCT 2015-004220-61 (https://eudract.ema.europa.eu/), ISRCTN46099296 (http://www.controlled-trials.com).

Sheep deficient in vitamin E preferentially select for a feed with a higher concentration of vitamin E.

Given the capacity of ruminants to modify diet selection based on metabolic needs, we hypothesised that, when given a choice, lambs experiencing a vitamin E deficiency would consume more of a vitamin E-enriched feed than lambs not deficient in vitamin E. Fifty-six Dohne Merino lambs were divided into two groups and fed either a vitamin E-deficient diet over 40 days to induce low plasma vitamin E or a vitamin E-enriched diet to induce high plasma vitamin E. The lambs were then offered a choice of vitamin E-enriched and vitamin E-deficient pellets. For half of the animals, the enriched diet was paired with strawberry flavour and the deficient diet was paired with orange flavour, while the reverse pairings were offered to the others. Lamb preference for the diets was measured daily for the following 15 days. There was a three-way interaction between the high and low vitamin E treatment groups×vitamin E content and type of flavour in the feed×time (days). The lambs preferred pellets flavoured with strawberry but this preference changed to orange flavour in vitamin E-deficient lambs if the orange flavour was paired with high vitamin E. Lambs without a deficiency continued to prefer strawberry-flavoured pellets, regardless of the vitamin E concentrations in the pellets. It is possible that self-learning contributed to the low vitamin E group of lambs changing preference to orange flavour in order to consume more vitamin E, presumably to remediate the deficiency.

Feed intake of sheep when allowed ad libitum access to feed in methane respiration chambers.

The patterns of feed intake when animals are allowed ad libitum access to feed in a respiration chamber is not known, nor are the potential effects of the artificial environment of chambers on voluntary feed intake. The objectives of the study were to describe the pattern of hourly feed intake of sheep when fed for ad libitum intake in respiration chambers and determine the repeatability of this pattern and the correlation between feed intake and methane production calculated at hourly intervals. Daily and hourly measurements of methane production and feed intake of 47 Merino wethers were measured in respiration chambers twice, 4 wk apart. We found that hourly feed intake of sheep with ad libitum access to feed in respiration chambers showed a repeatable pattern over the 2 measurement periods (r = 0.89, P < 0.001). During both measurements, sheep ate continuously throughout the 23 h period, but most of the eating occurred during the first 8 h in the respiration chambers. There was a significant linear correlation (r = 0.22) between hourly feed intake and hourly methane production (P < 0.001). An unexpected result from this study was that despite using an accepted and published acclimatization procedure to habituate the animals to the respiration chambers, sheep had 15 to 25% lower feed intake in the respiration chambers compared with their feed intake during the previous week in the animal house pens. In addition, daily feed intake in the respiration chamber was not correlated with feed intake in any of the 7 d before entering the chamber (P > 0.05). Future methane research may consider using feed intake and changes in intake levels as a quantitative indicator of habituation to the methane measurement procedure and environment, which, given the tight association between feed intake and methane production, will be crucial in providing accurate values for methane production.

Consumption of a high-salt diet by ewes during pregnancy alters nephrogenesis in 5-month-old offspring.

Maternal nutrition during pregnancy can affect kidney development in the foetus, which may lead to adverse consequences in the mature kidney. It was expected that high-salt intake by pregnant ewes would lead to a reduction in foetal glomerular number but that the ovine kidney would adapt to maintain homoeostasis, in part by increasing the size of each glomerulus. Merino ewes that were fed either a control (1.5% NaCl) or high-salt (10.5% NaCl) diet during pregnancy, as well as their 5-month-old offspring, were subjected to a dietary salt challenge, and glomerular number and size and sodium excretion were measured. The high-salt offspring had 20% fewer glomeruli compared with the control offspring (P < 0.001), but they also had larger glomerular radii compared with the control offspring (P < 0.001). Consequently, the cross-sectional area of glomeruli was 18% larger in the high-salt offspring than in the control offspring (P < 0.05). There was no difference in the daily urinary sodium excretion between the two offspring groups (P > 0.05), although the high-salt offspring produced urine with a higher concentration of sodium. Our results demonstrated that maternal high-salt intake during pregnancy affected foetal nephrogenesis, altering glomerular number at birth. However, the ability to concentrate and excrete salt was not compromised, which indicates that the kidney was able to adapt to the reduction in the number of glomeruli.

Nutritional restriction of pre-pubertal liveweight gain impairs ovarian follicle growth and oocyte developmental competence of replacement gilts.

The objective was to investigate the effects of moderate restriction of pre- and peri-pubertal liveweight gain on ovarian development and oocyte meiotic competence. At 70 d of age, and 27.7 ± 0.4 kg liveweight (LW), 64 Large White/Landrace crossbred gilts were allocated to two treatment groups (n = 32 gilts/treatment); one group was fed to attain a LW of 70 kg at 161 d of age (LIGHT), while the other group was fed to reach 100 kg LW (HEAVY). At 161 d of age, half of the gilts in each group (n = 16) were fed to gain LW at 0.5 kg/d (LOW), while the remaining half (n = 16) were fed to gain LW at 1.0 kg/d (HIGH) between 161 and 175 d of age, at which point they were killed and ovaries collected. For each gilt, surface antral follicles were counted and aspirated according to three size categories: 1-2.9 mm (small); 3-6 mm (medium); and > 6 mm (large). Follicles were pooled for each size class and treatment. Cumulus-oocyte-complexes (COC) recovered from small and medium follicles were matured in vitro (IVM) for 44 to 46 h, and meiotic maturation assessed. There was an effect of treatment (LIGHT versus HEAVY) on the number of medium sized follicles: 25.1 ± 2.59 versus 34.3 ± 2.60 (P < 0.05). The ovaries of LOW gilts had more small follicles and fewer medium follicles compared to those of HIGH gilts: 92.8 ± 8.35 versus 59.8 ± 5.24, and 25.1 ± 2.59 versus 32.5 ± 2.86 (P < 0.05). Target LW at 161 d did not affect meiotic progression of oocytes. However, LOW compared to HIGH LW gain between 161 and 175 d resulted in fewer oocytes reaching MII (0.40 versus 0.54; P < 0.05). In conclusion, moderately restricting feed intake impaired follicle growth beyond 3 mm and reduced oocyte meiotic competence. Further, although a carry-over effect of long-term feed restriction on follicle growth was evident, acute changes in feed intake during the 14 d prior to ovary collection had the greatest effect on oocyte nuclear maturation in vitro.

Animal grazing selectivity and plant chemistry issues impact on the potential of Rhagodia preissii as an anthelmintic shrub.

Rhagodia preissii had shown significant in vitro anthelmintic activity in a previous study, we examined the effect of including this shrub in the diet of sheep infected with Trichostrongylus colubriformis. Worm-infected merino wethers were grazed for 7 weeks on either R. preissii or annual pasture, and faecal egg counts (FECs) were conducted weekly. Plant material was collected weekly from eaten and uneaten plants, and analysed for levels of plant secondary metabolites (tannins, oxalates, saponins) and in vitro anthelmintic activity. While mean FECs were consistently lower in sheep grazing R. preissii compared to pasture (reductions of 20-74%), the differences were not significant. There was no relationship between grazing preference (eaten or uneaten) and in vitro anthelmintic activity of plant extracts. The levels of saponins and oxalates did not correlate with grazing preference or in vitro anthelmintic activity, while tannins were not responsible for the anthelmintic activity. While the identity of the grazing deterrent and in vitro anthelmintic compounds remain unknown, the presence of plants which were both highly preferred by the sheep and showed in vitro anthelmintic activity indicates a potential to develop the species as an anthelmintic shrub through selection of shrub populations dominated by such plants.

Offspring born to ewes fed high salt during pregnancy have altered responses to oral salt loads.

Prenatal growth is sensitive to the direct and indirect effects of maternal dietary intake; manipulation can lead to behavioural and physiological changes of the offspring later in life. Here, we report on three aspects of how a high-salt diet during pregnancy (conception to parturition) may affect the offspring's response to high oral salt loads: (i) dietary preferences for salt; (ii) response to salt and water balance and aldosterone and arginine vasopressin (AVP) concentrations after an oral salt challenge; (iii) concentrations of insulin and leptin after an oral salt challenge. We used two groups of lambs born to ewes fed either a high-salt (13% NaCl) diet during pregnancy (S lambs; n = 12) or control animals born to ewes fed a conventional (0.5% NaCl) diet during pregnancy (C lambs; n = 12). Lambs were subjected to short- (5 min) and long-term (24 h) preference tests for a high-salt (13% NaCl) or control diet, and the response to an oral challenge with either water or 25% NaCl solution were also carried out. Weaned lambs born to ewes fed high salt during pregnancy did not differ in their preference for dietary salt, but they did differ in their physiological responses to an oral salt challenge. Results indicate that these differences reflect an alteration in the regulation of water and salt balance as the metabolic hormones, insulin and leptin, were not affected. During the first 2 h after a single salt dose, S lambs had a 25% lower water intake compared to the C lambs. S lambs had, on average, a 13% lower AVP concentration than the C lambs (P = 0.014). The plasma concentration of aldosterone was higher in the S lambs than in the C lambs (P = 0.013). Results suggest that lambs born to ewes that ingest high amounts of salt during pregnancy are programmed to have an altered thirst threshold, and blunted response in aldosterone to oral salt loads.

Iron content of eggs from hens given diets containing organic forms of iron, serine and methyl group donors, or phytoestogens.

1. This investigation included three experiments to determine whether the iron content of egg yolks could be enriched by supplementation of the laying diet with iron bound to organic compounds (Experiment 1), serine and methyl group donors methionine and choline (Experiment 2) or phytoestrogens (Experiment 3). 2. Hens at 34, 54 and 56 weeks of age were given experimental diets for 6, 4 and 4 weeks, respectively, in Experiments 1-3. Yolks from eggs laid over three successive days in the final week of feeding were pooled for each hen for analysis of iron by inductively coupled plasma mass spectrometry. 3. Iron concentration in egg yolk averaged 68-70, 66-71 and 62-69 microg/g in the respective experiments. 4. The addition of bloodmeal (1.22 mg Fe/egg) or phytoestrogens (1.25 mg Fe/egg) increased the total iron content of yolks by over 15% compared with the control diet (1.10 mg Fe/egg), and although this increase was not statistically significant it suggests that the iron content of eggs could be sufficiently manipulated to justify a nutritional claim of iron enrichment.

Exploring the anthelmintic properties of Australian native shrubs with respect to their potential role in livestock grazing systems.

We measured in vitro anthelmintic activity in extracts from 85 species of Australian native shrub, with a view to identifying species able to provide a degree of worm control in grazing systems. Approximately 40% of the species showed significant activity in inhibiting development of Haemonchus contortus larvae. The most active extracts showed IC50 values of 60-300 microg/ml. Pre-incubation with polyvinylpolypyrrolidine removed the activity from some extracts, implicating tannins as the bioactive agent, while in other cases the pre-incubation had no effect, indicating the presence of other anthelmintic compounds. Plant reproductive maturity (onset of flowering or fruiting) was associated with increasing anthelmintic activity in some species. Variability was observed between plants of the same species growing in different environments, while variation between individual plants of the same species within a single field suggests the existence of distinct chemotypes. Significant activity against adult H. contortus worms in vitro was also demonstrated in a limited number of extracts tested against this life stage. Our study indicates that there is potential for Australian native shrubs to play an anthelmintic role in grazing systems, and highlights some plant biology factors which will need to be considered in order to maximize any anthelmintic effects.

Dietary exposure of pregnant ewes to salt dictates how their offspring respond to salt.

We investigated changes in salt preference, food and water intake, renin activity and salt excretion in adult offspring from ewes that were fed a high-salt diet (14% NaCl, high-salt offsrping) or grazed saltbush (saltbush offspring) from day 60 of pregnancy until day 21 of lactation. High-salt offspring were compared to offspring born to ewes consuming a control diet (2% NaCl) and saltbush offspring were compared to offspring from ewes which grazed a control diet of dry pasture. All offspring were weaned at 3 months of age and grazed the same clover-based pasture until testing started at 8 months of age. The preference for a low-salt diet (0.5% NaCl) when offered with an alternative (7% NaCl) did not differ between the offspring groups. High-salt offspring and saltbush offspring had a lower food intake (14% and 27% respectively) and lower water intake (35% and 20% respectively) than their control offspring. Both high-salt offspring and saltbush offspring had lower basal renin activity than their respective controls. After consuming salt, the renin activity of the saltbush offspring continued to be lower than controls whereas the renin activity of the high-salt offspring became similar to controls. In general, the saltbush offspring excreted an oral salt load more rapidly, though this depended on the extent of the salt load. This important adaptation of offspring born to ewes that consumed saltbush during pregnancy may improve their ability to cope with high-salt diets such as saltbush when they consume it themselves. However, the high-salt offspring did not possess such beneficial adaptations.

Feeding pregnant ewes a high-salt diet or saltbush suppresses their offspring's postnatal renin activity.

If ewes consumed a high-salt diet or saltbush during the last 3 months of pregnancy and for 3 weeks after birth, we expected the renin activity of their lamb to be suppressed at birth and at 3 weeks of age. We also expected an increase in the concentration of cations other than sodium in the ewe's milk and an increase in the plasma Na concentration of the lamb at birth. To test these hypotheses, Merino ewes were fed a high-salt diet (14% NaCl) in an animal house and compared to control ewes eating a control diet (2% NaCl). In addition, we compared ewes grazing saltbush (about 13% salt in diet) to ewes grazing pasture from day 60 of pregnancy to 3 weeks after birth. Lambs born to ewes consuming saltbush had 85% lower (P < 0.001) renin activity than offspring from ewes consuming pasture at 3 weeks of age. Similarly, lambs born to ewes consuming a high-salt diet had 20% lower renin activity at birth and 3 weeks (P = 0.07). Feeding ewes a high-salt diet or saltbush altered the mineral composition of the milk; the largest change was a 10% increase in K levels (P 0.05). Consuming a high-salt diet or saltbush lowered the plasma Na of ewes at 130 days of gestation (by 3-5 mmol/l; P < 0.001), but only lambs from ewes fed the high-salt diet had a lower plasma Na at birth (P < 0.05). Suppression of the renin activity of lambs could lead to permanent physiological changes in salt balance in later life.

Reproductive capacity of Merino ewes fed a high-salt diet.

An option to increase the productivity of saline land is to graze sheep on salt-tolerant plants, which, during the summer/autumn period, can contain 20% to 25% of their dry matter as salt. This study assessed the impact of coping with high dietary salt loads on the reproductive performance of grazing ewes. From the time of artificial insemination until parturition, 2-year-old maiden Merino ewes were fed either a high-salt diet (NaCl 13% of dry matter) or control diet (NaCl 0.5% of dry matter). Pregnancy rates, lamb birth weights, milk composition and the plasma concentrations of hormones related to salt and water balance, and energy metabolism were measured. Leptin and insulin concentrations were lower (1.4 ± 0.09 v. 1.5 ± 0.12 ng/ml; (P < 0.05) and 7.2 ± 0.55 v. 8.2 ± 0.83 ng/ml; P < 0.02) in response to high-salt ingestion as was aldosterone concentration (27 ± 2.7 v. 49 ± 5.4 pg/ml; P < 0.05), presumably to achieve salt and water homeostasis. Arginine vasopressin concentration was not significantly affected by the diets, but plasma concentration of T3 differed during gestation (P < 0.02), resulting in lower concentrations in the high-salt group in the first third of gestation (1.2 ± 0.18 v. 1.3 ± 0.14 pmol/ml) and higher concentrations in the final third of gestation (0.8 ± 0.16 v. 0.6 ± 0.06 pmol/ml). T4 concentration was lower in ewes ingesting high salt for the first two-thirds of pregnancy (162 ± 8.6 v. 212 ± 13 ng/ml; P < 0.001). No substantial effects of high salt ingestion on pregnancy rates, lamb birth weights or milk composition were detected.

Increasing the age of gilts at first boar contact improves the timing and synchrony of the pubertal response but does not affect potential litter size.

One hundred and ninety-two Large White/Landrace crossbred gilts were used in this study. The study was conducted in two blocks, with 16 gilts allocated to each of six treatments in each block. The treatments compared the effects on puberty attainment of commencing boar exposure at 161, 182 or 203 days of age, and the effect of first mating gilts at either the pubertal or second oestrus on ovulation rate and early embryo survival. Boar contact took place in a detection-mating area (DMA), and consisted of 20 min/day of full contact with a vasectomized boar greater than ten months of age. Gilts were artificially inseminated at the allocated oestrus, with the reproductive tracts collected at 22.8+/-0.4 days after first mating (mean+/-S.E.M.), and the numbers of corpora lutea and viable embryos recorded. The age at which gilts attained puberty increased with the age at which boar exposure commenced. Mean gilt ages at puberty were 179.5+/-1.6, 191.7+/-1.2 and 210.3+/-0.9 days, respectively, when boar contact commenced at 161, 182 and 203 days of age, P<0.01. Mean days-to-puberty was significantly shorter when boar contact began at 182 and 203 days of age compared to 161 days of age (10.4+/-1.2 and 8.3+/-0.9 days versus 18.9+/-1.5 days, respectively, P<0.01). Similarly, commencing boar exposure at 182 or 203 days of age as opposed to 161 days of age significantly increased the proportion of gilts attaining puberty within 10 days of start of boar exposure (0.67 and 0.70 versus 0.24, P<0.01). Mean days-to-puberty and the synchrony of puberty attainment were similar when boar contact commenced at 182 and 203 days of age. There was no significant effect of mating oestrus or age of gilts at mating on ovulation rate, embryo number or embryo survival. In conclusion, the current data indicate that the timing and synchrony of puberty attainment is significantly improved when first boar exposure of gilts is delayed until 182 days of age. Further, it is evident that within the age range investigated, delaying first mating until the second oestrus does not significantly increase either ovulation rate or embryo number at day 20 post-mating.

Discovery of nonxanthine adenosine A2A receptor antagonists for the treatment of Parkinson's disease.

During a program to investigate the biochemical basis of side effects associated with the antimalarial drug mefloquine, the authors made the unexpected discovery that the (-)-(R,S)-enantiomer of the drug is a potent adenosine A2A receptor antagonist. Although the compound was ineffective in in vivo animal models of central adenosine receptor function, it provided a unique nonxanthine adenosine A2A receptor antagonist lead structure and encouraged the initiation of a medicinal chemistry program to develop novel adenosine A2A antagonists for the management of Parkinson's disease (PD). The authors have synthesized and screened more than 2,000 chemically diverse and novel adenosine A(2A antagonists. Early examples from two distinct chemical series are the thieno[3,2-dy]pyrimidine VER-6623 and the purine compounds VER-6947 and VER-7835, which have high affinity at adenosine A2A receptors (K(i) values 1.4, 1.1, and 1.7 nmol/L, respectively) and act as competitive antagonists. In particular, VER-6947 and VER-7835 demonstrate potent in vivo activity reversing the locomotor deficit caused by the D2 receptor antagonist haloperidol, with minimum effective doses comparable with that of KW6002 (0.3 to 1 mg/kg). In conclusion, the authors have discovered potent, selective, and in vivo active nonxanthine adenosine A2A antagonists that have considerable promise as a new therapy for PD.

Shearing during pregnancy--review of a policy to increase birthweight and survival of lambs in New Zealand pastoral farming systems.

Over the past decade, lambing percentages have risen in conjunction with a rise in the percentage of multiple lambs born. Multiple-born lambs are smaller than their singleton counterparts and are particularly susceptible to starvation-exposure. Any technique that can increase the birthweight or thermoregulatory capability, or both, of otherwise lightweight lambs has the potential to substantially increase survival of multiple-born lambs. In the United Kingdom under housed conditions, shearing during pregnancy has been shown to increase both the birthweight and thermoregulatory capability of newborn lambs. However, shearing during pregnancy under pastoral conditions has failed to consistently affect the newborn lamb's thermoregulatory capability. In contrast, under New Zealand's pastoral conditions shearing during pregnancy has been found to increase birthweight, but results have been inconsistent in both magnitude and birth-rank specificity. Increase in feed intake by the dam, types of shearing comb used and changes in gestation length do not explain the variation observed. When studies involving shearing during pregnancy are collated it becomes apparent that there are two criteria that must be met to achieve a birthweight response. Firstly, the dam must have the potential to respond and secondly, the dam must have the means to respond. Any increase in lamb survival through shearing during pregnancy would be predominantly through an increase in birthweight of otherwise lightweight lambs. However, in the few studies conducted to date, an increase in birthweight has not resulted in a statistically significant increase in survival. For an increase in birthweight to have a positive effect on lamb survival, lambs must be otherwise destined to be born within a birthweight range in which survival rate is below optimum (<4.0 kg), and the increase in birthweight observed must move a significant proportion of otherwise lightweight lambs into a higher range of survival rate.

Agonist-induced functional desensitization of recombinant human 5-HT2 receptors expressed in CHO-K1 cells.

The desensitization characteristics of recombinant human 5-HT(2A), 5-HT(2B), and 5-HT(2C) receptors (VSV and INI isoforms) stably expressed in CHO-K1 (Chinese hamster ovary) cells was investigated by calcium fluorimetry. Comparative desensitization characteristics of the agonists 5-HT, m-chlorophenylpiperazine (mCPP), and 2,5-dimethoxy-4-iodoamphetamine hydrobromide (DOI) were performed. Human 5-HT(2C (INI)) receptors exhibited a greater degree of desensitization to all agonists tested than edited 5-HT(2C (VSV)) receptors. A 2-hr exposure to 5-HT resulted in a significantly larger reduction in response upon re-exposure to 5-HT at 5-HT(2C (INI)) receptors, as compared to 5-HT(2C (VSV)) receptors (72% and 47% respectively, P < 0.01). Both receptor isoforms were expressed at similar densities. Human 5-HT(2B) receptors exhibited the most dramatic degree of desensitization, with prior exposure to 5-HT reducing subsequent response to 5-HT by 80%, with an extremely rapid time-course (t(1/2) < 5 min). The response at 5-HT(2A) receptors was reduced by 54%. The partial agonists mCPP and DOI also elicited desensitization, generally in line with their relative efficacies at each receptor, but exhibited more rapid kinetic profiles than 5-HT. Heterologous desensitization of an endogenously expressed G(q/11)-coupled purinergic receptor was also examined following preincubation of the cell lines with 10 microM 5-HT. Only stimulation of 5-HT(2C (VSV)) receptors resulted in a profound attenuation of subsequent ATP mediated responses. These results demonstrate differing degrees of both homologous and heterologous desensitization of 5-HT(2) receptors. Additionally, the different desensitization profiles of 5-HT(2C (INI)) and 5-HT(2C (VSV)) receptor may be due to signal transduction differences caused by RNA editing.

Characterisation of agonist binding on human 5-HT2C receptor isoforms.

The 5-HT2C receptor is expressed in different isoforms as a result of mRNA editing. Both INI (unedited) and VSV (a fully edited version) isoforms are abundant in rat brain. The VSV isoform lacks the high affinity recognition site for 5-HT, which may be caused by low efficiency coupling to G-proteins. In this study we have investigated the pharmacology of the agonist binding site of these two isoforms of the 5-HT2C receptor. The VSV isoform was expressed in Chinese hamster ovary cells (CHO) and the INI isoform in both Chinese hamster ovary cells and human embryonic kidney cells (HEK-293). Saturation analysis using [3H]5-HT revealed high and low affinity recognition sites on the INI isoform in both cell types whilst the VSV isoform did not have the high affinity binding site for [3H]5-HT. Displacement studies were undertaken using [3H]5-HT to label the receptors. In these studies the affinity of agonists (5-HT, Ro600175 ((S)-2-(6-Chloro-5-fluoroindol-1-yl)-1-methylethylamine), MK212 (6-Chloro-2-(piperazinyl) pyrazine), mCPP (1-(m-chlorophenyl)-piperazine), TfMPP (N-(m-trifluoromethylphenyl)piperazine), DOI (1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane), DOB (1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane) and 8OH-DPAT (8-hydroxy-2-(di-N-propylamino)tetralin) was higher at the INI isoform, whilst antagonist affinity (ketanserin and mesulergine) did not change between the two receptor isoforms. There were no differences between the INI isoform expressed in the CHO and HEK-293. This suggests that the INI isoform of the 5-HT2C receptor is pharmacologically similar to the VSV form of the 5-HT2C receptor but that it couples more efficiently to G-proteins.

Metabolic responses to mid-pregnancy shearing that are associated with a selective increase in the birth weight of twin lambs.

Previous studies have shown that shearing pregnant ewes at mid- or late-pregnancy is associated with an increase in lamb birth weight. The present study was designed to investigate metabolic responses that may underlie this response. Single- and twin-bearing ewes were either unshorn or shorn at mid-pregnancy (Day 69 of pregnancy; P69), and insulin, glucose and epinephrine challenges were conducted on P109-111 and P132-134. Shearing increased the birth weight of twin lambs by over 1 kg (P < 0.001) without having any effect on singleton birth weight. This response was associated with a 10-20% reduction in the insulin response to a glucose challenge (P < 0.05) without a change in glucose clearance following either glucose or insulin challenges. The lipolytic response to epinephrine challenge increased as pregnancy progressed, but was not associated with the increased birth weight of twin lambs born to shorn ewes. By late pregnancy, a 25% reduction in maternal IGF-I concentration and a two- to threefold increase in maternal IGFBP-1 concentration (P < 0. 05) associated with shearing were observed. The increase in lamb birth weight associated with mid-pregnancy shearing may have been associated with an increase in the non-insulin dependent uptake of glucose by the placental-fetal unit.

Functional characterization of agonists at recombinant human 5-HT2A, 5-HT2B and 5-HT2C receptors in CHO-K1 cells.

1. The goal of this study was to characterize the agonist pharmacology of human 5-HT2A, 5-HT2B and 5-HT2C (VSV) receptors expressed in CHO-K1 (Chinese hamster ovary) cells. 2. We used a fluorometric imaging plate reader (FLIPR) which allows rapid detection of rises in intracellular calcium levels upon the addition of agonists. 3. Stimulation of all three receptors by 5-HT caused a robust concentration dependent increase in intracellular calcium levels. No such effect was observed from non-transfected control CHO-K1 cells. 4. The rank order of potency of agonists at the different receptor subtypes varied. Tryptamines, BW-723C86, d-norfenfluramine, Ro 60-0175 and LSD exhibited the following rank order of potency; 5-HT2B>5-HT2C>5-HT2A. Piperazines such as m-Chlorophenylpiperazine (mCPP), ORG-12962, MK-212 and also ORG-37684 exhibited a rank order of potency of 5-HT2C>5-HT2B>5-HT2A. The phenylisopropylamines DOI and DOB had a rank order of 5-HT2A>5-HT2B>5-HT2C. 5. Many agonists tested had partial agonist actions when compared to 5-HT, and a wide range of relative efficacies were exhibited, which was cell line dependent. For example, mCPP had a relative efficacy of 65% at 5-HT2C receptors but <25% at either 5-HT2A or 5-HT2B receptors. 6. Interpretation of literature values of functional assays using different cell lines, different receptor expression levels and different receptor isoforms, is complex. Species differences and the previous use of antagonist radioligands to characterize agonist potency in binding assays emphasizes the importance of studying agonists in the same experiment using the same assay conditions and parental cell lines.